Dissertations / Theses on the topic 'Communicable diseases'

La prevenció i el control de les malalties transmissibles depenen, en gran mesura, de la detecció ràpida i eficaç. Els mètodes convencionals per a la detecció d'un patogen, com ara el cultiu microbiològic, generalment requereixen molt de temps, són laboriosos, necessiten personal qualificat i no són aptes com a eines de diagnòstic en el punt d'atenció. El desenvolupament de mètodes de diagnòstic ràpid en el marc dels criteris ASSURED, de l'anglès (A) Affordable, (SS) Sensitive i Didàctiques, (O) User-friendly, (R) Rapid and Robust, (I) Equipment free, and (d) Deliverable to those who need it, Affordable, descrits per l'Organització Mundial de la Salut (OMS), es troben en l'actualitat sota intens estudi. Per tant, la present tesi aborda el disseny i desenvolupament d'estratègies, mètodes i materials per millorar les prestacions analítiques i simplificar el procediment en proves de diagnòstic ràpid, incloses noves estratègies de preconcentració en fase sòlida, mètodes d'amplificació i materials avançats, així com la seva integració en diferents plataformes (principalment biosensors basats en detecció electroquímica i proves en paper amb lectura òptica). En tots els casos, les aplicacions seleccionades es centren en malalties transmissibles, inclosos els patògens transmesos pels aliments i els micobacteris. Amb aquesta finalitat, es comparen dues plataformes basades en paper en diferents configuracions (flux lateral i vertical) en termes de rendiment analític per a la detecció de Mycobacterium. Per aconseguir una millora addicional en el límit de detecció, s'estudia la preconcentració prèvia dels bacteris per separació immunomagnètica. En segon lloc, s'avaluen i es comparen en termes del seu rendiment analític la detecció simultània de Salmonella i E. coli mitjançant flux lateral d'àcid nucleic amb lectura visual i genosensors electroquímics. Si bé aquests mètodes requereixen PCR de doble etiquetatge per a l'amplificació, es poden adaptar fàcilment a termocicladors portàtils que funcionen amb bateries per poder ser realitzats en entorns amb recursos limitats per satisfer les demandes de diagnòstic ASSURED. A més, també es presenta en aquesta tesi la síntesi de polímers magnètics impresos molecularment, per tal de reemplaçar les partícules magnètiques biològicament modificades, i prenent com a model la detecció de biotina i de molècules biotinilades. A més, es realitza la caracterització del material mitjançant diferents tècniques analítiques i es compara, en tots els casos, amb el polímer no imprès. Aquest material biomimètic mostra un gran potencial per a la preconcentració i detecció d'una àmplia gamma d'analits. Malgrat tot els progressos, les tècniques d'amplificació d'àcid nucleic segueixen essent necessàries per assolir els límits de detecció requerits en algunes malalties transmissibles. En aquest sentit, les tècniques d'amplificació isotèrmiques són bons candidats per dur a terme proves de diagnòstic en entorns on la PCR pot ser una barrera. En concret, es descriu en aquest treball la detecció d' E.coli mitjançant un genosensor electroquímic basat en l'amplificació isotèrmica. En aquest cas, s'optimitza la lectura electroquímica per voltamperometria d'ona quadrada en elèctrodes d'un sol ús comparant dues estratègies de marcatge del producte amplificat. És important ressaltar que totes aquestes estratègies apunten a ser utilitzades com a eines per millorar les proves de diagnòstic ràpid en entorns de baixos recursos, per interrompre la cadena d'infecció de malalties transmissibles i permetre, per tant, un tractament precoç.<br>La prevención y el control de las enfermedades transmisibles dependen, en gran medida, de la detección rápida y eficaz. Los métodos convencionales para la detección de un patógeno, como el cultivo microbiológico, generalmente requieren mucho tiempo, son laboriosos, necesitan personal cualificado y no son aptos como herramientas de diagnóstico en el punto de atención. El desarrollo de métodos de diagnóstico rápido en el marco de los criterios ASSURED, del inglés (A) Affordable, (SS) Sensitive and Specific, (U) User-friendly, (R) Rapid and Robust, (E) Equipment free, and (D) Deliverable to those who need it, Affordable, descritos por la Organización Mundial de la Salud (OMS), se encuentran en la actualidad bajo intenso estudio. Por lo tanto, la presente tesis aborda el diseño y desarrollo de estrategias, métodos y materiales para mejorar las prestaciones analíticas y simplificar el procedimiento en pruebas de diagnóstico rápido, incluidas nuevas estrategias de preconcentración en fase sólida, métodos de amplificación y materiales avanzados, así como su integración en diferentes plataformas (principalmente biosensores basados en detección electroquímica y pruebas en papel con lectura óptica). En todos los casos, las aplicaciones seleccionadas se centran en enfermedades transmisibles, incluidos los patógenos transmitidas por los alimentos y las micobacterias. Con este fin, se comparan dos plataformas basadas en papel en diferentes configuraciones (flujo lateral y vertical) en términos del rendimiento analítico para la detección de Mycobacterium. Para lograr una mejora adicional en el límite de detección, se estudia la preconcentración previa de las bacterias por separación inmunomagnética. En segundo lugar, se evalúan y se comparan en términos de su rendimiento analítico la detección simultánea de Salmonella y E. coli mediante flujo lateral de ácido nucleico con lectura visual y genosensores electroquímicos. Si bien estos métodos requieren PCR de doble etiquetado para la amplificación, se pueden adaptar fácilmente a termocicladores portátiles que funcionan con baterías para poder ser realizados en entornos con recursos limitados para satisfacer las demandas de diagnóstico ASSURED. Además, también se presenta en esta disertación la síntesis de polímeros magnéticos impresos molecularmente, con el objeto de reemplazar las partículas magnéticas biológicamente modificadas, y tomando como modelo la detección de biotina y moléculas biotiniladas. Además, se realiza la caracterización del material mediante diferentes técnicas analíticas y se compara, en todos los casos, con el polímero no impreso. Este material biomimético muestra un gran potencial para la preconcentración y detección de una amplia gama de analitos. A pesar de todo este progreso, las técnicas de amplificación de ácido nucleico siguen siendo necesarias para alcanzar los límites de detección requeridos en algunas enfermedades transmisibles. Las técnicas de amplificación isotérmica son buenos candidatos para llevar pruebas de diagnóstico en entornos donde la PCR puede ser una barrera. En concreto, se describe en esta disertación la detección de E. coli mediante un genosensor electroquímico basada en la amplificación isotérmica. En este caso, se optimiza la lectura electroquímica por voltamperometría de onda cuadrada en electrodos desechables comparando dos estrategias de marcaje del producto amplificado. Es importante resaltar que todas estas estrategias apuntan a ser utilizadas como herramientas para mejorar las pruebas de diagnóstico rápido en entornos de bajos recursos, para interrumpir la cadena de infección de enfermedades transmisibles y permitir, por tanto, un tratamiento precoz.<br>The prevention and control of communicable disease rely, to a large extent, on effective and early detection approaches. Conventional methods for the detection of a pathogen, such as microbiological culture, are usually time-consuming, laborious, need skilled personnel and are non-amenable to point-of-care diagnostic tools. The development of rapid diagnostic methods in the framework of the ASSURED criteria as (A) Affordable, (SS) Sensitive and Specific, (U) User-friendly, (R) Rapid and Robust, (E) Equipment free, and (D) Deliverable to those who need it, outlined by the World Health Organization (WHO), are under intensive study. Therefore, the present dissertation addresses the design and development of strategies, methods and materials to improve the analytical performance and to simplify the analytical procedure in rapid diagnostic tests, including novel solid-phase preconcentration strategies, amplification methods and advanced materials, as well as their integration in different platforms (mainly biosensors based on electrochemical detection and paper-based strips for optical readout). In all instances, the applications selected are focused on communicable diseases, including foodborne pathogens and mycobacteria. Therefore, two paper-based platforms in different configurations (nucleic acid lateral and vertical flow) are compared in terms of the analytical performance for the detection of Mycobacterium. In order to achieve a further improvement in the limit of detection, the preconcentration of the bacteria is performed by immunomagnetic separation. Secondly, the simultaneous detection of Salmonella and E. coli by nucleic acid lateral flow with visual readout and electrochemical genosensing are evaluated and compared in terms of their analytical performance. Although these methods required double-tagging PCR for amplification, portable, battery-powered thermocyclers can easily be adapted for resource-constrained settings to meet the demands for ASSURED diagnosis. Furthermore, the synthesis of Magnetic Molecularly Imprinted Polymers, in order to replace biological-modified magnetic particles is also presented in this dissertation, taking as a model the detection of biotin and biotinylated molecules with outstanding performance. Moreover, the characterization of the material is performed by different analytical techniques and compared, in all instances, with the non-imprinted polymer. This biomimetic material shows a great potential for the preconcentration and detection of a huge range of analytes. Despite all these progress, nucleic acid amplification techniques are still necessary to reach the challenging limits of detection required in some communicable disease. Isothermal amplification techniques are good candidates to bring sensitive diagnostic tests in places where the PCR can be a barrier. In detail, the electrochemical genosensing of E. coli based on isothermal amplification is also described in this dissertation. In this approach, the electrochemical readout by square-wave voltammetry on disposable electrodes is optimized comparing two different labelling approaches. It is important to highlight that all these strategies aim to be used as tools for the improvement of rapid diagnostic test in low resource settings, to interrupt the chain of infection of communicable diseases and enabling the rapid treatment.

BACKGROUND:Over the past two decades, international health policies focusing on the fight against the human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS), tuberculosis (TB), malaria, and those diseases that address maternal and child health problems, among others, have skewed disease control priorities in China and other Asian countries. Although these are important health problems, an epidemic of chronic, non-communicable diseases (NCDs) in China has accounted for a much greater burden of disease due to the ongoing rapid socioeconomic and demographic transition.DISCUSSION:Although NCDs currently account for more than 80% of the overall disease burden in China, they remain very low on the nation's disease control priorities, attracting marginal investment from central and local governments. This leaves the majority of patients with chronic conditions without effective treatment. International organizations and national governments have recognized the devastating social and economic consequences caused by NCDs in low- and middle-income countries, including China. Yet, few donor-funded projects that address NCDs have been implemented in these countries over the past decade. Due to a lack of strong support from international organizations and national governments for fighting against NCDs, affected persons in China, especially the poor and those who live in rural and less developed regions, continue to have limited access to the needed care. Costs associated with frequent health facility visits and regular treatment have become a major factor in medical impoverishment in China. This article argues that although China's ongoing health system reform would provide a unique opportunity to tackle current public health problems, it may not be sufficient to address the emerging threat of NCDs unless targeted steps are taken to assure that adequate financial and human resources are mapped for effective control and management of NCDs in the country.SUMMARY:The Chinese government needs to develop a domestically-driven and evidence-based disease control policy and funding priorities that respond appropriately to the country's current epidemiological transition, and rapid sociodemographic and lifestyle changes.

Non-communicable diseases (NCDs) have reached epidemic proportions globally and in South Africa. This thesis is situated within the health equity framework. The aim is to assess the extent of wealth related inequalities in NCDs and to assess the impact of the social determinants of health in mediating these inequalities. Data from the first South African National Health and Examination Survey (SANHANES-1) and wave 4 of the South African National Income Dynamics Study (NIDS) were used. The methods used include the concentration curve, concentration index and decomposition analysis to assess the drivers of socioeconomic inequality in NCDs and some causes of NCDs including smoking, obesity, high blood pressure; use of screening services and effective coverage for hypertension management. The prevalence of smokers is 18.7%, the population average BMI is 26.38 kg/m2, and the prevalence of hypertension is 29.7%. The distribution of these risk factors is pro-wealthy with concentration indices ranging from 0.048 for hypertension, 0.057 for smoking prevalence to 0.115 for obesity. While these risk factors are prevalent amongst the wealthy, the outcomes are worse amongst the poor. The concentration index for expenditure on cigarettes is strongly pro-poor, (-0.130) compared to the prowealthy smoking prevalence. The hypertensive poor suffer more severe hypertension with a concentration index of -0.054 for depth and -0.079 for severity, respectively. Obesity affects the wealthiest the most. However, the overweight adults who are poor tend to suffer more severe obesity as shown by a relatively smaller concentration index of depth (0.015) and severity (0.033) respectively. The overall utilisation of screening services is below 50% for eligible respondents. The two wealthiest quintiles benefit disproportionately more than they should, given their share of the population. This is particularly true for diabetes and cholesterol with a concentration index of 0.27 for cholesterol, 0.129 for diabetes and 0.052 for hypertension. Adults that do not take up screening services are predominantly the black race group, poor, rural, male, unemployed and uninsured. Only 23% of those with hypertension are diagnosed, on treatment and are controlled. Wealth-related variables such as education, wealth, health insurance coverage and province of residence drive most of the observed pro-wealthy inequalities in this thesis. Wealthier adults benefit to a larger extent from the care cascade, compared to the poor. Therefore, until there is a substantial increase in early diagnosis and effective treatment, high levels of mortality from NCDs will persist in South Africa. And until the poor are prioritised through radical policy change in all economic sectors, the observed inequalities will continue.

Mosquito-borne diseases account for hundreds of thousands of deaths each year, highlighting the need for successful intervention methods, which can be targeted at either the pathogen, mosquito vector, or human host. This thesis aims to contribute to better intervention methods focused against malaria and dengue by either (i) improving available research tools, (ii) enhancing the understanding of a promising intervention method or (iii) designing new intervention candidates. Firstly, a superior method for studying in vitro malaria infection of the liver is shown, with implications for vaccine and drug interventions. Secondly, the biology of Wolbachia infection in Anopheles gambiae mosquitoes in the context of the target of rapamycin signalling cascade is investigated in an attempt to improve our understanding of its malaria inhibitory phenotype and inability to stably infect An. gambiae mosquitoes. Finally, an algorithm is developed for the design of a hypothesis driven conservation-based vaccine against viral mosquito diseases with a particular focus on dengue.

The thesis is based on studies performed at Department of Medicine and Public Health during XXXIV doctoral course, curriculum “Epidemiology, preventive, rehabilitative and occupational medicine’ (Coordinator Prof. Claudio Ferri) from 2018-2021, under supervision Prof. Domenico Passafiume and, Prof. Dina Di Giacomo. Doctoral Programme expected learning of the experimental techniques and methodologies of scientific research, through access to different types of patients by age, clinical pathology, psychopathology and, need for intervention. Doctoral Course was divided in two dimensions: a) Clinical research training b) Scientific thematic study of the clinical area Clinical research training was performed by participation in research projects on patients with chronic diseases with collaboration in two Academic Laboratories: the Psychology and Psychoncology Laboratory (Resp. Prof. Dina Di Giacomo), Neuropsychology Laboratory (Resp. Prof. Domenico Passafiume) and, San Salvatore Hospital ASL1 Abruzzo; the activities have been finalized in scientific productivity reported in the appendix of the publications and in the appendix of the presentation at national and international congresses. Research activities was conducted on young, adult and, old population affected by chronic diseases to explore emotional characteristics, protective and health risk factors during medical treatments and/or survivorship. Person-centered approach was applied by clinical interviews, measurements in one-to-one interaction, and/or online survey. Studies can be grouped into 3 research lines: a) Exploration of the emotional trigger and strengths for better psychological adaptation during survivorship in the management of chronic diseases; b) Promotion of the Healthy Aging in the third generation; c) Investigation of the risk factors for post-traumatic stress symptoms during the global Covid-19 pandemic Studies conducted in research field on the exploration of the emotional triggers and the strengths for better psychological adaptation during survivorship in the management of chronic diseases aimed to: a) highlight the emotional impact and long-term effects of surgical interventions (kidney transplant, vascular access surgery) on the adult population diagnosed with chronic kidney diseases; b) identify the strengthens (personality traits, cognitive reserve) and risk factors for the development of psychological well-being after a diagnosis of chronic disease with the possible presence of other clinical comorbidities; c) to examine modifications in satisfaction degree and Quality of Life after surgery and intensive clinical treatments; d) detect the long-term effects of chronic diseases (disorders of the cardiovascular, digestive and locomotor systems) on the psychological adaptation; e) evaluate impact on mental health and body image perception following surgery. Studies conducted in research field on promotion of the Healthy Aging in the third age generation aimed: a) to examine the digital confidence on old population and to identify behavioral models for the technology, related at the cognitive ability; b) to identify the role of the cognitive reserve in adhering to digital solutions among the elderly; c) to investigate the impact of the technophobia on the Quality of Life in inclusion perspective; d) to experiment intervention protocols, cognitive stimulation and Montessori’s method in subjects with Alzheimer's type dementia. Studies conducted in the field of research on investigation of the risk factors for post-traumatic stress symptoms during the global Covid-19 pandemic aimed to: a) investigate the risk of developing post-traumatic stress disorder (PTSD) among health workers (medical doctor, nurses, and mental health professionals); b) investigate the relationships among emotional distress and personality traits; c) analyze the emotional dimensions of the health workers during the COVID-19 pandemic; d) to determine the psycholog

Background: Rapid economic growth in mainland China has been accompanied in recent years by rising levels of inequality and a growing burden of non-communicable disease (NCD), though little is known at present about the relations between these forces. This thesis makes use of data from a large sample of older men and women in Guangzhou, one of China’s most developed cities, to examine the relations between inequality, inequity and non-communicable disease. Objectives: This thesis addresses two research questions: what is the relationship between inequality/inequity and non-communicable disease in China; and what are the implications of this relationship for health policy in China. These two questions lead to two working hypotheses: first, that inequalities may be both a cause and consequence of NCDs in China, potentially creating a vicious cycle which reinforces inequality and inequity; and second, that reducing dependence on out of pocket payments as a source of healthcare finance may help to prevent the continuation of the inequality-NCD cycle. Methods: I used data from the Guangzhou Biobank Cohort Study (GBCS), including 30,499 men and women aged 50 or over from Guangzhou and multi-variable regression methods to examine associations of socioeconomic position at four life stages (childhood, early adulthood, late adulthood and current) with several health outcomes: self-rated health, chronic obstructive pulmonary disease, metabolic syndrome and markers of immunological inflammation (white blood cells, granulocytes and lymphocytes). These analyses related to the hypothesis that inequalities may be a cause of non-communicable disease in China. I also examined whether inequity may be a consequence of non-communicable disease by measuring whether horizontal inequity (deviation from the principle of equal access to healthcare for equal need) was greater for treatment of NCDs than for general healthcare. I tested this using both concentration index methods and multi-variable regression models. For comparative purposes, I conducted these analyses in data from three settings: Guangzhou, Hong Kong and Scotland (UK). Results: I found that socioeconomic deprivation across the life course was associated with poorer self-rated health, higher risk of COPD, higher white cell and granulocyte cell counts and (in women only) higher risk metabolic syndrome and higher lymphocyte cell counts. I also found evidence of pro-rich inequity in utilisation of treatment for three major non-communicable conditions (hypertension, hyperglycaemia and dyslipidaemia) in Guangzhou, whilst there was no evidence of inequity in general healthcare utilisation (doctor consultations and hospital admissions) or treatment of gastric ulcer. Conclusion: My findings gave qualified support for the idea that socioeconomic inequalities may contribute to some, though not all, non-communicable diseases in China. Moreover, the mechanisms which link socioeconomic inequality to NCDs in China remain unclear. My results also supported the suggestion that a rising burden of non-communicable disease may contribute to greater pro-rich inequity in healthcare utilisation, especially for conditions which are chronic and asymptomatic. As rates of NCDs continue to rise in China and other developing countries, policies to prevent and treat common NCDs may be improved by a clearer understanding of how inequality is related to non-communicable disease.<br>published_or_final_version<br>Community Medicine<br>Doctoral<br>Doctor of Philosophy

In the human gastric mucosa, an inflammatory response stimulated by H. pylori infection can lead to gastric cancer and peptic ulcer disease. Expression of the i1 active variant of Vacuolating Cytotoxin A (VacA) by the colonising bacterial strain has been identified as an independent risk factor for disease. VacA skews the adaptive immune response towards a regulatory phenotype to promote persistent H. pylori colonisation. In H. pylori-infected individuals, regulatory T cells (Tregs), which suppress inflammation through mechanisms including interleukin-10 (IL-10) production, are thought to play a role in protection against extra-gastric diseases such as multiple sclerosis and oesophageal cancer. IL-10 is an immunomodulatory cytokine which is expressed by several immune cell types including regulatory B cells (Bregs), whose role in H. pylori infection is unclear. Blood was donated by uninfected and infected patients, and those who underwent successful eradication of their H. pylori infection. A flow cytometry antibody panel was developed to quantify the relative frequencies of peripheral blood Bregs and Tregs, and investigate differences according to H. pylori status. Mice were also infected with H. pylori to determine VacA i1 versus i2 differences in the induced regulatory B and T cell frequencies. Stool samples were collected from patients to develop a VacA i-region PCR-based diagnostic test. Results showed that compared to during H. pylori infection, the proportion of IL-10-producing Tregs in the peripheral blood of patients declined after successful eradication therapy. A pilot study in mice revealed B lymphocytes to be another important source of IL-10, and the population expanded after H. pylori infection. In a study of H. pylori-positive, H. pylori-negative and H. pylori-eradicated patients, there were no significant differences in peripheral blood Breg or IL-10+ Breg frequencies. Data from an expanded mouse study using blood and spleen showed that VacA variants in a colonising H. pylori strain did not induce differences in Breg or Treg frequencies 9 weeks after wildtype or mutant H. pylori SS1 infection. The H. pylori 16S gene was successfully detected in stool DNA samples and could be used to determine infection status, but the development of a vacA i-region PCR-based typing stool test was unsuccessful. Previous work in the research group has identified how Treg frequencies are associated with H. pylori infection and disease. While Bregs are capable of producing IL-10 after stimulation, their role in H. pylori infection in mice and humans appears to be limited. The consistency of peripheral blood Treg frequencies in patients from their infected state until two years post-eradication is a start to understanding whether H. pylori-induced extra-gastric protection may also be maintained after eradication. While stool remains a promising resource for non-invasively diagnosing H. pylori infection worldwide, there are strong concerns about contamination and reproducibility which are unlikely to be overcome for use in a clinical setting.

Background: The 2009-10 influenza A(H1N1) pandemic was the first pandemic influenza of the twenty-first century and presented the first major opportunity for the use of influenza vaccines en-masse during a pandemic scenario. National anticipatory policies of pandemic influenza vaccine preparedness were implemented, and vaccine guarantee agreements were activated. Large quantities of vaccines were purchased and made available to identified citizens over the course of the pandemic. The use of pandemic influenza vaccines has been examined in this research. Methods: A comparative health policy approach in five study countries (Sweden, New Zealand, Japan, Singapore, and Canada) was conducted. Qualitative interviews (n= 36) were undertaken in each country with key pandemic influenza response personnel (n = 39). Participants included public health officials, policy makers and clinicians engaged at national country response level. Interviews facilitated discussions surrounding the 2009-10 influenza A(H1N1) pandemic response and use of vaccines. Documentary examination of available records supplemented the analysis of the interview data. Results: Several interview themes were identified following data analysis of the use of pandemic vaccines in the study countries. Themes of the vaccine use included: single or multiple vaccine supplier routes; hemisphere variation; historical pandemic legacy; targeted populations; setting vaccination priorities; side effect concerns; perceived effectiveness of vaccines during the pandemic influenza response. The themes which were most prominent comprised the sourcing and distribution of the vaccines during the response and the associated communication challenges. The necessary prioritisation of vaccines caused extensive discussions and uneasiness by the pandemic influenza response personnel as the initial vaccines arrived in small quantities and required allocation, especially in circumstances where country’s intended for all/most citizens to eventually have access to the vaccine. The variation in timing of the vaccination campaigns and disease activity would suggest that subsequent influenza wave morbidities and mortalities could have been reduced if vaccines had been available more promptly. The southern hemisphere country, New Zealand, exemplified the circumvention of vaccine safety concerns through the use of a trivalent vaccine inclusive of H1N1. Conclusions: Pandemic vaccines were the cornerstone of two countries responses and were associated with high uptake rates. Vaccine discussions, such as prioritisation and essential workers estimates, can be established during interpandemic phases by pandemic influenza response personnel. The use of annual seasonal influenza vaccines that are inclusive of the novel pandemic influenza strain should play a greater role in future pandemic influenzas, should the vaccination campaign timing be appropriate, as this may reduce public anxiety concerning the perceived safety of novel vaccines. The use of the 2009-10 influenza A(H1N1) pandemic vaccines had varied in success and the lessons learnt from this event have important implications for future policy. Pandemic influenza response personnel are recommended to prepare as fully as possible during this interpandemic period.

Epstein Barr Virus (EBV) is a major risk factor in Multiple Sclerosis (MS), via as yet unclear mechanisms. Several hypotheses have been proposed to explain how EBV infection could cause MS and the aim of this thesis was to better understand the mechanisms of action of EBV in the context of MS studying a) the role of EBV in myelin antigen presentation by B cells and b) the association of HERVs with MS. In a non-human primate experimental autoimmune encephalomyelitis (EAE) model, an EBV-related lymphocryptovirus enables B cells to protect a proteolysis-sensitive immunodominant myelin oligodendrocyte glycoprotein (MOG) peptide (residues 35- 55) against destructive processing. This facilitates its cross-presentation to autoaggressive cytotoxic MHC-E-restricted cytotoxic T cells. The present study extends these observations to human B cells and identifies a key role of autophagy. EBV infection upregulated antigen presentation-related markers on B cells and activated the cross-presentation machinery. Although human MOG protein was degraded less in EBV-immortalized B-lymphoblastoid cell lines (LCL) than in uninfected B cells, induction of cathepsin G activity by EBV led to total degradation of the immunodominant peptides. Inhibition of cathepsin G or citrullination of the arginine residue within a LC3-interacting regions (LIR) motif of immunodominant MOG peptides abrogated their degradation. Internalized MOG co-localized with autophagosomes, which may protect it from destructive processing. Thus, EBV infection switched MOG processing in B cells from destructive to productive possibly facilitating cross-presentation of disease-relevant epitopes to CD8+ T cells. This mechanism could facilitate presentation of myelin autoantigens that may be involved in MS induction and progression. The first part of this thesis shows a possible EBV-mediated mechanism involved in MS pathogenesis, but it is likely that different mechanisms act alternatively or cumulatively in different individuals based on environmental and genetic differences. A further mode of action of EBV is through the activation of Human Endogenous Retroviruses (HERVs). In normal conditions HERVs are silenced or expressed at low levels, but in some pathological cases, like MS, their expression is higher than in the healthy population. We performed a systematic review and meta-analysis of the literature on the association between HERVs and MS. The systematic review suggested a strong association between HERV expression and MS, in particular with the HERV-W family. The meta-analysis showed odds ratios of 22, 44, and 6 for the expression of MSRVpol in serum/plasma, MSRVenv in PBMC and MSRVpol in CSF respectively. Furthermore, we confirmed the association experimentally. An increased expression of MSRV/HERV-Wenv and TLR4 RNA was detected in blood of MS patients compared with control groups and the viral protein Env was expressed mainly by B cells and monocytes, but not by T cells. Our finding that EBV infection can induce the expression of MSRV/HERV-Wenv is consistent with previous reports in the literature. We also established that such increased expression was not due to a repression of retroviral restriction factors in LCL. A further connection between HERVs and MS is supported by the observation that people infected by HIV may have a lower risk of developing MS than the HIV non- infected, healthy population. We found that the expression of MSRV/HERV-Wenv RNA in HIV-infected people was lower than in MS patients and similar to healthy controls. Nevertheless, there was no difference in MSRV/HERV-Wenv expression between antiretroviral drug -treated and -untreated HIV patients. The expression of MSRV/HERV-Wenv was also detected in vitro in LCL treated with different classes of antiretroviral treatments (ART) and only Efavirenz (NNRI) reduced MSRV/HERV- Wenv expression. In conclusion, taking in consideration the multifactorial aetiology of MS, it is likely that EBV infection and increased expression of MSRV/HERV-W are significant contributing factors in genetically predisposed individuals. This thesis helps to better understand the mechanisms of action of EBV and HERVs in the context of MS.

Background Community acquired pneumonia (CAP) is a common illness in patients admitted to hospital, accounting for nearly 10% of acute medical admissions. Despite widespread use of antimicrobial therapy, morbidity and mortality from this disease remains high. In recent years in the UK, there have been significant developments in both preventative and treatment strategies for this illness. To understand the impact of these changes and direct future management strategies, it is important that the epidemiology of this disease is fully understood. This thesis investigates changes in epidemiology and outcomes in adult patients admitted to UK hospitals with a primary diagnosis of CAP, in recent years and with reference to the herd protection effect of the conjugate Streptococcus pneumoniae vaccine. Methods There are 3 study populations presented in this thesis. Data were derived from (a) the British Thoracic Society national CAP audit database, (b) a longitudinal cohort study of adults hospitalised with CAP, within the Greater Nottingham area, and (c) an observational study of adults admitted to four hospitals within the East Midlands area with a diagnosis of CAP. The specific methods used for the identification of study participants, laboratory and statistical analysis are described in detail in ensuing chapters. Results Across the UK, there was a significant reduction in 30-day mortality between 2009 and 2014; this improvement in outcome may be attributable in some part to improved processes of care. Whilst data derived from coding databases have previously been used to report CAP related mortality trends, this thesis has demonstrated significant variation in coding accuracy across UK institutions and that miscoded cases of pneumonia had lower odds of 30 day mortality compared to those individuals with CAP. Consequent to herd effects from national infant vaccination programmes and changes in nasopharyngeal carriage of S pneumoniae, this thesis shows that (a) school holiday periods were associated with increased incidence of pneumococcal CAP in hospitalised adults, (b) older adults at high risk of pneumococcal disease were less likely to be hospitalised with vaccine serotype CAP compared to non-vaccine-serotype pneumococcal CAP, and (c) there was a decrease in the overall burden of vaccine-serotype pneumococcal CAP compared to non-vaccine-serotype pneumococcal CAP. Conclusion Important changes in the epidemiology of adult CAP in the UK over recent years are reported in this thesis. This includes temporal decreases in mortality rates of all cause CAP, as well as a significant ongoing burden of non-vaccine serotype pneumococcal CAP. This data on the current burden of disease and associated outcomes should help inform future CAP management strategies.

Background Malaria and pneumonia are the leading causes of under-five mortality in sub-Saharan Africa especially in Nigeria. The Integrated Management of Childhood Illness (IMCI) guidelines were developed by the World Health Organisation (WHO)/United Nations Children’s Fund (UNICEF) as a strategy to reduce the burden of these and other preventable childhood diseases. However, there appears to be a paucity of evidence on the diagnostic performance of the revised IMCI guidelines and whether they offer an advantage over lay diagnosis (caregiver) for malaria and pneumonia management in Nigeria. Aim and specific objectives This study evaluates early diagnostic approaches (IMCI guidelines and lay diagnosis) for malaria and pneumonia in children under-five at the primary healthcare level. To address the overarching aim of the study, the following four specific objectives were studied: I. To assess the diagnostic accuracy of the IMCI guidelines and lay diagnosis (caregiver) for malaria and pneumonia in comparison to reference diagnostic approaches (microscopy and chest X-ray for malaria and pneumonia respectively). The extent of agreement between caregivers’ and health workers’ diagnosis of malaria and pneumonia is also assessed. II. To estimate the burden of malaria and pneumonia among children under-five presenting to study primary healthcare centres (PHCs) according to various diagnostic approaches. III. To determine the clinical outcomes in children diagnosed with malaria and pneumonia according to the IMCI guidelines and risk factors for severe outcomes. IV. To qualitatively explore caregivers’ and health professionals’ perspectives on lay diagnosis and IMCI guidelines as diagnostic approaches for childhood malaria and pneumonia. Methods A mixed methods approach was used for this study. The quantitative component used a consecutive sampling approach to recruit 903 children aged 2–59 months who met study eligibility criteria for malaria and pneumonia assessment according to the IMCI guidelines at presentation to five study PHCs in Benin City, Nigeria. Caregivers of these children were also asked what they thought the diagnosis was (lay diagnosis). Diagnostic accuracy was assessed in terms of sensitivity, specificity, positive and negative predictive values, Area under the Receiver Operating Characteristic Curves (AUROC) values and 95% Confidence Intervals (C.I). The extent of agreement was assessed in terms of Cohen’s kappa statistic (k) and 95% CI. The estimated burden of malaria and pneumonia during the study period was assessed using proportions and 95% C.I. Clinical outcomes in children diagnosed with malaria and pneumonia by the IMCI guidelines were described in terms of frequency and percentages, while the potential risk factors associated with clinical outcomes were assessed using odds ratios (ORs) and 95% C.I. For the qualitative component, health stakeholders (17 health professionals and 13 caregivers) who met the study eligibility criteria were purposively recruited and interviewed using semi-structured interviews. An inductive approach to thematic analysis was used for data analysis. Results Compared to microscopy, the diagnosis of malaria by health workers using the IMCI guidelines was poorly accurate with an AUROC value of 0.57 (with sensitivity and specificity of 51.8% and 61.3% respectively). Similarly, caregivers’ diagnosis of malaria was poor with an AUROC value of 0.55 (with sensitivity and specificity of 31.1% and 79.5% respectively) as compared to microscopy. Using the IMCI guidelines as the reference diagnostic test, caregivers’ diagnosis of malaria was more accurate (AUROC 0.60) in comparison to that of pneumonia (AUROC 0.54). There was a slight or minimal level of agreement (k=0.14; 95% CI: 0.09-0.19) between caregivers and health workers in the diagnosis of malaria and pneumonia. The estimated burden of malaria and pneumonia was relatively low, varying by the study local government areas, PHCs and seasonality, irrespective of the diagnostic approach. Where follow-up data were available, approximately 57% (172/304) and 78% (81/104) of the children diagnosed with malaria and pneumonia, respectively, recovered without complications within 30 days. Self-medication prior to presenting to study PHCs and use of preventive measures against malaria were independently and significantly associated with improved clinical outcomes. In contrast, exposure to solid fuels increased the odds of severe illness following malaria or pneumonia diagnosis. The qualitative component of the study found that caregivers rely on lay diagnosis despite the awareness of its limitations. The perceptions of malaria and pneumonia appeared to influence caregivers’ home management practices and health seeking behaviours. Caregivers showed willingness to be trained in the IMCI guidelines for improved home-based management of malaria and pneumonia. Health professionals believed that the IMCI guidelines were useful for managing both malaria and pneumonia. However, there are some recurring challenges to the wide-scale and sustainable implementation of the IMCI strategy in Nigeria. These include inaccurate diagnosis of malaria and inadequate funding. Conclusion The IMCI guidelines are crucial in the effective management (diagnosis and treatment) of malaria and pneumonia at the primary healthcare level in Nigeria. Although not perfect, lay diagnosis has an important contribution in the early detection and management of malaria and pneumonia at the community level in Nigeria. However, there is need for further investment in the training of both health professionals and caregivers in the IMCI guidelines for better health outcomes in under-five population. The training of caregivers in the IMCI guidelines and potential for a scale-up will benefit from careful design, piloting, implementation, and monitoring.

Thesis (M.S. in Modeling, Virtual Environments and Simulations (MOVES))--Naval Postgraduate School, March 2005.<br>Thesis Advisor(s): Chris Darken. Includes bibliographical references (p. 47-48). Also available online.

Le sfide lanciate dalle malattie non trasmissibili sono accolte da tecnologie sempre più all'avanguardia. Nonostante questo, ancora oggi gestire e monitorare gravidanze a rischio rimane un problema. La simulazione di condizioni come quella data dal diabete gestazionale, può aiutare a capire quali sono i principali fattori che influenzano l'andamento della malattia in modo da poterne evitare l'insorgenza e, in questo modo, migliorare la salute di madri e generazioni future. Questa tesi ha come obietto lo studio e il miglioramento di un sistema Agent-Based pensato per il trattamento del diabete di tipo 1 e la modellazione di una sua estensione per il diabete gestazionale. Al termine della tesi è stato migliorato il sistema rendendolo più fedele ai cambiamenti fisiologici che avvengono durante il metabolismo del glucosio e la modellazione della placenta e relativamente delle modifiche che apporta all'intero sistema getta le basi per nuovi sviluppi legati al trattamento di malattie durante il periodo di gestazione.

There is currently no effective tool available to quickly and economically measure a change in landmass in the setting of biomedical professionals and environmental specialists. The purpose of this study is to structure and demonstrate a statistical change-detection method using remotely sensed data that can detect the presence of an infectious land borne disease. Data sources included the Texas Department of Health database, which provided the types of infectious land borne diseases and indicated the geographical area to study. Methods of data collection included the gathering of images produced by digital orthophoto quadrangle and aerial videography and Landsat. Also, a method was developed to identify statistically the severity of changes of the landmass over a three-year period. Data analysis included using a unique statistical detection procedure to measure the severity of change in landmass when a disease was not present and when the disease was present. The statistical detection method was applied to two different remotely sensed platform types and again to two like remotely sensed platform types. The results indicated that when the statistical change detection method was used for two different types of remote sensing mediums (i.e.-digital orthophoto quadrangle and aerial videography), the results were negative due to skewed and unreliable data. However, when two like remote sensing mediums were used (i.e.- videography to videography and Landsat to Landsat) the results were positive and the data were reliable.

Helicobacter pylori is regarded as the most important risk factor for peptic ulcer disease and gastric cancer. In Kurdistan region, northern Iraq, gastric cancer is rare (5/100,000). To investigate some possible reasons for this, the prevalence of H. pylori infection, gastric mucosal histopathological changes in H. pylori infected subjects, and virulence factor genotypes (especially dupA) of colonising strains were studied. The immune response to H. pylori infection, focusing on genes associated with T-helper (Th) and regulatory T-cell (Treg) cells, was also investigated. It was found that 79% of 163 adults and 37% of 120 children were seropositive for H. pylori (p<0.0001). For infected people, gastric lymphocyte infiltration was more prominent in the antrum (p=0.01). 71% of Iraqi H. pylori strains were positive for cagA and its presence was significantly associated with peptic ulcer disease (PUD) (p<0.01). cagA genes encoding four or more tyrosine phosphorylation motifs could not be found in any of the Iraqi strains. Isolates possessing the i1 form of vacA were significantly associated with GU (p<0.02). 32% of Iraqi H. pylori isolates were dupA-positive and presence of this gene was associated with PUD (p<0.01). The levels of IFNγ, IL-12 p35, IL-10, IL-4 and FOXP3 mRNA were found to be elevated in gastric mucosal samples from H. pylori-infected patients compared to those from H. pylori-negative patients (median increase 7-fold p=0.001; 17-fold p=0.002; 1320-fold p=0.001; 1184-fold p=0.001; and 3-fold p=0.01, respectively), indicating a predominant IL-4 and IL-10 (Th2) response. Interestingly, IFNγ mRNA levels were 16-fold higher in tissues taken from 17 infected smokers than found in tissues taken from 18 infected non-smokers (p=0.009). IL-4 mRNA levels in tissues from 20 infected females were 40-fold higher than in tissues from 15 males (p=0.005). Nucleotide sequencing of the dupA 3' region from 32 strains showed that dupA commonly had additional single base insertions or deletions that either truncated or extended the open reading frame (ORF). We have therefore classified dupA into two main groups: the common extended ORF within jhp0917-19 (dupA1), and dupA with an early stop codon to truncate the ORF (dupA2). ELISA performed on supernatants from H. pylori-infected gastric epithelial cell lines found no significant differences in IL-8 production between strains that possessed or lacked dupA. In comparison to wild-type H. pylori, disruption of dupA significantly reduced IL-12, IFNγ, TNFα and IL-8 production by peripheral blood mononuclear cells (PBMCs) in 2/4 strains. For the remaining 2 strains, where gene sequencing revealed a frame shift resulting in truncated dupA in the wild-type, the level of these cytokines was unchanged by dupA mutation. H. pylori infection is common in Kurdistan region and acquired at a young age. The low cancer rate may be partially explained by a predominant lymphocyte infiltration in the antrum rather than the corpus, which has been reported to be associated with reduced risk of gastric adenocarcinoma. An absence of the more toxic cagA genotype with four or more tyrosine phosphorylation motifs in the Iraqi strains, and the predominance of Th2 cytokine expression rather than a more pro-inflammatory Th1 response to H. pylori could also contribute to a reduced incidence of cancer. dupA1 appears to play an important role in promoting the inflammatory response of leukocytes to H. pylori.

Background: Allergic disease is uncommon in developing countries, especially in rural areas. A protective effect of helminth infection has been implicated as a potential explanation. Objectives: To determine whether reduced exposure to helminth infection is associated with a higher risk of allergen skin sensitisation and allergic disease, and whether such an association could be explained by a helminth-induced up-regulation of certain cytokines, in particular anti-inflammatory IL-10. Methods: We invited 1,742 rural Vietnamese schoolchildren to take part in a cross-sectional baseline survey followed by a randomised, double blind, placebo-controlled trial of anti-helminthic therapy at 0, 3, 6, and 9 months to compare the change in exercise-induced bronchospasm (primary outcome), wheeze, rhinitis, eczema, and allergen skin sensitisation (secondary outcomes) at 12 months. 244 secondary schoolchildren also had venous blood taken to measure helminth induced IL-10, IFN-gamma, IL-5, and IL-13. Out of these 244 children, 144 were infected with hookworm and had bloods taken again at 12 months. Results: Baseline survey 1,601 schoolchildren (92% of those eligible) in grades 1-9 aged 6-18 participated in the baseline survey. 0.4% (6/1601) of children had a fall in peak flow after exercise of at least 15%. Doctor-diagnosed asthma was equally rare (0.4%, 6/1601), while 5.0% (80/1601) of children had experienced wheezing over the past 12 months. 6.9% (110/1601) of parents reported that their children had suffered of hay fever in the past 12 months, and in 2.6% (41/1601) of cases this diagnosis was confirmed by a doctor. 5.6% of children (89/1601) reported an itchy rash over the past 12 months. 0.9% (14/1601) had a history of flexural involvement and on examination 0.5% (8/1601) proved to have flexural eczema on the day of the survey. Skin prick test positivity was commoner than allergic disease. 33.5% (537/1601) of children had at least one positive skin prick test (dustmites 14.4%, cockroach 27.6%). The cross-sectional analysis yielded only significant results for allergen skin sensitisation. In univariate analysis, sensitisation was less frequent in children with hookworm or Ascaris infection, and increased in those with better santitation, including flush toilets and piped drinking water. In multivariate analysis, the risk of allergen skin sensitisation to house dust mite was reduced in those with Ascaris lumbricoides infection (adjusted OR=0.28, 95% CI 0.10-0.78) and in children with higher hookworm burden (adjusted OR for 350+ versus no eggs per gram faeces=0.61, 0.39-0.96), and increased in those using flush toilets (adjusted OR for flush toilet versus none/bush/pit=2.51, 1.00-6.28). In contrast, sensitisation to cockroach was not independently related to helminth infection but was increased in those regularly drinking piped or well water rather than from a stream (adjusted OR=1.33, 1.02-1.75). Intervention study 1,566 children in grades 1-8 completed the baseline survey and all consented to be randomised to either anti-helminthic treatment or placebo. 1487 children (95%) completed the intervention study. There was no effect of therapy on the primary outcome, exercise-induced bronchoconstriction (within-participant mean % fall in peak flow from baseline after anti-helminthic treatment 2.25 (SD 7.3) vs placebo 2.19 (SD 7.8, mean difference 0.06 (95% CI -0.71-0.83), p=0.9), or on the prevalence of the secondary clinical outcomes questionnaire-reported wheeze (adjusted OR=1.16, 0.35-3.82), rhinitis (adjusted OR= 1.39, 0.89-2.15), or flexural eczema (adjusted OR=1.17, 0.39-3.49). However, anti-helmithic therapy was associated with a significant allergen skin sensitisation risk increase in the treatment compared to the placebo group (adjusted OR=1.31, 1.02-1.67). In post-hoc analysis this effect was particularly strong for children infected with Ascaris lumbricoides at baseline (adjusted OR=4.90, 1.48-16.19), the majority of whom were co-infected with hookworm. Cytokine profiles Hookworm-induced IL-10 was inversely related to allergen skin sensitisation (any positive skin prick test) at baseline, but this result missed conventional statistical significance (univariate OR=0.70, 0.48-1.03; adjusted OR=0.72, 0.44-1.18). No other cytokine response was associated with skin prick test positivity at baseline (univariate OR IFN-gamma=1.15, 0.71-1.85; univariate OR IL-5=0.84, 0.53-1.33). Similary, no significant changes in any of the cytokine profiles were observed following anti-helminthic therapy in the treatment compared to the placebo group (p=0.3 for all three cytokines). Conclusion The baseline study suggested that hookworm and Ascaris infection, sanitation and water supply independently reduce the risk of allergic sensitisation. The intervention study confirmed that helminth infection and allergic sensitisation are inversely related and that the effect of Ascaris and hookworm infections on skin prick test responses is additive. However, we found little evidence to suggest that this effect was mediated by IL-10. There was also insufficient evidence to suggest that loss of exposure to gut worms for 12 months results in an increase in clinical allergic disease. The effect of more prolonged de-worming warrants further research.

The gastric pathogen Helicobacter pylori infects more than half of the population worldwide. H. pylori manage to establish persistent infection, which would be life-long if not treated. In order to establish such an infection, this pathogen has to deal with the host immune system. H. pylori has certain characteristics which make the bacteria less announced to the host immune system. Additionally, for remaining in the harsh and acidic environment of the stomach with peristaltic movements and a high frequency of turnover of epithelial cells, H. pylori has developed different binding modes to structures present both in the mucus and on the surface of gastric cells and also to extracellular matrix proteins. Evidently, adhesion has a determinant role for a successful colonization by H. pylori. It has been shown that a small fraction of the H. pylori infection is in intimate contact and attached to the host epithelium. Despite its small proportion, this group maintains the persistency of infection. As there is no suitable in vitro system to mimic the human stomach for studies of H. pylori infection, we have developed the In Vitro Explant Culture technique (IVEC). By using this model we could show that H. pylori use the Lewis b blood group antigen to bind to the host gastric mucosa, during experimental conditions most similar to the in vivo situation. Furthermore, we could show that the host tissue responses to the bacterial attachment by expression of Interleukin 8 (IL- ), which will guide the inflammatory processes. Interestingly, by inhibition of bacterial adhesion through receptor competition i.e., by use of soluble Lewis b antigen, IL-8 production was hampered in the IVEC system, which further validates the presence of a tight relation between bacterial adhesion and induction of host immune responses. One of the inflammation signaling cursors in vivo is the upregulated sialylated Lewis x (sLex) antigen, an inflammation associated carbohydrate structure well established as a binding site for the selectin family of adhesion molecules. We could show that during chronic gastric inflammation, which is actually caused by the persistent H. pylori infection, the bacterial cells adapt their binding mode, and preferentially bind to sLex, which will provide an even more intimate contact with the host cells. This interaction is mediated by SabA, the H. pylori adhesin for sialylated oligosaccharides/glycoconjugates. By employing red blood cells as a model we could further demonstrate that SabA is identical to the “established” H. pylori hemagglutinin. We could also show that SabA binds to sialylated glycolipids (gangliosides) rather than glycoproteins on cell surfaces. Our result also revealed that SabA also binds to and activates human neutrophils. Such effect was unrelated to BabA and the H. pylori Neutrophil Activating Protein (HP- AP), which were not directly involved in the activation of neutrophils. Furthermore, phagocytosis of bacteria by neutrophils was demonstrated to be mainly dependent on presence of SabA. Interestingly, HP-NAP showed a possible role in guiding the bacterial adhesion during conditions of limited sialylation, i.e. equivalent to mild gastritis, when the tissue would be less inflamed and sialylated. In conclusion, H. pylori adhesion causes host tissue inflammation, then the bacteria will adapt to the new condition and bind to epithelial cells in a tighter mode by synergistic activities of BabA and SabA. Additionally, SabA bind to and activate human neutrophils, which will exacerbate inflammation responses and cause damage to host tissue. Thus, BabA and SabA are potential candidates to be targeted for therapeutic strategies against H. pylori and gastric disease.

Frontline healthcare workers (HCWs) in the UK have been prioritised for free occupational immunisation against seasonal influenza since 1999. During the 2009-10 influenza pandemic, they were identified as a priority group to receive the strain-specific vaccine. Nevertheless, take-up rates among HCWs have rarely exceeded 50%, even during the pandemic. Most attempts to change this situation have been predicated on the assumption that these low rates are the result of reluctance or resistance by individual HCWs, who must be persuaded or coerced to comply with employer directives. To gain a novel understanding of this immunisation programme, an actor-network theory approach is adopted to trace the journeys of vaccines through two Local Health Boards in Wales during the 2009-10 H1N1 influenza pandemic and in the following winter influenza season (i.e. during 2010-11). The research reported shows that low uptake is largely the result of complex social, organizational and cultural processes. Only when these have been changed will it be appropriate to frame the remaining problem as reluctance or resistance by individual HCWs. The study reveals that this immunisation programme is inherently unstable and subject to ambivalence from actors at all levels. Suggestions for practical improvement are given.

Community-acquired pneumonia (CAP) is a leading cause of adult morbidity and mortality worldwide despite decades of effective antibiotics and vaccination initiatives. There have been no recent significant improvements in outcomes, including 30-day mortality. The bacterium Streptococcus pneumoniae is the most prevalent causative pathogen in CAP, being found in up to half of cases. In September 2006 a childhood pneumococcal vaccine (PCV-7) was introduced, leading to reductions in vaccine-type (VT) pneumococcal disease in infants, with possible additional benefits reported in adults. However, the effect that infant PCV-7 vaccination has on adult disease has to date been inadequately described in a small fraction of patients with invasive CAP, almost exclusively in populations in the US. These issues are explored fully in the literature review, encompassing chapters 1, 2 and 3. New strategies for CAP are therefore required. The outcome of CAP can be improved by a) preventing the disease by vaccination and herd immunity, and b) ameliorating the course of the disease after it has been acquired. This thesis presents a collection of studies that aim to acquire observational data to investigate these two issues. The majority of the included studies are drawn from a two year prospective cohort study of consecutive adults with CAP admitted to a large UK teaching hospital trust between September 2008 and September 2010. After obtaining informed consent, the presence of pneumococcal disease in each participant was established by testing urine samples for pneumococcal capsular polysaccharide, a test which has a high sensitivity and specificity. The urine samples were subsequently tested for pneumococcal serotype. A full record of care processes, investigations, and clinical outcomes was made, and child contact in the month preceding admission was assessed. These methods are described more fully in chapter 4. Chapter 5 presents the data on the pneumococcal serotypes found in the cohort over a two year period, and links them to epidemiological characteristics in the study population. The most prevalent serotypes were 14, 1, 8, 3 and 19A, with VT serotypes less frequent in the second year of the study. Chapter 6 examines the association that infecting serotype has with disease manifestation and patient characteristics. Infection with a serotype not contained within PCV-7 (NVT) was associated with younger and fitter patients, a higher rate of complications such as para-pneumonic effusion, and hypotension at admission. The effect of child contact on pneumococcal disease is reported in chapter 7. Prior contact with a child aged ≤8 years was particularly associated with pneumococcal aetiology, and contact with a PCV-7 vaccinated child independently associated with NVT CAP. The findings from these three chapters are unique in that they relate individual pneumococcal serotype to specific clinical disease patterns, epidemiology and transmission in both invasive and non-invasive pneumococcal CAP for the first time. They show a change in serotype distribution in adults following the introduction of PCV-7 in infants, which is important to inform future vaccine development for both adults and children. Furthermore, different serotypes are associated with different clinical disease patterns, which may have a significant impact on the disease that clinicians see at the “front door” given that the serotype distribution of pneumococcal CAP may be changing. Finally, the link between child vaccination and adult disease provides more direct evidence for the transmission of pneumococci from children to adults as a mechanism for the development of CAP in adults. The second part of this thesis looks at current care processes, and how these might be improved. Chapters 8, 9 and 10 relate to efforts to better predict prognosis, and chapters 11 and 12 with how patents with CAP may be better managed at the “front door”. Symptoms are clearly important to patients, but the role of symptoms in management and outcome is unclear. Chapter 8 presents a study validating a symptom score that has not yet entered routine use, but which is shown to correlate with clinical outcomes, and may be useful in assessing outcome in low severity CAP. The influence that oxygenation status at admission has on outcome is poorly understood. Chapter 9 describes a study showing that whilst hypoxaemia does positively predict adverse outcome, it is not as predictive as existing severity scores. The presence of hypoxaemia may however identify a subset of patients who are classified as low severity by existing severity scoring, but are nevertheless at increased risk of adverse outcome. Severity scoring is the cornerstone of management in adult CAP, and is explored in chapter 10. Current severity scores adequately predict mortality in CAP, but often generate a group of “moderate severity” where appropriate management is often unclear. This study looked at the effect of pre-admission functional status on outcome in conjunction with existing severity scores in this difficult group, and validated a novel severity score for predicting need for escalation of care, SMART-COP. Incorporation of functional status does marginally improve the performance of existing severity scores, but may be of more use as a post-severity score test to identify sub-groups of patients with moderate severity CAP who are at increased risk of death. Chapter 11 looks at the influence that making a prompt diagnosis (rather than prompt treatment with antibiotics, as has previously been studied) has on outcome, using the time between admission and first chest radiograph as a surrogate measure. Whilst an early chest radiograph was not associated with an improvement in mortality, it was associated with a shorter length of hospital stay, and may therefore be regarded as a marker of good quality care. There is current debate as to the role of the speciality physician in the front-door early assessment of patients, and whether early review of patients with CAP may improve outcome compared with management by a non-specialty physician. Chapter 12 looks at the effect that early specialist senior respiratory review has on outcome for adults with CAP, showing a clear benefit on length of hospital stay to early consultant review. In conclusion, this thesis provides an up-to-date picture of the circulating pneumococcal serotypes in non-invasive adult CAP, and correlates infecting serotype to clinical and epidemiological parameters. It also identifies five areas of clinical care where management processes could be improved. By addressing of these aspects the outcome of CAP may be improved in the future.

The epidemiology and natural history of Hepatitis C has been studied in a large geographically determined population (Trent HCV study). It has previously been suggested that patients with Hepatitis C and a persistently normal Alanine aminotransferase (PNALT) represent a group of patients with mild disease and at low risk of disease progression. Patients with PNALT were, therefore, compared to those with an elevated ALT. The majority of patients initially fulfilling the definition of a PNALT had an abnormal ALT within 3 years of follow-up. They also demonstrated similar rates of fibrosis progression as a sub-group of HCV infected patients with an elevated ALT who were re-biopsied prior to any institution of therapy. They, therefore, warrant the same consideration with regard to treatment. The morbidity and mortality associated with Hepatitis C with severe fibrosis was assessed in a group of patients with a liver biopsy demonstrating Ishak fibrosis stage  4. A worse prognosis than previously reported was observed for this patient population. Once decompensation develops, HCV infection is associated with a high mortality rate. Indicators of poor synthetic liver function and hypergammaglobulinaemia were important prognostic factors for mortality, while combination antiviral therapy was associated with improved survival. The majority of HCV infected patients (75%) diagnosed with hepatocellular carcinoma (HCC) were known to have cirrhosis at least 6 months prior to diagnosis of HCC and were, therefore, amenable to surveillance. There was a variable application of surveillance, however, and no significant improvement in survival was demonstrated. Age, duration of infection and immunoglobulin G levels were associated with an increased risk of HCC in cirrhotic patients in the univariate analysis. Achieving an SVR was associated with a reduced risk. No variable in cirrhotic patients was shown to be independently associated with HCC in the multivariate analysis. A comparison of disease progression and treatment outcome in White and Asian (Indian subcontinent) patients was made. Asian patients generally presented at an older age and with more severe disease on biopsy. The patient’s ethnic group was not associated with the likelihood of either an SVR or completion of therapy. Instead cirrhosis and a raised GGT were associated with a failure to achieve SVR in the multivariate analysis. The platelet count is a surrogate marker for the severity of liver fibrosis and correlates with the Ishak fibrosis stage. An analysis of factors associated with an SVR was performed. In the multivariate model, age at start of treatment was the only independent predictor of SVR in Genotype 1, while estimated duration of infection and Ishak stage were predictors in genotype 2/3 patients. The platelet count was not an independent predictor of SVR or completion of therapy.

Limited understanding of influenza transmission has been a frequent obstacle during the development of pandemic influenza infection prevention and mitigation strategies. The science is hotly debated, especially the relative importance of transmission via large droplets or aerosols. Clarification of the relative importance of different modes of transmission is critical for the refinement of evidence-based infection control advice and has been called for by the European Center for Disease Control (ECDC), the World Health Organization (WHO), and the US Institute of Medicine. The primary aims of this thesis were to investigate influenza transmission; i) by obtaining data concerning viral shedding and the presence of influenza virus in the near environment of infected individuals and ii) through the exploration of a human challenge model to study transmission. Two major clinical studies have been performed; • Shedding and environmental deposition of novel A (H1N1) pandemic influenza virus. The primary aims of the study were to correlate the amount of virus detected in a subject’s nose with that recovered from his/her immediate environment (on surfaces and in the air) and with symptom duration and severity. Adults and children, both in hospital and from the community, who had symptoms of influenza infection were enrolled. Information about symptoms was collected and samples were taken including nose swabs, swabs from surfaces and air samples. Forty two subjects infected with influenza A(H1N1)pdm09 were recruited and followed up. The mean duration of nasal viral shedding was 6.2 days (by PCR) and 4.6 days (by culture). Over 25% of cases remained potentially infectious for at least 5 days. Symptom scores and viral shedding were poorly correlated. From surface swabs collected in the vicinity of 40 subjects, 15 (38%) subject locations were contaminated with virus. Overall 36 of 662 (5.4%) surface swabs taken were positive for influenza, two (0.3%) yielded viable virus. Subjects yielding positive surface samples had significantly higher nasal viral loads on illness Day 3 and more prominent respiratory symptom scores. Room air was sampled in the vicinity of 12 subjects and PCR positive samples were obtained from five (42%). Particles small enough to reach the distal lung (≤4µm) were found to contain virus. • Use of a human influenza challenge model to assess person-to-person transmission: Proof-of-concept study. The primary aim of this study was to establish that an experimentally induced influenza infection is transmissible. Healthy subjects deemed sero-susceptible to influenza A/H3N2/Wisconsin/67/2005 were intranasally inoculated (Donors) and when symptoms began, further sero-susceptible subjects (Recipients) were exposed to Donors during an ‘Exposure Event’. Subjects were in close contact, e.g. playing games and eating meals together, for a total of 28 hours during a 2 day period. Samples were collected to confirm infection status. Among 24 healthy adult subjects, nine were randomised to the ‘Donor’ group and 15 to the ‘Recipient’ group. Following inoculation 5 out of 9 Donors (55%) developed illness and 7 out of 9 (78%) were proven to be infected. After exposure, 5 out of 15 Recipients developed symptoms and 3 out of 15 were proven to be infected. Three others were found to be non sero-susceptible prior to exposure. The overall attack rate in Recipients was 20% but was 25% after adjustment for pre-exposure immunity. The contact, droplet and aerosol routes of influenza transmission are all likely to have a role. This thesis shows that transmission of influenza via surfaces may be less important than current infection control policies and public guidance documents imply. Air sampling results add to the accumulating evidence that supports the potential for aerosol transmission of influenza. The human challenge model could be used to investigate routes of influenza transmission further and a study funded by the Centers for Disease Control (CDC) is planned.

N,N-Diethyl-m-toluamide (DEET) is one of the most effective and commonly used mosquito repellents. However, during laboratory trials a small proportion of mosquitoes are still attracted by human odours despite the presence of DEET. In this study behavioural assays identified Aedes aegypti females that were insensitive to DEET. The selection of either sensitive or insensitive groups of females with males of unknown sensitivity over several generations resulted in two populations with different proportions of insensitive females. Crossing experiments showed the ‘DEET-insensitivity’ trait to be dominant. In addition to the finding of heritable DEET-insensitivity, unselected culture mosquitoes were shown to change their sensitivity to DEET after brief pre-exposure to the repellent. Female mosquitoes that were sensitive to DEET when first tested became insensitive when retested. Electroantennography showed that mosquitoes that were insensitive to DEET had a reduced response to DEET compared with mosquitoes that were sensitive to it. This was the case both for culture mosquitoes displaying insensitivity to DEET after brief pre-exposure to it, and for the sensitive and insensitive lines selected for several generations. Single sensillum recordings of the selected lines identified DEET-sensitive sensilla in the sensitive line that did not respond to DEET in the insensitive line. This study suggests that behavioural insensitivity to DEET in Ae. aegypti is a genetically determined dominant trait, which can also be temporarily induced by pre-exposure, and resides in changes in sensillum function. These results highlight the necessity for careful monitoring of DEET-insensitivity in the field, and caution when designing laboratory methods for repellency assays.

This thesis aims to examine the effectiveness of a mobile phone based SMS game as a learning intervention for the Peer Educators of the Males having Sex with Males (MSM) groups in Kolkata, India. MSM groups are marginalised and are at higher risk of HIV/AIDS, falling under the core groups for the National AIDS Control and Prevention programmes in India. Peer to peer education for behaviour change in HIV/AIDS prevention projects is a bottom up approach to reach out to this marginalised population for HIV prevention. Training is in place for MSM peer educators but research shows gaps in their support and learning needs. This project developed a mobile game based learning tool to address the peer educators’ learning and support needs. Using a participatory research approach a multiplayer SMS based simulation game was developed, deployed and evaluated, using an existing game engine called ‘Day of the Figurines’. In an effort to enhance experience sharing and peer learning the real life experiences of the peer educators were captured and incorporated through a participatory and iterative process as scenarios of the game. A SMS game on mobile phones was chosen to be in keeping with the marginalised, secretive nature of the MSM identity of the peer educators as well as be in keeping with the mobile nature of their work. The SMS game was piloted in Nottingham and Kolkata and the final intervention was deployed and evaluated in Kolkata with a group of sixteen peer educators from MANAS Bangla, a network of community based MSM organisations in Kolkata, India. Evaluation of the game showed it to be useable, relevant to peer education, interesting and entertaining but in some cases slow, uninteresting and confusing. The game play was affected by technical faults but players still exchanged SMS messages with the game and communicated between players using the ‘chat’ feature of the game. Playing the game enabled players to acquire better communication skills and increased confidence, it gave them a feeling of self-efficacy and influenced their work practices. The intervention was instrumental in increasing the peer educators’ critical consciousness, it created a space to address the practical barriers faced by the peer educators by providing dialogic methods for developing knowledge, encouraging and facilitating collaboration, developing communication skills and increasing access to learning opportunities. This research contributes an exploration of peer educators’ problems, evaluation of mobile game based learning and account of participants’ experiences in a mobile-health development context in resource constrained settings.

Background: Hepatitis C virus infection affects more than 170 million people worldwide and is frequently associated with chronic liver disease and hepatocellular carcinoma. No protective vaccine is yet available and the current standard of care, consisting of pegylated interferon alpha and ribavirin, has limited efficacy. Ribavirin is a key component of any effective anti-HCV regimen. However, accumulation of ribavirin in the red cell compartment not only reduces drug efficacy as a result of diversion to extra-hepatic sites but also produces haemolytic anaemia which can lead to dose reduction or discontinuation of treatment. Lipid or polymer based nanoparticles can be used to deliver therapeutic agents, such as drugs or small interfering RNAs (siRNAs) directly to their site of action. We therefore elected to develop new antiviral strategies based on the targeted delivery of ribavirin to hepatocytes, coupled with the identification of new therapeutic targets. In order to inform the rational use of direct intracellular delivery of ribavirin, we enquired whether variation in expression of the ribavirin transporter may determine drug uptake and permit the identification of individuals who would benefit from these alternative approaches to treatment. Aims: The aims of this study were to: • identify host proteins involved in virus replication • demonstrate reduction of viral replication by modulation of host gene expression • develop and test a nanoparticle based system for the delivery of therapeutic molecules, including siRNAs either alone or in combination with ribavirin. • assess the relationship between ribavirin uptake by primary human hepatocytes and expression of ribavirin receptors Methods: A subgenomic HCV replicon system was established to study the virus-host relationship and identify host proteins supporting viral replication by using stealth siRNA. Viral RNAs were in vitro transcribed and transfected into Huh7 cells and expression assessed using engineered GFP as a reporter gene. siRNAs were co-transfected with viral RNAs using a nucleofector. Modulation of host gene expression was measured by both quantitative RT-PCR and protein blotting. Liposomal nanoparticles containing ApoB-100 duplexes were supplied by Lipoxen. Primary human hepatocytes were isolated by a modified two step collagenase perfusion method and cultured on collagen coated plates. HPLC and real time PCR conditions were used to measure and correlate drug uptake and receptor expression respectively. Equilibrative nucleoside transporter (ENT1) gene was analysed by direct sequencing. Results: A JFH1 (HCV genotype 2a) virus based subgenomic replicon system was successfully established. Using this model system, host proteins VAP-A and STAT3 were shown to positively regulate virus replication while ACTN1 had no effect. Liposomes failed to deliver either siRNA targeted at apoB-100 or ribavirin and this was found to be due to structural instability of the delivery vehicle. In contrast, fluorescently labelled liposomes were stable and could be taken up by human hepatocyte cell lines under optimised conditions. A protocol capable of efficient isolation and culture of hepatocytes from human donor was validated. Data from primary human hepatocytes show that ENT1 expression was highly variable in different sets of primary livers and correlated strongly with ribavirin uptake. Strikingly, Huh7 cells did not take up ribavirin despite expressing wild type ENT1. It was also found that interferon alpha does not modulate ENT1 expression and therefore ribavirin uptake, suggesting it to be a highly unlikely mode of synergism between the two drugs. Conclusion: Modulation of host proteins VAP-A and STAT3 inhibited viral replication, confirming that host genes can be used as a potential target to inhibit viral replication. Liposomes used in this study were, however, found to be ineffective vehicles for the delivery of ribavirin or siRNA, as the majority of drug leaked before cellular uptake. Polymer based nanoparticles are currently being assessed for antiviral drug delivery. Variation in ENT1 expression may account for differences in response rate in patients receiving anti-HCV therapy. Results in the Huh 7 cell line suggest that, while ENT1 is necessary, other factors are also required to mediate ribavirin uptake.

Pneumonia is a common diagnosis in general practice and is associated with significant morbidity and mortality. Current estimates of pneumonia incidence in the UK are based on studies more than a decade ago and little is known about longer term outcomes in pneumonia patients. Though much is known about the aetiology of pneumonia and predictors of mortality, an emerging area for research is the relationship between commonly prescribed drugs in general practice and pneumonia. The aims of this thesis were first, to determine overall incidence and mortality for pneumonia and how these vary by socio-demographic characteristics like age, sex, deprivation; and second, to investigate whether statins, angiotensin converting enzyme inhibitors (ACEIs) and gastric acid suppressants like proton pump inhibitors (PPIs) and histamine 2 receptor antagonists (H2RAs) modify the risk of acquiring pneumonia and its prognosis. This study used data from The Health Improvement Network (THIN) database, a longitudinal database of anonymised computerised medical patient records from 330 United Kingdom (UK) general practices at the time of data extraction in 2006. A cohort design was used to determine pneumonia incidence and mortality in the UK. Case-control, case-series and cohort study designs were used to investigate associations between the various drug exposures and pneumonia. The overall incidence of pneumonia was 237 per 100,000 person-years (95 % confidence interval (CI): 235 to 239) and this rate was stable between 1991 and 2003. Pneumonia was more common in men and in children under the age of four years and adults over the age of 65 years. There was an increased incidence of pneumonia with higher levels of socioeconomic disadvantage. Pneumonia cases showed much higher all-cause mortality as compared to the general population, both in the short and long-term and this increase was independent of underlying comorbidity. After adjusting for potential confounders, current prescriptions for statins and ACE inhibitors were associated with a significant reduction in the risk of acquiring pneumonia. Current prescriptions for PPIs were associated with an increased risk of pneumonia. With regards the impact on mortality: the use of statins was associated with a lower risk of short and long-term mortality following pneumonia whereas the use of ACEIs was associated with a decreased mortality risk only in the short-term. No relationship was observed between prescriptions for PPIs, H2RAs and pneumonia mortality. This study shows that caution must be exercised while prescribing proton pump inhibitors especially in patients known to be at high risk of pneumonia. There is also a potential role for statins in preventing pneumonia in at-risk patients and improving pneumonia outcomes but this will necessitate clinical trials to determine adequate dose, duration and safety profiles before any prescribing policy recommendations are made.

There are many surgical implanted devices in current use and all are prone to biomaterial-related infections (BRI) associated with staphylococcal biofilm formation. BRI are usually associated with S. epidermidis or S. Aureus and are characterized by treatment failure and chronicity resulting in reoperation, removal of the implant, and loss of function or death. Staphylococcal small colony variants (SCVs) may be generated by exposure to sublethal concentrations of antibiotics or nutrient limitation which may occur in biofilms. Although the characteristics of S. aureus SCVs have been well studied, little information on SCVs of S. epidermidis and their potential role in BRI is currently available. This study was designed to investigate the biochemical and phenotypic characteristics of S. epidermidis SCVs to further identify characteristics which may contribute to their ability to cause these increasingly important infections. Exposure to two to four times the gentamicin MIC led to the emergence of stable S. epidermidis SCVs, and the ability to produce SCVs was strain dependent. These variants were isogenic by PFGE and less immunogenic by western blotting, and SDS-PAGE analysis of whole cell preparations and cell wall fractions showed altered protein profiles when compared to wild type strains. S epidermidis SCVs were resistant to aminoglycosides such as amikacin and/or netilmicin and they were thiamine and/or menadione auxotrophs. Chemiluminescence assays showed a decreased ATP content reflecting the deficiency in electron transport systems which results in a growth rate – all characteristics similar to those of S. aureus SCVs. Analysis of virulence factor production indicated that S. epidermidis SCVs showed increased lipolytic and proteolytic activity when compared to those of S. aureus. Some S. epidermidis SCVs showed phase variation in exopolysaccharide production which enabled them to be more adherent to uncoated plastic -a property that may also be important for the later stages of development of biofilms. Invasion assays demonstrated that some S. epidermidis and S. aureus SCVs were internalised by HUVECs by a receptor-mediated mechanism which differed from that of the wild type strains. Interaction of staphylococci with HUVECs induced cytokine production but SCVs stimulated production of IL1, IL-6 and IL-8 at lower concentrations than their related wild type parents in the first 6 hours of co-incubation. SCVs were also less damaging to the HUVEC cell line after 24 hours when compared to wild type strains. This study supports the suggestion that a switch to the S. epidermidis SCV phenotype could be a mechanism exploited by the wild type strains to facilitate their survival inside the host. The chronicity and increased antibiotic resistance associated with BRI could in part, be explained by the characteristics of SCVs identified in this study. In particular the ability to survive intracellularly combined with reduced immunogenicity and resulting decreased cytokine production, may contribute to persistence of infection. Although SCVs are resistant to some antibiotics, surviving intracellularly may further protect staphylococci from other drugs which are unable to enter mammalian cells. Resistance may be further enhanced for some strains in biofilms where enhanced polysaccharide production may also limit antibiotic access.

Background: The immunological as well as the psycho-social impact, of living with HIV/AIDS transform HIV/AIDS into a multidimensional process. Stigma and discrimination against people living with HIV/AIDS (PLWHA) are proposed as hostile scenarios increasing hopelessness and reducing perceived and real social support affecting people’s health status. Peer support strategies are proposed as key factors for dealing with this scenario; additionally, socio-cultural variables may determine the provision and perception of social support. Objectives: To enhance the understanding of the process of living with HIV/AIDS and the role played by social support and to suggest cooperative strategies for dealing with stigma and discrimination against PLWHA to improve people’s health. Sample and method: Five studies were conducted studying 37 HIV positive members and non-members of peer support organisations (PSOs) in Chile and England; nine healthcare professionals working with PLWHA; and three spokes persons from PSOs of PLWHA from Romania, England and Chile. Results: PSOs of PLWHA, which reflect a cooperative strategy used by PLWHA to deal with stigma and self-provide social support, appear to play an important and underexplored role in PLWHAs’ health status; this relationship is also affected by socio-cultural characteristics. A measure of PSS was developed and theoretical analysis lead to a linkage with Maslow’s hierarchy of needs. Personality characteristics were found critical for the success of PNI based interventions. Conclusions: Living with HIV/AIDS involves psychological and social complications. PSOs are a powerful cooperative strategy improving quality of life and general health; however, further research is needed to establish the real impact of PSOs over HIV+ people. Implications: The peer-support strategy of PSOs is a powerful but underused clinical strategy. Healthcare teams and PLWHA may benefit from including this strategy if cooperative work is carried out with PSOs.

Background: After three decades, Human Immunodeficiency Virus (HIV) continues to pose significant threats globally. The efforts to curb the HIV epidemic have required investment in research, with clinical trials being a major focus, to develop HIV prevention, treatment, and cure interventions. A large portion of such research has been undertaken within low income settings, due to the high burden of HIV and the availability of willing volunteers within this setting. HIV research calls for the implementation of ethical research practice which is informed by policy guidelines. However, current policies are largely informed by inputs from high income countries, and lack the voices of those closely involved in research implementation. In order to contribute to ethics policy development in HIV research, it is essential to involve different stakeholders by exploring their experiences/views on the issue. Existing research in this field has mainly explored experience of recruitment and trial conduct, while very little has been done on trial closure, indicating a significant evidence gap worth exploring. This research therefore sought to illuminate, explore and understand the significant issues regarding the care of HIV positive drug trial participants during closure of HIV clinical trials, within a low income setting, specifically, Uganda. Study aim: The study aimed to explore how care is perceived and enacted in HIV drug trial closure in Uganda, by addressing the following specific objectives: 1. From the perspective of research participants and research staff, to explore the views, opinions and understandings of the ethical/legal/moral post-trial obligations in HIV drug trials. 2. From the perspective of research staff, to explore the experiences, practices and processes related to care for HIV drug post-trial participants in a low income setting. 3. From the perspective of research participants, to explore the experiences of care at trial closure. 4. From the perspective of research participants, to explore the experiences of transitioning from HIV research to care/community. Methodology: The study adopted an interpretive-constructivist approach, and employed a social constructivist grounded theory methodology. The study included a total of 21 trial participants and 22 research staff from three different HIV drug trials, in two Ugandan research institutions. In addition, relevant ethical documents were reviewed from two of the included trials. Data collection and analysis followed the principles of grounded theory, with data collection and preliminary analysis being undertaken concurrently, and earlier data informing subsequent data collection. Data collection strategies included individual interviews, focus group discussions, and key informant interviews. Data was collected over a period of 10 months, from October 2014 to August, 2015. NVivo10 software was used to manage the data. Ethical approval was received from the University of Nottingham UK and The AIDS Support Organization (TASO) Uganda, Research Ethics Committees (RECs). The study was registered with the Uganda National Council for Science and Technology (UNCST), as SS 3608. Permission to conduct the research was granted by the respective research institutions, and written informed consent was received from all respondents. Findings: The findings showed that trial closure was often stressful for HIV positive participants in Uganda, and often resulted in negative psychological, socio-economic and health impacts. The negative effects mainly resulted from being stopped from accessing research related health care, which was of a significantly higher quality, and the inability to find alternative care to match the research standards. The main concerns which arose during the transition process of participants from HIV drug trials to usual care facilities include: the loss of the quality care and valued relationships in research, the need to find and link to alternative care facilities, the need to meet the increased financial needs, and worries about the effects/outcomes of research participation. These concerns demanded a range of additional care and supportive strategies from researchers (and other stakeholders). A conceptual model, the model of ‘Facilitated Transition’ was developed, which summarises the findings of this research and provides a diagrammatic representation of the research findings, showing the links and relationships between the different elements. The research established that the transition of HIV positive trial participants from research to usual care facilities is a process, which appears to consist of three overlapping phases. These phases include: The pre-closure phase which represents events occurring before the actual trial closure but that underpin post-trial care, the trial closure phase which is the active phase of the closure, in which trial participants are prepared and exited from the trials, and the post-trial phase which represents the events occurring after trial participants have been linked to post-trial care facilities until 12 months later. These phases are demarcated by specific time points, which reflect how the transition process evolves, proceeds and concludes. At the various phases of the process, specific concerns (care needs) arise, being influenced by the participants’ previous care experiences and perceptions, plus their health and socio-economic positions. Specific actions are required to proactively facilitate trial participants during these phases. These actions are underpinned by the perceived ethical and moral responsibilities of the researchers, and are principally aimed at establishing a continuum of HIV care and treatment after trial closure, promoting positive care experiences for trial participants during the transition, and enabling the settlement and adaptation of trial participants to care in the public healthcare system. Conclusions: This is the first known study to investigate perspectives on post-trial care among HIV positive trial participants in a low income setting, from those closely engaged in the research process. This study has provided novel contributions in the area of HIV research ethics and post-trial care in general. The study has established that trial closure involving HIV positive participants raises significant ethical, moral and practical concerns in the Ugandan context. The findings further demonstrated that current post-trial care practice does not meet all the care needs of the HIV positive trial participants. Existing ethical recommendations on post-trial care place an emphasis on the need to ensure access to trial drugs and provision of trial results, where as less attention is given to other important aspects, as revealed in this research. To meet the post-trial care needs of HIV positive participants in Uganda, a comprehensive trial closure strategy is required. In addition to the already existing aspects of post-trial care, the new strategy should aim to: (i) address the financial needs of trial participants through financial assessment, support and empowerment, (ii) provide practical support during linkage to post-trial care, and (iii) offer post-trial follow-up to monitor and support the participants. Implementing these recommendations may require involvement of various stakeholders, including researchers, ethics authorities, research funders and donors, public healthcare workers, families, trial participants, and the community. Recommendations for future research: Further research is required to ascertain the rates of linkage to care, and to assess the health outcomes of post-trial participants following trial exit. In addition, a study to target the views of other stakeholders, such as the public healthcare facility workers, the family, and ethics authorities on post-trial care may be essential to understand better the ways in which to support HIV positive trial participants in Uganda. Furthermore, a longitudinal prospective study on a larger sample is required to test the model proposed in this research. And finally, there is need to deliberate more on the ethical and moral implications of financial benefits in HIV research involving HIV positive participants in a low income setting.

Since discovery H. pylori has been one of the most intensively researched bacteria. Although the majority of those infected remain asymptomatic it can lead to serious diseases which carry significant morbidity and mortality. The most serious complication of H. pylori infection is gastric cancer and one of the most effective ways to reduce the associated mortality is to detect pre-malignant disease, as this develops in a step wise manner. Using advanced endoscopy is one way to detect pre-malignant conditions but due to the variety in endoscopic techniques and mucosal classifications the diagnosis is often dependent on histology. This work aimed to develop simple, accurate classification systems to detect H. pylori associated disease. The sensitivity and specificity of magnification Narrow Band Imaging for detecting H. pylori gastritis was 69% and 67%, intestinal metaplasia 87% and 97% and dysplasia 92% and 98% respectively. H. pylori is also associated with iron deficiency anaemia but the mechanisms remain unclear. In children it has been proposed that H. pylori disrupts iron regulatory mechanisms via the peptide hepcidin but this has not been extensively researched in adults. Serum hepcidin was significantly lower in H. pylori infected anaemic individuals and anaemic individuals without evidence of infection when compared to controls (9-fold, p=0.009 and 5-fold, p < 0.0001 respectively). These results are opposite to data from children, possibly explained by the presence of gastric atrophy. The cellular localisation of ferroportin was different in the H. pylori infected group which could be due to local cytokine production. Gaining a better understanding of this mechanism could aid the development of more targeted investigation and treatment. However, with regards to allergic and autoimmune conditions, there is growing evidence to suggest H. pylori is inversely associated. It is believed that any benefit associated with H. pylori is confined to childhood when the immune system is developing. A significant reduction was seen in IL10+ Tregs (p=0.0029) after successful eradication suggesting the removal of H. pylori may have systemic consequences on the immune system that are still not fully understood. This work has highlighted the use of endoscopic techniques to identify individuals at risk of disease. It has also described the effects of eradication on the immune system which potentially could have implications for individuals with allergic conditions with regards to eradication therapy.

Hepatitis C virus (HCV) causes acute and chronic liver diseases in humans. Its two envelope proteins, E1 and E2, are heavily glycosylated. They interact with host cell receptors and provide a target for host immune recognition. The host virus interactions determine the pathogenesis and outcome of HCV infection. L-ficolin is a soluble pattern recognition molecule of importance in innate immune defence against microorganisms. It activates the lectin complement pathway upon binding to carbohydrate recognition patterns on microorganisms. It was hypothesised that L-ficolin could interact with HCV glycoproteins. Both recombinant and serum derived L-ficolin were investigated for binding to the envelope glycoprotein E1E2 of HCV. Specific, dose-dependent binding of L-ficolin to HCV glycoprotein E1E2 was observed. The interaction between L-ficolin and HCV particles in infected sera was also demonstrated. Binding of L-ficolin to HCV pseudoparticles expressing E1E2 glycoproteins resulted in neutralisation of virus infectivity. The serum L-ficolin level was significantly higher in patients with mild HCV liver fibrosis compared to patients with severe HCV liver fibrosis. These results suggest a potential protective effect of L-ficolin, as an innate immune defence, against HCV infection. To study the role of anti-HCV E1 and E2 (anti-E1E2) in HCV disease, the levels of anti-E1E2 antibodies were evaluated in 230 sera of patients with chronic hepatitis C by enzyme-linked immunosorbent assay. The antigens used were recombinant HCV glycoproteins derived from genotype 1 (H77c) and genotype 3 (UKN3A1.28). Seroreactivity was greater when sera were tested against antigen derived from their homologous genotype than against heterologous antigen. The seroreactivity was inversely proportional to the viral load and to the degree of liver fibrosis. These results demonstrate that seroreactivity against E1E2 depends upon the genotypic origin of the E1E2 antigens and the infecting genotype, and suggest a possible protective effect of anti-E1E2 against disease progression.

Background: Globally, the number of adolescents living with perinatally-acquired HIV continues to rise including in Malawi. To date, this group has received relatively less attention in the field of HIV care; yet they are increasingly surviving into adulthood. There is a growing need for the development of appropriate care and support services for this group; however their sexual and reproductive health (SRH) needs remain poorly addressed. Research Aim: This study aimed to explore perinatally-infected young women’s experiences of growing up with HIV in order to understand their SRH outcomes within their wider socio-cultural and structural context. Methods: A qualitative case study design was adopted whereby each ‘case’ comprised a female adolescent (15-19 years), a nominated caregiver and a service provider. Data was collected for 14 cases through in-depth interviews. The interviews with adolescents were based on an innovative visual method known as ‘my story book’. Results: The study found that young women endured multiple losses that negatively impacted upon their sense of self and belonging. Emotional, material and social support were essential in helping them to build a sense of identity, but their access to such resources was highly variable. Young women’s strategies to seek love, acceptance or support often led them to take sexual risks and left them with little control over their reproductive health. Both the service providers and caregivers often ‘turned a blind eye’ to young women’s sexual activities, leading to poor SRH outcomes. Lack of open discussion on SRH issues was related to cultural and religious norms hindering young women’s access to information and contraception. Conclusion: Addressing the complex needs of the young women poses a key challenge for Malawi’s HIV services. One way forward is to explore ways in which services could develop integrated models of care, offering a ‘one-stop shop’ to this vulnerable group.

Background: The global policy focus of today’s HIV efforts and strategies is to reverse the spread of HIV/AIDS and provide care, treatment and support. A key component of this strategy is to increase individual HIV status awareness through expansion of HIV testing and counselling (HTC). However, the numbers tested still remain low and evidence suggests that there are significant missed opportunities for HIV testing in clinical settings. One key strategy to expand HTC in clinical settings has been to implement a policy of ‘provider initiated counselling and testing’ (PITC) in which all patients accessing health facilities for treatment are routinely offered a HIV test. The introduction of PITC has brought with it a ‘dilution’ of the previously lengthy and stringent testing process by doing away with signed informed consent and extensive pre and post test counselling. The previous process was recognised as a barrier to public health gains of HIV testing expansion, particularly as it differentiated an HIV test from other routine medical tests resulting in a sense of HIV exceptionalism. In its place, the PITC policy recommends an opt-out approach and replaces the extensive pre test counselling with an information giving session placing more emphasis on post test counselling in cases where the result is positive. This change has given rise to debates about the potential for PITC to infringe patients’ rights to informed consent and counselling especially in developing countries. Emerging evidence from the exploration of the PITC process within antenatal settings in the Sub Saharan Africa has revealed some of the complexities of implementing PITC guidelines in different cultural and healthcare contexts. These studies suggest that information giving and consent are difficult to apply in contexts characterized by healthcare worker dominance, lack of sufficient resources and time constraints. This study aimed to specifically investigate how patients and counsellors co-construct informed consent and perform counselling during the PITC consultation. It examined ‘real time’ patient-counsellor interaction within hospital outpatient and inpatient settings in Kenya, explored the patient’s experience of a routine HIV test and evaluated how stigma and patient – provider interaction norms influence the PITC process in this context. Methods: In order to explore the context of the routine testing consultation and the way the interaction played out, a qualitative research approach was adopted, utilizing multiple data collection methods (interviews, observations and audio recording of consultations). The study was carried out in two government run health facilities in Kenya’s capital, Nairobi. The intention was to follow patients through the PITC process, i.e. before testing, during the HIV test and (whenever possible) after the HIV test. To get a broader picture of the events during the routine HIV testing consultation, additional interviews were conducted with five nurse-counsellors whose consultations had been observed. Ethical approval was obtained from the Kenya National Research Council, Kenya Medical Research Institute and the Aga Khan University Ethics Committee. The data were analysed using Charmaz’s constructivist grounded theory approach which allowed for a systematic yet flexible approach to analysis. This method facilitated immersion and engagement with the data, and provided a means of managing the different data sets in the study and undertaking a process of constant comparison within and between data sets. Findings: Results from the study suggest that HIV remains a highly stigmatised illness in Kenyan society and is associated with death and immorality. This is still the case in spite of years of health promotion and high profile media campaigns raising awareness about HIV and the availability and effectiveness of treatment. The context of stigma shaped the consultation so that both patients and counsellors worked together to help patients to maintain a ‘moral face’. Patients tended to withhold information on risky sexual behaviour whilst the counsellors avoided inquiring into this domain. The PITC consultation was characterised by a counsellor dominated approach to communication and health promotion. Counsellor inputs were generic, highly scripted and didactic rather than patient-centred. As a result, the counsellors’ style of communication allowed little space for personalised risk assessment or for patients to ask questions or to express concerns. The findings suggest that informed consent enabling explicit refusal of the test offer was difficult to achieve in an environment where the HIV test was not framed as a choice and patients came to the health facility expecting to be told what to do. Nevertheless, in spite of the obvious lack of explicit informed consent and the counsellor dominated interaction, post test interviews revealed that patients were satisfied with the nature of the interaction. The study concludes that there is a considerable distance between the policy recommendations and their implementation on the ground due to the complexity of real world practice. Lay constructions about HIV (HIV stigma) and the existing norms of patient-provider interaction that are characterised by a passive patient and a dominant health care provider shape the way the consultation unfolds. PITC training programs and manuals need to include skills and strategies that can support counsellors manage an uncomfortable interaction and emphasis the need to ensure an individualized post test counselling is carried out. The thesis makes several contributions to knowledge. The study pays attention to the operationalization of PITC recommendations thus illuminating how the PITC policy is translated into practice within a developing country like Kenya. It informs the existing debates on how informed consent and counselling should be implemented. The study findings suggest that in spite of the global debates on what constitutes ideal informed consent and counselling, in practice, sociocultural norms shape how these issues are translated and implemented. However, the study indicates that diversion from the PITC policy recommendations does not necessarily constitute a disregard for the recommendations but, rather, is an attempt to adapt to the prevailing environment. The study methodology enabled unique insights to be gained on how counselling and consent are constructed and managed in the PITC setting through the use of observations / audio recording to examine ‘real time’ interactions. The research study has been able to illuminate barriers that are posed by sociocultural and organisational structures in the real world implementation of the PITC policy. Therefore, my study suggests that the national PITC policy needs to consider the practical problems faced on the ground in developing contextually appropriate recommendations for the conduct of PITC and implementation of key guidelines.

Background Papular pruritic eruption (PPE) of human immunodeficiency virus (HIV) infection is common HIV-infected populations who live in tropical and subtropical regions. It is characterized by chronic and intensely itchy papules that are usually more highly concentrated on the extremities, adversely impacting on quality of life. Its aetiology has been postulated to be an altered and exaggerated immunological response to insect bites or stings. It has been reported to diminish in severity or resolve with antiretroviral therapy (ART). Its presence after at least six months of ART has been proposed as one of several clinical markers of failure of antiretroviral treatment. Objectives 1. To systematically summarise the evidence of interventions for PPE 2. To translate, culturally adapt, and test oral administration of a Runyankore-version of Skindex-16 for use in dermatology research in Mbarara, Uganda 3. To determine factors associated with PPE in HIV-infected Ugandan adults receiving ART for at least 15 months 4. To describe the natural history of PPE in HIV-infected Ugandan adults over two years from the time of ART initiation and explore the association between recurrent or persistent PPE and antiretroviral treatment failure Methods Systematic review of interventions for PPE Electronic searches of Medical Literature Analysis And Retrieval System Online (Medline), Excerpta Medica Database (Embase), Cumulative Index To Nursing And Allied Health Literature (CINAHL), Global Health Library, Cochrane Library, World Health Organization (WHO) International Clinical trials registry and National Library Of Medicine (NLM) gateway were carried out from January 1980 to July 2014. Studies of any design were included. The primary outcome measure for this review was resolution of skin disease. The quality of evidence was assessed using the Newcastle-Ottawa quality assessment scale and Grading Of Recommendation, Assessment, Development And Evaluation (GRADE) approach, where appropriate. Two authors carried out data extraction and quality assessment of studies independently. Runyankore-version of Skindex-16 for oral administration in Mbarara, Uganda Skindex-16 in English was translated to Runyankore, and then back-translated to English. The original and back-translated versions of Skindex-16 were compared for fidelity of translation. The Runyankore-version was administered orally to 47 dermatology patients and 47 random hospital visitors. Study participants were also asked about the characteristics of their skin condition including its duration, presence of skin colour change and ease or difficulty of concealment as well as an open question on how their skin condition has affected them. Case control study examining factors associated with PPE in the ART era This is a case–control study nested within a 515-person cohort receiving care at the HIV clinic of a teaching hospital in Mbarara, Uganda. Forty-five cases and 90 controls were enrolled. Cases had received ART for ≥15 months, fulfilled the clinical case definition of PPE and had skin biopsy findings consistent with PPE. Each case was individually matched with two controls for age, sex and ART duration. Cohort study describing the natural history of PPE over two years from ART initiation This is a cohort study of HIV-infected Uganda adults initiating ART and receiving care at the HIV clinic of a teaching hospital in Mbarara, Uganda who fulfilled the clinical case definition of PPE and had skin biopsy findings consistent with PPE. Standardised interviews, clinical photography, HIV viral load, CD4 counts and CD8+ T-cell activation markers were measured at three-month intervals for two years. Results Systematic review of interventions for PPE No randomised controlled trials were identified. Thirteen studies with a total 188 participants were included. ART was associated with resolution of PPE in a prospective observational study that had high loss to follow-up rates. Two observational studies reported positive responses of PPE to oral antihistamines (promethazine and cetirizine). Pentoxifylline was associated with diminished signs and symptoms of PPE in an uncontrolled open trial and superior to dapsone and a combination of antihistamine and topical corticosteroids in a parallel group non-randomised trial. Resolution of PPE was reported with a combination of topical corticosteroids and oral antihistamines in a case report. The efficacy of ultraviolet B (UVB) phototherapy was reported in an observational study with eight participants and three case studies with a total of five participants. Runyankore-version of Skindex-16 for oral administration in Mbarara, Uganda Oral delivery was feasible, taking ≤10 minutes per subject. High Cronbach α values (0.86, 0.88 and 0.85 for Symptoms, Emotions and Functioning subscales, respectively) demonstrated internal consistency reliability. As hypothesised, subjects with reported skin problems, dyspigmentation and difficulty in concealment had higher mean Skindex-16 scores, indicating construct validity. A large proportion (72.4%) of responses to the open-ended question were addressed in Skindex-16, indicating content validity. Case control study examining factors associated with PPE in the ART era Twenty-five of 45 cases (56%) had histological findings consistent with PPE (known as PPE cases). At skin examination and biopsy (study enrolment), a similar proportion of PPE cases and their matched controls had plasma HIV RNA <400 copies/ml (96% vs. 85%, p=0·31). The odds of having PPE increased four-fold with every log increase in viral load at ART initiation (p=0.02) but not at study enrolment. CD4 counts at ART initiation and study enrolment, and CD4 gains and CD8 T-cell activation measured 6 and 12 months after ART commencement were not associated with the presence of PPE. Study participants who reported daily insect bites had greater odds of being cases [odds ratio (OR) 8.3, p<0.001] or PPE cases (OR 8.6, p=0.01). Cohort study of natural history of PPE over 2 years from ART initiation Seventeen (15 female and 2 male) participants with a median age of 29.8 years were enrolled. Median CD4 count and HIV viral load at ART commencement was 108 cells/mm3 and 114,442 copies/ml, respectively. Resolution of PPE occurred in 13 of 17 (76%) study participants at a median time of 8.5 months after ART initiation, although PPE recurrence was observed at seven participants during the study period. Two participants had persistent PPE. Virological failure was not detected in any study participant. HIV RNA was less than 400 copies/ml at a median time of three months from ART initiation in all study participants. Conclusions 1. The evidence base of interventions for PPE is of low quality. There is some evidence of the efficacy of ART in the management of PPE. Pentoxifylline and phototherapy may have a role in its management but are unlikely to be available in resource-limited settings. Oral antihistamines and topical corticosteroids may be helpful in some individuals affected by PPE. 2. The orally administered Runyankore-version of Skindex-16 is reliable, with construct and content validity, and feasible for use in dermatology research in Mbarara, Uganda. 3. PPE in HIV-infected Ugandan adults receiving ART for at least 15 months was associated with reported daily insect bites and greater HIV viraemia at ART commencement, independent of CD4 count. 4. Recurrent or persistent PPE in HIV-infected Ugandan adults observed over two years from initiation of ART was not associated with virological failure in participants of this study.

Background (Kenya) Sub-Saharan Africa (SSA) is in the midst of experiencing an unprecedented increase in non-communicable diseases (NCD), specifically diabetes and hypertension. This shift has required public sector health systems, which have historically focused on managing acute diseases, to redesign their services to appropriately serve chronic disease needs. Issue Addressed In order to provide a description of our efforts to bring up comprehensive services for NCDs in rural Kenya within this thesis, I have specifically selected publications which target different aspects of the healthcare system. This includes our efforts related to clinical training for pharmacists, screening for NCDs, medication supply chains, remote phone-based care services, and care delivery based in the community. Prior to the implementation of the programs mentioned in these domains, access to these services was largely not available in western Kenya. Furthermore, the publication of our research from this western Kenyan cohort is designed to supplant the relatively limited research which emanates from rural sub-Saharan Africa. Research Questions For each of these selected publications, we defined a set of primary and, in some cases, secondary research questions focused on identifying the contextualized attributes of service delivery in this setting while also assessing the impact. For the first publication on training for clinical pharmacists, we assessed the impact of Kenyan Bachelor of Pharmacy interns and North American Doctor of Pharmacy interns while providing clinical care in an inpatient setting in Kenya. Our primary research question assessed whether there was a significant difference in the number of clinical interventions documented by interns from the two countries. In the second paper, we shifted our focus to outpatient care and wanted to address the uptake of different strategies of screening for diabetes and hypertension. Our primary research question assessed whether there were any significant differences in follow-up at the public sector clinic after screening positive via home-based screening (community health volunteer provides screening at your home) versus community-based screening (a community wide event is established where people voluntarily show up to receive screening) in a rural setting. In the third paper, we sought to continue to improve aspects of outpatient care by describing our model for improving access to medications. Our primary research question focused on descriptively assessing the change in availability of essential medications before and after implementation of this model. In the fourth paper, we described and assessed our model for providing intensive diabetes follow-up remotely. Our primary research question focused on whether patients experienced statistically significant improvements in blood glucose control after participating in this service for six months. In the fifth paper, we brought together various elements of our prior activities to design and evaluate the community-based model of care called BIGPIC - Bridging Income Generation through grouP Integrated Care. The primary research question for this investigation was to identify the frequency with which patients who screened positive for diabetes or hypertension linked to care. Secondary research questions compared the linkage frequency observed with this model compared to a historical control, along with a descriptive assessment of the loss to follow up, and an assessment of whether this model led to statistically significant reductions in blood pressure after 1 year of implementation. Short Summary of the Individual Papers with Results Linking Them Together Within our assessment of pharmacy training, we found that the Kenyan pharmacy interns provided statistically significantly more clinical interventions per day than their North American counterparts. This result highlighted the potential for Kenyan pharmacy providers to provide clinical services which were largely unavailable in western Kenya prior to this research. Despite the lack of the clinically focused Doctor of Pharmacy curriculum in Kenya, Kenyan pharmacy interns within the Bachelors in Pharmacy program were able to make an average of 16.7 consultations per day with the medical team compared to 12.0 per day for the North Americans. In the second paper we shifted our focus to the outpatient setting and were surprised to find that there weren’t any statistically significant differences in follow-up between home-based versus community-based screening for NCDs. This highlighted the reluctance of rural patients to travel to public sector facilities for care regardless of the screening method utilized. This realization led us to simultaneously focus on improving the reliability of services available in public sector while also trying to implement solutions to facilitate the provision of remote services for care. Within our efforts to improve medication access in paper 3, we were able to demonstrate how our revolving fund pharmacy model was able to improve access to medications from < 40% to > 90%. In paper 4, we were able to implement a self-monitored blood glucose program and demonstrate a dramatic improvement in the blood sugars of patients enrolled in the self-monitored blood glucose program with a statistically significant 31.6% absolute decline in HbA1c. The culmination of these efforts and learnings is described in paper 5, where we implemented the BIGPIC care delivery model which resulted in a statistically significant improvement in linkage to care for screened patients, a retention in care frequency of 70.3%, and a statistically significant mean decline in the systolic blood pressure of 21mmHg (95% CI 13.9-28.4, P < 0.01).