Human papillomavirus infection is regulated by multiple factors including methylation, viral integration and aberrations in host and viral gene expression. In most patients, the infection is transient and cleared effectively by the host’s immune system but in a minority of cases, the Human papillomavirus infection progresses to neoplasia and cancer if not detected and treated. Assays that detect the presence of Human Papillomavirus infection alone are not good prognostic markers of clinical outcome. Alternative clinical biomarkers that can measure other regulatory factors of Human Papillomavirus infection are required to more accurately predict patient outcome and help direct treatment options specifically to patients at risk of neoplasia and cancer progression. This study aimed to examine several Human Papillomavirus regulatory factors to determine if they would be suitable as clinical biomarkers and explore further the link between molecular changes and associated pathology. This involved development of assays, application to samples obtained from different cohorts of women to ascertain prognostic validity and development of an in vitro system to model the in vivo pathology. Initial work focused on investigating Human Papillomavirus 31, 33, 35 and 51 integration and methylation assays as prognostic biomarkers in young women attending their first routine cervical smear. Results indicated that Human Papillomavirus E2 gene disruption and methylation were not common events and the assays investigated were not suitable biomarkers for predicting clinical outcome in xx young women. The assays were then applied to clinical samples taken from patients with varying grades of cervical disease and high levels of viral methylation were present in women with high grade disease (Cervical Intraepithelial Neoplasia II+) and a correlation between methylation and HPV gene disruption was shown. Novel in vitro organotypic raft cultures of cells from Vulval Intraepithelial Neoplasia and Vaginal Intraepithelial Neoplasia were employed to understand how molecular changes in viral methylation and gene expression correlated with observed pathology. The organotypic raft cultures showed diverse differentiation patterns. No correlation of pathology with viral integration status was detected. However, organotypic raft cultures with high-grade disease morphology all had a higher level of methylation and expression of regulatory genes p16, Ki-67 and Sonic Hedgehog in comparison cultures displaying a histology consistent with low-grade disease. p16 and Ki-67 are already being examined as part of cervical screening triage. The findings presented in this thesis, highlight a need for further research into Human Papillomavirus infection and the molecular changes associated with Sonic Hedgehog gene expression and viral methylation as these show promise as prognostic biomarkers.

Dengue infection can present with a broad spectrum of clinical phenotypes. The hallmark of severe disease is increased vascular permeability, sometimes leading to hypovolaemic shock. Microvascular/endothelial dysfunction may underlie this, but have not been assessed clinically. I performed a prospective observational study in two hospitals in Vietnam recruiting 300 children ( > 3 years) and adults presenting to a) outpatient department with fever for < 72 hours and suspected dengue, b) patients hospitalized with warning signs or severe disease. Clinical and laboratory assessments were performed daily for 5 days, and 2 weeks later. Microvascular imaging using Sidestream dark field Imaging (SDF), and endothelial function testing using peripheral artery tonometry (EndoPAT) were performed at enrolment, defervescence/hospital discharge and follow-up. Plasma endothelial biomarkers were performed at the same time-points. Echocardiogram derived parameter of intravascular volume and cardiac function were also assessed at the same time-points and daily in patients admitted to ICU. We demonstrated firstly, that moderate microcirculatory alterations occur in dengue and are most prominent in the critical phase and worst in patients with plasma leakage. In the outpatient arm, these microcirculatory alterations were similar in other febrile illnesses. The flow alterations correlated with VCAM-1 and Angiopoietin-2 in dengue patients. Significantly elevated levels of syndecan-1 were shown in dengue patients during the critical phase, associated with the degree of plasma leakage, suggesting there is widespread endothelial glycocalyx degradation. In addition, we have shown patients with dengue have endothelial dysfunction with impaired endothelium-dependent vasodilation, worst in patients with severe plasma leakage, which is apparent early in the febrile phase. Endothelial dysfunction correlated with high levels of arginase-1 and low L-arginine levels. In severe dengue, myocardial impairment was associated with respiratory distress but not recurrent shock. High lactate levels at admission to intensive care and lower stroke volumes were associated with recurrent shock. Overall, this work has shown microvascular and endothelial dysfunction are associated with disease progression and severe outcomes in dengue, and may provide useful future biomarkers and therapeutic targets.

The Epstein-Barr virus (EBV) is characterised by the ability to infect human B lymphocytes in vitro and to transform and immortalise them into lymphoblastoid cell lines (LCLs). EBV strains can be divided into type 1 and type 2, according to the sequence of the viral EBNA-2 protein. Type 1 strains exhibit a superior capacity to efficiently transform B cells and this was mapped to the type 1 EBNA-2 protein. Substitution of the serine residue located in the transactivation domain (TAD) of type 2 EBNA-2 by the equivalent aspartate-442 of type 1 protein was previously shown to confer a type 1-like cell growth phenotype to type 2. In the present study, a stronger ability to transactivate gene expression was demonstrated for type 1 EBNA-2 TAD in comparison to type 2 EBNA-2 and this was determined by aspartate-442. This residue is located next to the interaction site of the transcriptional repressor BS69 with EBNA-2. BS69 inhibits gene transactivation by type 1 and type 2 EBNA-2 but a higher BS69 affinity for type 2 EBNA-2 protein has been shown in this study. Selective EBNA-2 activation of target promoters is also involved in the differential induction of target genes by type 1 and type 2 EBNA-2. Biotinylated oligonucleotide pulldown assays showed that efficient type 1 EBNA-2 binding at the LMP-1 promoter is dependent on the PU.1 and octamer motifs. Greater binding of type 1 EBNA-2 at the differentially regulated promoters, in conjunction with its stronger transactivation ability, conferred by aspartate-442, may determine the higher induction of the LMP-1 and cellular genes required for cell growth proliferation and therefore result in superior B cell transformation by type 1 EBV.

The prevalence of human herpesvirus 8 (HHV-8) in patients with Kaposi's sarconna (KS) or people at risk of developing KS is high, while that in low-risk populations remains unclear. Use of disparate serological assays for anti HHV-8 detection contributes to this uncertainty. Hypothesising that in populations at low risk of HHV-8, the detection of HHV-8 genome enhances the specificity of and lends sensitivity to estimations of HHV-8 prevalence, studies were conducted to compare the genoprevalence and molecular epidemiology of HHV-8 in UK subpopulations at varying risk of HIV infection or KS: blood donors, human immunodeficiency virus (HIV)-infected individuals, bone marrow transplant (BMT) recipients, and patients with chronic fatigue syndrome (CFS). A protocol for amplifying sub-genomic HHV-8 DNA was first developed using blood originating from HIV-seropositive patients, from which CD45+ cells were immunomagnetically separated and their extracted DNA submitted to nested PCR. It was determined that such an approach afforded greater sensitivity to HHV-8 DNA detection than approaches based on PCR applied to separated peripheral blood mononuclear cells. Using the improved protocol, DNA from open reading frame (ORF) 26 of the HHV-8 genome could be amplified from 24% of blood donor samples. In a subsequent donor group, DNA from ORFs 26 and K1 was detectable in 8% and 9%, respectively. ORF K1 sequences could be classified as belonging to genotypes A1, A4 and C3. HHV-8 seropositivity in the second group was 12% and 24%, as determined by two antibody assays, and The Genotypic characterisation of Human Herpes Virus 8 in Different Groups. herpes simplex virus-2 seropositivity was 0%. No associations were found between HHV-8 genome and anti HHV-8 detection. The findings in the BMT and CFS groups further substantiate the hypothesis that HHV-8 infection is more widespread than previously thought, carriers may not mount antibody responses detectable by current serological assays, and the principal HHV-8 transmission route is not sexual.

The rapid increase in malarial drug resistance and the severe side effects related to high doses of the current drugs have contributed to the urgent need for a new drug strategy to treat malaria. In situ gel implantation is used to increase the solubility of drugs, leading to higher absorption in the body. In addition, entrapping antimalarial drugs in nanocapsules has the potential to restore the use of older drugs with toxic side effects by modifying their bio-distribution, reducing their toxicity, and increasing their efficacy. The techniques used tested the effects of free drug, in situ gel systems, and nanocarrier formulations against malaria parasites. A formulation of 30% polymer to solvent was used as an in situ gel, holding one of the three malarial drugs tested. Our experiments demonstrate that the single dose in situ gel preparation leads to similar release as multiple doses of free drug. However, a 73%, 27%, and 100% cure rate was achieved with silver sulphadiazine, halofantrine, and chloroquine phosphate in situ gel, respectively. In contrast, a 0%, 0%, and 66.7% cure rate was achieved with the same drugs alone, respectively. However, late treatment did not result in the same effects observed for immediate treatment. In nanocarrier system, water-soluble chloroquine phosphate was prepared via double emulsion solvent evaporation, while nonwater-soluble halofantrine and silver sulphadiazine were prepared via interfacial polymer deposition. In vitro release studies showed slower silver sulphadiazine and chloroquine phosphate release in phosphate buffer saline media, when administered in nanocapsules than when administered in the free formulation. The antimalarial experiments showed 100% cure rate in both immediate and late infections. Nanocarriers are more efficacious than in situ gelling systems against malaria. This phenomenon is attributable to slow in vitro release as well as nanocarrier size, charge, and cellular uptake properties.

Hospital acquired infection (HAI) is one of the most frequent adverse event in healthcare worldwide, affecting hundreds of millions of patients annually. In both developed and developing countries, this burden falls mainly on the critically ill. In Vietnam, HAI data are limited, particularly from the intensive care units (ICUs). Robust data are needed to evaluate these problems. This thesis addresses this need. A point prevalence survey (PPS) of HAI and antibiotics use was conducted monthly at 15 ICUs of 14 tertiary and provincial hospitals across Vietnam from October 2012 to September 2013. Subsequently an observational study focused on ventilator associated pneumonia (VAP) and ventilator associated respiratory infections (VARI) at the ICUs of three referral hospitals in Hanoi and Ho Chi Minh City from November 2013 to November 2015. Analysis of 3287 patients in the PPS showed that 29.5% patients had at least one HAI on the surveyed day of which 80% was hospital acquired pneumonia. Antibiotics were being administered to 84.8% patients. <i>A. baumannii, P. aeruginosa</i>, and <i>K. pneumoniae</i> accounted for > 50% of HAI pathogens with carbapenem resistance rates of 89.2%, 55.7%, and 14.9% respectively. The presence of an invasive device was associated with significantly higher risk for HAI. The incidence of VAP and VARI in 374 patients analysed as part of the second study was 9.9% and 24.6% respectively with corresponding incidence densities of 7.6 and 21.4 episodes/1000 ventilation days respectively. Patients with VARI had an excess ICU stay of 11 days, ventilation duration 12.5 days, antibiotic consumption 11 DOT; and ICU cost 2189 US$ compared with those without VARI. Given an estimated 22,570 patients admitted to the ICUs of 14 surveyed hospitals in 2012 and VARI prevalence of 24.6%, we would expect 5552 patients developed VARI leading to an extra 69,403 ventilation days, 61,074 ICU days, 61,074 DOT antibiotic consumption, and an extra ICU cost of 12,153,810 US$. With a total of 40 tertiary and 304 provincial acute care hospitals across Vietnam, the extra cost for VARI nationally would be many times higher. In conclusion, this thesis provides compelling evidence that the burden of HAI in Vietnamese ICUs, particularly VAP/VARI, is substantial. There is also a high level of antibiotic consumption and widespread bacterial resistance to carbapenem antibiotics. Effective infection control measures and antibiotic stewardship programmes are urgently needed to address these problems in Vietnamese ICUs.

The observation that individuals living in malaria endemic regions who are repeatedly infected with <i>P. falciparum</i> can acquire immunity, first to severe, then to uncomplicated clinical episodes of malaria, and finally to high parasite densities, provides hope that a vaccine is achievable. Immunoglobulins have been identified as a key component of naturally acquired immunity and identifying the targets and mechanisms by which this protection is achieved is a clear research priority. To date, only a small proportion of the parasite proteome has been evaluated in the context of naturally acquired immunity. This thesis was aimed at contributing to this knowledge gap by identifying novel potential antigen targets of protective antibodies and validating these in samples from Tanzanian adults. First, to identify merozoite antigens that were immunogenic, I resolved proteins extracted from <i>P. falciparum</i> merozoites by two-dimensional gel electrophoresis and tested these for reactivity with immunoglobulins from malaria immune adults. Immunoreactive proteins were then identified by mass-spectrometry. In complementary studies, purified <i>P. falciparum</i> merozoites were treated with proteolytic enzymes to release proteins localised on the surface of merozoites, which were subsequently identified by mass-spectrometry. Using stringent criteria, where I combined the data obtained from 2D-immunoblots and surface-trypsinization experiments with bioinformatics prediction (for the presence of a signal peptide and/or transmembrane domain), I narrowed down to 222 potential merozoite vaccine targets. These included known surface and/or immunogenic proteins such as the 6-cysteine proteins (Pf12, Pf38, Pf41), MSP-1, 3, 7, 9, 10, GLURP, AMA1, GAMA, MTRAP, LSA3 and RhopH3 as well as many unstudied proteins. From the set of unstudied proteins, I prioritised 27 antigens for immunoprofiling and identified 19 antigens that are targets of naturally acquired antibodies and potential novel vaccine candidates. Next, using a cohort of adults living in a village in Tanzania that experiences hyperendemic malaria transmission throughout the year, I examined antibody responses to the novel potential vaccine candidates to test whether they were correlated with protective immunity. I began by identifying a panel of antigens that were immunogenic and elicited a stable antibody response in adults. Subsequently, I identified six antigens that were individually associated with protection from clinical episodes of malaria. Individuals who became ill during the follow-up period had significantly lower levels of these antibodies compared to those who did not. These antigens were the pantothenate transporter (PfPAT), a putative amino acid transporter (PF3D7_0629500), PF3D7_0830500, PF3D7_1025300, PF3D7_1345100 and PF3D7_1401600. In addition, the breadth of antibody responses to the tested antigens was associated with protection from clinical malaria. Finally, four of these six antigens strongly correlated with protective effector functions. Antibody responses to PfPAT were strongly correlated to both the ability to fix soluble factor C1q (C1q-fixation) to merozoites as well as with their interaction with neutrophils to release reactive species (ADRB). In addition, antibody responses to the putative amino acid transporter, PF3D7_1345100 and PF3D7_1401600 were strongly associated with C1q-fixation ability. Recruitment of the soluble factor C1q onto the surface of merozoite results in lysis via the classical complement pathway. The release of reactive oxygen species by neutrophils is thought to be toxic to the intra-erythrocytic stages of the parasites and is associated with parasite clearance. This thesis shows that 19 antigens, some of which have been studied for the first time in this work, are targets of naturally acquired antibody responses. Six of these antigens appeared to be associated with protective immunity to malaria in adults and correlated strongly with immune effector mechanisms that are thought to be important for parasite clearance. These findings provide a set of antigens that warrant further evaluation for inclusion into the vaccine pre-clinical development pipeline.

Fasciola hepatica is a parasitic trematode capable of infecting a range of vertebrate species including livestock and humans. Both clinical disease (fasciolosis) and sub-clinical infections are of major economic and welfare importance in food producing animals. The life cycle of F. hepatica requires an intermediate host, which in the UK is the mud snail Galba truncatula. High levels of moisture and ambient temperatures between 10-30oC provide optimal conditions for the development of both parasite and snail. As a consequence of changing weather patterns, disease prevalence has increased in recent years, whilst an over-reliance on effective anthelmintics to control disease has resulted in the selection of drug resistance within parasite populations. Studies investigating vaccination as a potential method to control F. hepatica in cattle have identified specific components of the vaccine-induced immune response such as IgG2 antibody titre and avidity, which are associated with protection. Conversely, evidence from experimental infections indicate that F. hepatica modulates the host immune system towards a non-protective type-2 response, extending parasite survival within the host. The overall objective of this thesis was to analyse the immune response in calves and adult cattle naturally exposed to, and infected with, F. hepatica. This was achieved firstly through the validation of a herd level diagnostic test to identify infected beef and dairy herds, and secondly by evaluating the type of immune response present in infected animals. The outputs will be valuable in informing vaccine development, since the type of immune response present in naturally infected cattle, will ultimately have implications for how such vaccines are applied in the field. Chapter 3 describes the validation of a composite sample analysis for fluke egg counts. A total of 138 individual samples from 7 commercial beef herds in mid-Wales were sampled. Fluke egg counts were done on individual samples in addition to composite samples composed of ten 5g samples. These data together with individual counts from a further 22 dairy farms (638 individual samples) were fitted to negative binomial distributions at the farm level. These were stochastically re-sampled to generate a range of predicted composite counts from which confidence intervals and test sensitivity were determined. When referred back to the original counts, all composite counts were within the generated confidence intervals, with the lower confidence interval indicating a 95% test sensitivity at ≥0.4 eggs per gram of faeces compared to individual count data. With the exception of lactating dairy cattle, diagnosis of F. hepatica is limited to individual faecal and/or serum sampling. This analysis represents an important development, since a validated composite worm egg count for cattle provides a simple yet effective test for screening groups of animals for infection. In chapter 4, immune responses in naïve dairy heifers (n=42) naturally exposed to F. hepatica were evaluated. Calves on 3 commercial UK dairy farms were sampled monthly over the course of their first grazing season and analysed to determine fluke infection status and parasite specific immune responses. Where infection was present, this was associated with increases in type-2 associated responses, with increases in interleukin-4 production, interleukin-5 transcription and an eosinophilia. A reduction in the type-1 associated cytokine, interferon-γ was also observed over the course of infection. These findings suggest that a natural challenge with F. hepatica induces a non-proliferative type-2 response. This has implications for vaccine development and application, since current evidence suggests that stimulation of additional components such as IgG2 antibody and strong cell mediated responses are required for protection. Chapter 5 describes a study carried out on a commercial dairy farm, characterising the immune responses in adult dairy cattle (n=27) with chronic infections. The effect of treating infected animals with triclabendazole (12mg/kg) on the immune response was also assessed. Both parasite specific and mitogen stimulated interleukin-4 production were positively associated with F. hepatica antibody titres based on linear regression analysis, whilst no such correlation was found with interferon-γ. This suggests that modulation of the immune response towards a type-2 response is a feature in chronic infections. Additionally, increases in the regulatory cytokines Transforming Growth Factor-β and interleukin-10, associated with infection in pre and post treatment groups respectively may indicate that these cytokines play a role in parasite induced immune modulation, which has been described previously in experimentally infected cattle. Overall, these results show that cattle exposed to and infected with F. hepatica under natural grazing conditions develop a type-2 immune response. This has implications for future vaccination programmes, as the presence of immune modulation arising from natural infection suggests any vaccine induced immune response should be fully developed prior to natural exposure to ensure protection. These results also highlight the importance of the impact of fluke infections on the host’s immune system and the need to investigate and better understand the relationship between F. hepatica and other co-infecting pathogens.

Lymphatic filariasis (LF) is a public health problem in 73 countries and is associated with marked morbidity and disability. It is unique because of its transmission by five main genera of mosquitoes, including Culex, Aedes, Anopheles, Mansonia and Ochlerotatus. In Mali, LF endemicity mapping in 2004 found all eight administrative regions to be endemic for LF. Prior to the National LF Elimination Programme (NPELF), six pilot sentinel villages were selected for baseline research studies to inform the most appropriate strategy for monitoring the impact of the proposed elimination programme based on treatment with ivermectin in combination with albendazole. The following three objectives form the basis of my PhD studies:(i) investigate LF vector population and associated transmission patterns before, during and after the initiation of mass drug administration (MDA) (ii) assess efficacy of new entomological trapping tools for LF post-MDA xenomonitoring and (iii) determine transmission potential in a urban environment in Mali. The overall design is a descriptive study including cross sectional entomological surveys along with longitudinal human surveys to assess the MDA impact. I used standard infection status assessment methods as well as recently developed methods; including the antibody test for Wb123. I conducted these studies in both rural (Sikasso and Kolondieba districts) and urban areas (Bamako, the capital city). My thesis is the first report of the outcome of up to five years post-MDA annual assessment of W. bancrofti transmission using both entomological and parasitological data in an Anopheles transmission area where albendazole plus ivermectin is the recommended drug regimen and Anopheles gambiae s.l the main vector for LF transmission. These features are found mainly in the Western part of Africa. In the pilot sentinel sites in Mali, made of six neighbouring villages, seven MDA rounds with the albendazole plus ivermectin were successful not only at stopping LF transmission (infection rates within 6-7 years old children < 2%) in the short term, but also at sustaining it for up to five years after the last MDA. In contrast, impact assessment in another hyper endemic area (two neighbouring villages treated by the NPELF in the district of Kolondieba) did not demonstrate interruption of transmission after the sixth and seventh MDA rounds. The reasons of these different outcomes of MDA implementation in the different areas are discussed. Of note, the failure in the latter villages was detected using only the ICT card, a method that has been found to overestimate the infection rate in children when compared to the circulating filarial antigen test (Og4C3 ELISA) and the Wb123 antibody test in the pilot sentinel area. In Anopheles transmission areas, it has been observed that focal low-level transmission can exist without being a real threat for re-emergence of transmission, due to lower capacity of the vector to transmit when parasite density is low. Nevertheless, in areas that fail the Transmission Assessment Survey (TAS), adult populations should be checked in addition to the recommended 6-7 year-old age group. Additionally, this thesis showed very promising results for using the Ifakara tent trap type C (ITTC), a human baited trap alternative to the human landing catch. Anopheles yields and infection rates using ITTC were strongly correlated with results using human landing catch (HLC) overall, as well as monthly, in two villages with significantly different Anopheles densities. Finally, it appears that the current version of the TAS needs more tools and additional directions for human infection status determination especially when the baseline endemicity level is high. Further evaluation the ITTC after reducing its bulkiness is required to confirm its usefulness for LF entomological studies in Anopheles transmission areas. From the 6,174 Culex spp and the 16 Anopheles gambiae s.l processed and 1,002 volunteers tested, there was no evidence of LF transmission in the urban environment of Bamako in Mali.

No description available.