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Enteric fever caused by Salmonella enterica serovars Typhi and Paratyphi A are a major public health concern in Kathmandu. The aim of this thesis was to identify and assess the population most at risk by investigating epidemiologic trends of enteric fever within a subset population of Kathmandu. Therefore,the burden and incidence of enteric fever within the study population and the seasonal and gender distribution of enteric fever was assessed. Considerable burden of enteric fever, unrelated to population density, correlating with the seasonal fluctuations in rainfall was observed. This thesis also aimed to improve the understanding of enteric fever transmission by identifying probable transmission routes,hence various water and food samples were analysed and the extent of faecal contamination in them was determined. S. Typhi isolates were sequenced and genotyped and combined with GPS data to longitudinally study the local distribution and infer transmission of this human restricted bacterial pathogen. Extensive clustering of typhoid independent of population size and density and existence of an extensive range of genotypes within typhoid clusters including individual households with multiple cases was observed. These observations predict that indirect transmission had an overwhelming contribution for disease persistence, potentially through contaminated water. Consistent with this hypothesis, S. Typhi and S. Paratyphi A were detected in water supplies and it was observed that typhoid was spatially associated with public water sources and low elevation. A concurrent case-control study was also conducted which allowed for the determination of risk factors in the population at risk. These studies imply that resources should be allocated toward controlling the most important vectors of enteric fever, including food sold by vendors, chlorination of drinking water, construction of proper water distribution and sewage networks,vaccination campaigns and hygiene education.

Plasmodium vivax malaria threatens nearly half the world’s population. This relapsing disease may be more severe than previously recognised and is proving refractory to current malaria control measures. This thesis aimed to describe the burden of anaemia and mortality attributable to vivax malaria in Southern Papua, Indonesia, an area endemic for multidrug-resistant P. vivax and P. falciparum, and to determine the potential of currently available antimalarial drugs to reduce transmission of P. vivax in co-endemic regions. Approximately 0.5 million uniquely identified clinical records from patients presenting to Mitra Masyarakat Hospital between April 2004 and May 2009 were matched with corresponding laboratory and pharmacy data in order to determine the burden of anaemia in the hospital setting and the effectiveness of primaquine prescription for preventing P. vivax relapses. Clinical information extracted from patient notes was used to clarify the contribution of P. vivax malaria to a series of deaths detected by an active hospital-based surveillance system. Additional secondary sources of data used in this thesis included a large house-to-house survey and multiple clinical trials of antimalarial therapy from both Southern Papua and Northwestern Thailand. In Southern Papua, P. vivax malaria is an important cause of haematological morbidity both in the hospital and community setting. This morbidity is most significant in the first year of life when P. vivax infection accounts for 23% of all severe anaemia (haemoglobin <5g/dL) in the hospital and approximately 28% of all moderate-to-severe anaemia (haemoglobin <7g/dL) in the community. In this region concomitant P. vivax infection accentuates haematological impairment associated with P. falciparum malaria. Plasmodium vivax in Southern Papua rarely causes death directly but rather indirectly contributes to mortality through exacerbation of comorbid conditions. In Northwestern Thailand, 53.8% of patients with falciparum malaria who were treated with a rapidly eliminated drug between 1991 and 2005 had a recurrence of vivax malaria within two months making P. vivax infection the most common cause of parasitological failure in these individuals. Slowly eliminated artemisinin combination therapies (ACT) provided the greatest protection against recurrent P. vivax parasitaemia during 63 days of follow-up. In three randomised controlled trials from Papua and Thailand, P. vivax gametocytaemia was shown to mirror asexual parasitaemia closely and to have the same characteristics in acute and recurrent infections. This emphasises that the most important chemotherapeutic means of blocking P. vivax transmission is prevention of future relapse. Primaquine is recommended for this purpose but analyses in this thesis suggest that in Southern Papua, unsupervised primaquine at a dose of 0.5mg/kg/day for 14 days, does not reduce the risk of subsequent relapse (Adjusted Hazard Ratio = 1.01 [95% confidence interval 0.95-1.07]). Plasmodium vivax malaria should not be neglected. High priority must be given to new hypnozoitocidal drug discovery. In the interim, optimising the safety and effectiveness of primaquine and adoption of a unified ACT-based blood schizontocidal treatment strategy for malaria of any parasitological cause in co-endemic regions will be crucial for controlling P. vivax malaria.

Meningococcal disease, caused by the bacterium Neisseria meningitidis, is an important cause of morbidity and mortality in young children and adolescents worldwide. There are 12 serogroups with most disease due to meningococci expressing one of five capsular polysaccharide antigens corresponding to serogroups A, B, C, Y and W135. In Europe, the majority of disease-causing strains are of serogroups B and C. No comprehensive vaccine is available against the bacterium due to the difficulty in producing serogroup B vaccines. A number of countries, e.g. UK and the Republic of Ireland have implemented routine meningococcal conjugate C (MCC) vaccine strategies. Due to the high proportion of disease accounted for by serogroup B in Europe and other developed countries, much research is currently being carried out to unearth vaccine candidates that would be protective and give as wide coverage as possible. Such candidates include the antigens PorA, FetA and factor H-binding protein. Potential drawbacks with antigens such as these which are under immune selection are high degrees of variability and lack of cross-immunity. Determination of the distribution, both geographically and temporally, of antigens and their association with clonal complex can aid in the formulation of novel vaccines and assess their potential coverage across Europe. Serological typing schemes involving characterisation of the polysaccharide capsule (serogroup) and outer membrane proteins such as PorA (serosubtype) and PorB (serotype) have been used for a number of years with some success. However, drawbacks associated with these methods include insufficient discrimination, limitations in panels of monoclonal antibodies used in the typing procedures and difficulty in comparison of results among labs. Consequently, in recent years genotypic methods such as multi-locus enzyme electrophoresis (MLEE) and subsequently multi-locus sequence typing (MLST) have been developed. These methods measure the variation in slowly evolving housekeeping genes whereas serological methods measure variation in antigens which are under immune pressure and are therefore more diverse. Combination of phenotypic and genotypic typing methods can offer high levels of discrimination. Molecular studies into meningococcal diversity have offered many important insights into its population biology, which have implications for prevention and control of meningococcal disease. These have included the identification of hyperinvasive lineages and the correlation of genetic type with antigenic type and disease epidemiology. The EU-MenNet programme was established as a pan-European infrastructure for the research and surveillance of European meningococcal disease. Its aim was to coordinate and disseminate the latest molecular isolate characterisation techniques (MLST) and electronic data transfer via the Internet to exploit epidemiological and population genetic studies. Within the EU-MenNet, the European Meningococcal MLST Centre (EMMC) was set up to carry out molecular typing — MLST, PorA and FetA — of European disease isolates from 18 countries over three years 2000, 2001 and 2002. The output of this project will be the largest representative molecular epidemiological study of meningococcal disease in Europe. Assessment of the data produced will give insights into the geographic and temporal distribution and structuring of disease-associated clonal complexes and antigens and their associations. This will give an indication of the meningococcal disease population in Europe and will be invaluable for the current, and ongoing, development and introduction of new meningococcal vaccines.

Understanding the transmission dynamics of infectious diseases is important to well-informed public health policy, responsive infection control and individual patient management. The on-going revolution in whole-genome sequencing provides unprecedented resolution for detecting evidence of recent transmission and characterising population-level transmission dynamics. In this thesis, I develop and apply evolutionary approaches to investigating transmission, focusing on three globally important pathogens. Hepatitis C virus (HCV) is a major cause of liver disease affecting 150 million people and killing 350,000 annually. I conducted a meta-analysis of twentieth-century HCV epidemics, finding that the age of the epidemic can be predicted by genetic diversity. Using the coalescent, I fitted classic susceptible-infected (SI), susceptible-infected-susceptible (SIS) and susceptible-infected-recovered (SIR) epidemiological models. Most epidemics showed signatures of SI dynamics, but three, from Argentina, Hong Kong and Thailand, revealed complex SIR dynamics. Norovirus is the leading viral cause of diarrhoea, estimated to cost the NHS around £115 million annually. I analysed whole norovirus genomes via a stochastic transmission model, finding that up to 86% of hospital infection was attributable to transmission from another patient in the hospital. In contrast, the rate of new introductions to hospital by infected patients was extremely low (<0.0001%), underlining the importance of ward management during outbreaks. Campylobacter is the most commonly identified cause of bacterial gastroenteritis worldwide. I developed a zoonotic transmission model based on phylogeography approaches to test whether three strains previously associated with multiple host species were in fact aggregates of strongly host-restricted sub-strains, or genuine generalists. Members of the same strain isolated from different host species were often more closely related than those isolated from the same host species. I estimated 419, 389 and 31 zoonotic transmissions in ST-21, ST-45 and ST-828 respectively, strongly supporting the hypothesis that these strains are adapted to a generalist lifestyle.

Erythrocytes infected by mature forms of the Plasmodium falciparum parasite adhere to other components of the vascular space, a behavior considered critical to the pathogenesis of severe malaria. Adhesion is mediated by the P. falciparum erythrocyte membrane protein 1 (PfEMP1), a highly variant antigen expressed by the parasite and subject to switching during the course of an infection. The host ligands of PfEMP1 include CD36, ICAM-1 and the ABO antigens. By employing a series of population- and family-based association studies from multiple African populations, we examined whether variation in the genes underlying these molecules affects susceptibility to severe malaria. Our results suggest that a common frameshift mutation in the ABO glycosyltransferase, responsible for blood group O, is associated with protection from severe malarial phenotypes (P=2x10⁻⁷), particularly severe malarial anaemia. However, we found no significant disease associations with variation in either the ICAM1 or CD36 genes. We focused on two particular functional polymorphisms, the missense ICAM-1Kilifi and the CD36 nonsense mutation T1264G. We genotyped both markers in around 10,000 individuals, but neither demonstrated an association with severe malarial phenotypes. Malaria has been a profound selection pressure shaping human genetic diversity. The last decade has seen the development of several haplotype-based methods to detect signatures of recent positive evolutionary selection. These techniques are potentially invaluable tools in our hunt for genetic variants that protect from life threatening malaria. We used simulations and empirical data from the International HapMap Project to demonstrate the validity of searching for long regions of haplotype homozygosity, as an approach to finding alleles undergoing selective sweeps. We analysed genetic data from a range of populations, particularly those utilized by HapMap, to investigate whether our candidate genes were associated with signals of recent positive selection. We characterized the distribution of a selection event associated with the CD36 1264G allele, focused in Central-West Africa, and demonstrated a novel signal of low population differentiation at the ABO gene, suggestive of longstanding balancing selection. Our work confirms that variation in the host ligands of PfEMP1 modulates severe malaria susceptibility, and highlights the value of using signals of selection, along with functional experiments and genetic association studies, to dissect the biology of severe malaria.

During human immunodeficiency virus 1 (HIV-1) infection, the viral population is in constant battle with the host immune system. The cytotoxic T-lymphocyte (CTL) response, a branch of the adaptive immune response, is implicated in viral control and can drive viral evolution in the infected host population. Endogenous viral peptides, or ‘epitopes’, are presented to CTLs by human leukocyte antigen (HLA) class I molecules on the surface of infected cells where they may be identified as non-self. Mutations in or proximal to a viral epitope can result in ‘escape’ from CTLs targeting that epitope. The repertoire of epitopes which may be presented is dependent upon host class I HLA types. As such, reversion may occur after transmission due to changes in viral fitness and selection in the context of a new HLA background. Thus, parameters describing the dynamics of CTL escape and reversion are key to understanding how CTL responses within individuals relate to HIV-1 sequence evolution in the infected host population. Escape and reversion can be studied directly using biological assays and longitudinal viral sequence data, or indirectly by considering viral sequences across multiple hosts. Indirect approaches include tree based methods which detect associations between host HLA and viral sequence but do not estimate rates of escape and reversion, and ordinary differential equation (ODE) models which estimate these rates but do not consider the dependency structure inherent in viral sequence data. We introduce two models which estimate escape and reversion rates whilst accounting for the shared ancestry of viral sequence data. For our first model, we lay out an integrated Bayesian approach which combines genealogical inference and an existing epidemiological model to inform escape and reversion rate estimates. Using this model, we find evidence for correlation between escape rate estimates across widely separated geographical regions. We also observe a non-linear negative correlation between in vitro replicative capacity and escape rate. Both findings suggest that epistasis does not play a strong role in the escape process. Although our first model worked well, it had some key limitations which we address in our second method. Notably, by making a series of approximations, we are able account for recombination and analyse very large datasets which would be computationally infeasible under the first model. We verify our second approach through extensive simulations, and use the method to estimate both drug and HLA associated selection along portions of the HIV-1 genome. We test the results of the model using existing knowledge, and determine a collection of putative selected sites which warrant further investigation. Finally, we find evidence to support the notion that the CTL response played a role in HIV-1 subtype diversification.

Die Weltgesundheitsorganisation WHO schätzte die Zahl der an Malaria erkrankten Menschen im Jahr 2009 auf weltweit 225 Millionen. Auf dem afrikanischen Kontinent betrafen 85% der durch Malaria verursachten Todesfälle Kinder unter fünf Jahren. Obwohl die Inzidenzen der P. falciparum-Malaria in einigen Teilen des subsaharischen Afrika sinken und andere Erkrankungen mit ähnlichen Symptomen wie denen der Malaria an Bedeutung gewinnen, ist eine vorsorgliche medikamentöse Behandlung im Verdachtsfall weiterhin üblich. Ziel dieser Arbeit ist die Generierung eines auf das Lebensalter bezogenen klinischen Algorithmus, der mit einfachen klinischen Symptomen die Diagnose einer P. falciparum - Parasitämie ermöglicht. Die Studie wurde in einem ländlichen Krankenhaus in der Ashanti-Region in Ghana durchgeführt, welche über das ganze Jahr hinweg holoendemisch für Malaria ist. Insgesamt wurden 5447 ambulante Besuche von 3641 Patienten im Alter zwischen 2-60 Monaten analysiert. Alle Kinder wurden von einem Pädiater klinisch untersucht und es wurden ein kleines Blutbild sowie ein Malariaausstrich (‘Dicker Tropfen’) angefertigt. Mit Hilfe einesClassification and Regression Tree (CART) wurde ein klinischer Entscheidungsbaum für die Prädiktion einer Plasmodium-Parasitämie generiert und prädiktive Werte für alle erfassten Symptome berechnet. Eine Parasitämie wurde bei Kindern im Alter von 2-12 Monaten mit einer Prävalenz von 13.8% und bei Kindern im Alter zwischen 12 und 60 Monatenmit einer Prävalenz von 30.6% gefunden. Das CART-Modell ergab altersabhängige Unterschiede in der Fähigkeit der Variablen eine Parasitämie vorherzusagen. Während sich bei Kindern im Alter zwischen 2 und 12 Monaten die „palmare Blässe“ als das wichtigste Symptom herausstellte, gewannen die Variablen „Fieber in der Anamnese“ und „erhöhte Körpertemperatur ≥ 37.5°C“ bei Kindern im Alter zwischen 12 und 60 Monaten an Bedeutung. Die Variable „palmare Blässe“ war bei Kindern jedes Alters signifikant (p<0.001) mit niedrigeren Hämoglobinwerten assoziiert. Im Vergleich zum Algorithmus des Integrated Management of Childhood Illness (IMCI) hatte das CART-Modell eine deutlich höhere Spezifität sowie einen höheren positiven prädiktiven Wert für die Vorhersage einer Parasitämie. Die Anwendung von altersbezogenen Algorithmen erhöht die Spezifität der Vorhersage einer P. falciparum - Parasitämie. Selbst in einer Population mit einer hohen Prävalenz an Anämie ermöglicht der prädiktive Wert der „palmaren Blässe“ eine Erkennung von signifikant geringeren Hb-Werten. Die Bedeutung der „palmaren Blässe“ sollte daher in der Schulung von Gesundheitshelfern hervorgehoben werden. Mangels ausreichender Sensitivität kann allerdings weder auf Basis des besten Algorithmus noch mit „palmarer Blässe“ als einzelnem klinischem Zeichen eine Therapieentscheidung getroffen werden. Sie sind daher kein Ersatz für eine vorsorgliche medikamentöse Behandlung und einen Erregernachweis.

We investigate the biological constraints determined by the complex relationships between ecological and immunological processes of host-pathogen interactions, with emphasis on influenza viruses in human, which are responsible for a number of pandemics in the last 150 years. We begin by discussing prolegomenous reviews of historical perspectives on the use of theoretical modelling as a complementary tool in public health and epidemiology, current biological background motivating the objective of the thesis, and derivations of mathematical models of multi-locus-allele systems for infectious diseases with co-circulating serotypes. We provide detailed analysis of the multi-locus-allele model and its age-specific extension. In particular, we establish the necessary conditions for the local asymptotic stability of the steady states and the existence of oscillatory behaviours. For the age-structured model, results on the existence of a mild solution and stability conditions are presented. Numerical studies of various strain spaces show that the dynamic features are preserved. Specifically, we demonstrate that discrete antigenic forms of pathogens can exhibit three distinct dynamic features, where antigenic variants (i) fully self-organize and co-exist with no strain structure (NSS), (ii) sort themselves into discrete strain structure (DSS) with non-overlapping or minimally overlapping clusters under the principle of competitive exclusion, or (iii) exhibit cyclical strain structure (CSS) where dominant antigenic types are cyclically replaced with sharp epidemics dominated by (1) a single strain dominance with irregular emergence and re-emergence of certain pathogenic forms, (2) ordered alternating appearance of a single antigenic type in periodic or quasi-periodic form similar to periodic travelling waves, (3) erratic appearance and disappearance of synchrony between discrete antigenic types, and (4) phase-synchronization with uncorrelated amplitudes. These analyses allow us to gain insight into the age-specific immunological profile in order to untangle the effects of strain structures as captured by the clustering behaviours, and to provide public health implications. The age-structured model can be used to investigate the effect of age-specific targeting for public health purposes.