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.

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.

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.

Hepatitis C Virus currently affects up to 3% of the world’s population. There is no effective vaccine yet available and the natural immune response to infection is largely inefficient. Progress has been made in isolating several broad-acting neutralizing antibodies that target the viral envelope protein E2. However, a dominant element of the epitopes targeted is an overlap with the highly conserved CD81 binding sites. Various E2 constructs were investigated as possible targets to be used in phage display panning of a combinatorial library of the phagemid vector pComb3H. HVR2 deletion showed optimal exposure of the CD81 binding sites and D535A disrupted known CD81 epitopes. A selection technique was designed to improve exposure of conserved sites on an E2 construct target molecule that disrupts CD81 epitopes while remaining conformationally correct. Optimisation of the screening methodology was used to assess the quality of enrichment of the library panning along with more efficient selection of specific clones. The approach adopted in this project isolated Fab clones specifically reactive to the protein target, one of which also showed preferential binding in acidic environments. Taken together, the information gathered on E2 and the implementation of the phage display method described will contribute to more effective ways of isolating novel antibodies.

The human non-integrin laminin receptor (LamR) is a multifunctional protein which is localised to a number of sub-cellular locations. LamR is a component of the ribosome and has a number of intracellular functions; it also acts as an extracellular receptor for laminin, growth factors, pathogenic microorganisms, prion proteins, toxins and the anticarcinogen epigallocatechin gallate (ECGC). Although LamR is present in most cellular compartments, its overexpression in many types of cancer cells suggests a vital role for LamR in tumor-cell migration and invasion. There are two isoforms of laminin receptor: the monomeric 37-kDa laminin receptor precursor (37LRP) and the mature 67-kDa laminin receptor (67LR). Although the precise molecular nature of 67LR is unclear, accumulating evidence strongly suggest that 37LRP can undergo homo- and/or hetero-dimerization with Galectin-3 (Gal-3) to form mature 67LR. A recent study demonstrated that both homo- and heterodimer LamR forms are present on the cell surface, where they form distinct populations. Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae type b (Hib) are major causes of bacterial meningitis. The contribution of LamR in traversal of the blood brain barrier (BBB) by neurotropic viruses is well established and interaction with LamR was recently found to be critical for initiation of bacterial contact with the blood brain barrier (BBB). These bacteria bind LamR via the surface protein adhesins: meningococcal PilQ and PorA; pneumococcal CbpA; and H. influenzae OmpP2. Further investigations showed that the fourth and second extracellular loops of meningococcal PorA and OmpP2 of H. influenzae, respectively, are responsible for LamR binding. The work presented here consists of two complementary projects in which a number of approaches were taken to characterise the ligand-binding domains of LamR. The first project aimed to identify sites on LamR that are critical for binding the ligands of bacterial meningeal pathogens. The second project aimed to identify residues that contribute to the stability of LamR homodimers and the heterodimer with Gal-3. Several mutations were introduced into full-length human LamR, either by deletion mutations within the C-terminal domain (CTD) of LamR using inverse polymerase chain reaction (IPCR), or by single-amino acid substitution in the N-terminal domain (NTD) of LamR using site-directed mutagenesis. Protocols for large-scale expression of full-length and truncated LamR proteins in human cells were developed, as well as non-denaturing purification protocols. We hypothesised that bacteria-binding domains could be located on both the NTD and CTD of LamR. In vivo examination using ELISA assays, in which the interaction of LamR and whole bacteria or purified recombinant PorA or PilQ were tested identified several novel sites on LamR that contributed significantly to binding of the bacterial ligands. These sites include amino acids 206-229 and 263-282, located within the CTD, and Tyrosine 156 in the NTD, each of which contributed to the binding of meningococcal PorA, and more specifically it’s fourth extracellular loop. Furthermore, three sites on LamR comprising amino acids 206-229 and 263-282 within the CTD and Tyrosine 139 in the NTD were shown to contribute to binding pneumococcal CbpA, OmpP2 and Loop two of OmpP2 of H. influenzae. These results indicate that the three neuroinvasive bacteria share the same binding sites on LamR. Bimolecular fluorescence complementation (BifC) coupled with flow cytometry and confocal microscopy revealed the vital contribution of amino acid residues Arginine 155, Tyrosine 156 and Lysine 166 (all within the NTD of LamR) for the homodimerization and heterodimerization of LamR with Gal-3. The dimerization of two meningococcal host receptors, LamR and Gal-3, may help to extend spectrum of their bacterial adhesins, which may act cooperatively or synergistically at different stages of infection. Information about the residues in LamR that contribute to the stabilization of LamR dimers has implications for the role of LamR dimers in the pathogenesis of bacterial meningitis. Identification of bacteria-binding domains on LamR is of particular interest for development of vaccines or therapeutic interventions that provide protection against the three major meningitis-causing bacteria. The aim of the current work was first to localise the domains of LamR responsible for binding with PorA and PilQ of N. meningitidis; CbpA and OmpP2 of S. pneumonia, and OmpP2 of H. influenzae. Also, previous studies have shown conspicuous homodimerisation of 37LRP and heterodimerisation with Gal-3. Our second aim was to identify of amino acid residues involved in 37LRP self-association and heterodimer formation with Gal-3. In current work, several regions of LamR were hypothesised to constitute the binding site for the bacterial ligands; these predictions were based on previous studies on LamR binding domains and the crystal structure of laminin receptor. Also, to investigate both homo and heterodimerisation of LamR, the involvement of several putative amino acid residues within 37LRP in LamR dimerisation was was hypothesised.

Infection with hepatitis C virus (HCV) is cleared spontaneously in only 20% of cases with the majority of individuals developing a chronic infection. This discrepancy in disease outcome is incompletely understood but current understanding of the immune response to HCV suggests that rapid induction of a broadly neutralising antibody (nAb) response leads to resolution of acute infection. The majority of nAb identified target the envelope glycoproteins, particularly E2, and most appear to inhibit binding of E2 to the cellular receptor CD81. Antibodies targeting other interactions, such as those with the receptor CLDN or the fusion determinant, are underrepresented in the repertoire of anti-HCV antibodies. However, the antibody discovery process may have been biased by the nature of the assays used. Therefore new assays are needed to enable the discovery and characterisation of antibodies in an unbiased manner. To facilitate this, a novel insect cell display library was developed for mapping antibody-binding epitopes. Cells expressing specific E2 mutants provided the necessary proof-of-principle that loss of antibody binding could be detected in this system before a library expressing randomly mutated E2 was developed. Sorting experiments demonstrated that single cells could be isolated and enriched based on loss of antibody binding. Secondly, a method for characterising the immunoglobulin (Ig) genes of HCV infected patients was developed; Ig genes were isolated from small numbers of B cells and the sequences analysed. Finally, a patient cohort was studied with a view to investigating the evolution of the antibody response during early infection. The unreliable nature of the samples prevented such analysis; however a DNA fingerprinting method of testing the origin and relatedness of serum samples was developed. This will improve the reliability of future studies. Together these methods provide a work model for the assessment of samples and the isolation and characterisation of antibodies.