Clinical experience suggests that adults with dengue manifest a pattern of complications different from those observed in children, but direct comparisons among populations experiencing the same exposure have rarely been published. I conducted a large prospective descriptive study of dengue across all age-groups presenting to a single institution in an endemic country during a defined time-period. Vascular leakage was more severe in the paediatric patients and DSS developed much more frequently in this age-group. In contrast haemorrhagic manifestations and severe organ involvement were more common in adults. Similar to the established findings in children, typical coagulation abnormalities were apparent in the adults i.e. prolonged APTT with reduced fibrinogen levels but without evidence of true disseminated intravascular coagulation. However thrombocytopenia was significantly worse among the adults throughout the evolution of the disease, even after adjusting for the higher rate of secondary infections in this group, and platelet counts after recovery remained lower than in the children. Clinically severe liver involvement was seen only in adults and was infrequent but usually resulted in severe bleeding. Chronic hepatitis B co-infection was associated with modestly but significantly increased levels of alanine aminotransferase, but did not otherwise impact the clinical picture. To investigate the mechanisms underlying the increase in APTT I carried out APTT Mixing Studies confirming that deficiency of coagulation factors is a major contributory factor. Since there is little evidence for procoagulant activation the most likely mechanism for this would be leakage of coagulation proteins, many of which are of a similar size to albumin. An additional explanation for the increased APTT could be the presence of a circulating anticoagulant I found very high levels of heparan sulfate (HS) in the dengue plasma, but was not able to show that the HS exerts an anticoagulant effect. I also used FACS analysis to demonstrate that circulating endothelial cells (CECs) are increased during dengue infections and that percentage CECs correlate with the severity of the coagulopathy and with bleeding. Parallel increases in both CECs and HS levels support the theory that disruption of the endothelial cell/glycocalyx complex occurs during dengue infections i.e. CECs appear to be shed from the endothelial layer while HS may be shed from the surface glycocalyx. These disruptions likely affect the function of the complex and could contribute to the pathogenesis of the systemic vascular leak syndrome. Acknowledgements The completion of this dissertation reminds me to acknowledge and remember all that I owe to the wonderful people who have helped and contributed in so many ways. I cannot thank them enough. Among them, first I would like to express the deepest gratitude to Dr Bridget Wills, my Director of Studies, for her kind encouragement, invaluable advice and help throughout the process. Without her guidance, my doctoral work would never have been completed. I am also sincerely grateful to my supervisors, Professor Cameron Simmons, who has given me valuable advice and support and Professor Edward Tuddenham, whose advice has been very useful for my research. My grateful thanks also go to Professor Jeremy Farrar, who has provided me with the opportunity to study in a dynamic and professional research environment. In addition, my appreciation goes to the following people who have helped me with the performance of this research:Dr Marcel Wolbers gave me helpful guidance on statistical issues. Ms Huynh Thi Le Duyen, Mr Huynh Le Anh Huy and Mr Nguyen Thanh Vinh performed the dengue diagnostics, including the capture IgM and IgG ELISA assays and DENV RT-PCR. Ms Huynh Thi Le Duyen, Ms Nguyen Thi Hanh Tien and Ms Nguyen Than Ha Quyen were also very kind in assisting me to carry out the heparan sulfate measurements, heparinase experiments and CEC enumeration. Ms Nguyen Tan Thanh Kieu, Ms Pham Thi Doan and all of the dengue group staff at OUCRU assisted and encouraged me in my work. Their kindness will not be forgotten. The research herein would not have been possible without the participation of the patients who consented to be involved in my studies, and to them, I am very grateful. My gratitude also extends to:The Director Board, all of the physicians and other staff of the clinical departments of the HTD who assisted me in doing this PhD. The staff in the Haematology and Biochemistry departments of the HTD, and Dr Le Huu Tai, who have all helped me with the basic laboratory tests and hepatitis diagnostics. I sincerely appreciate the generous financial support of the Wellcome Trust of Great Britain and Oxford University Clinical Research Unit. I would also thank:Dr Andrew Blann who kindly provided me with the Standard Operating Procedure for CEC enumeration. Professor Nguyen Tran Chinh and my colleagues at the Infectious Disease Department, University of Medicine and Pharmacy (HCMC) for their support for my PhD. Professor Tran Tinh Hien for his useful advice for my research. Finally, I would like to express my love for and special gratitude to my parents, who have made endless sacrifices for their children and had confident expectations of me. Also, thanks to my wife for her endurance and patience during my work on this PhD, and to my daughter who provided hugs every time I came home.
Monitoring antimalarial drug resistance is a useful epidemiologic tool and provides early detection of resistance foci. Using DNA extracted from the head/thorax of wild mosquitoes collected from Bagamoyo Coastal Tanzania, samples infected by Plasmodium falciparum (N = 89, in 2002 and N = 249 in 2004) were screened by nested polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay for mutations at Pfcrt76 and Pfmdr1-86 associated with chloroquine (CQ) resistance. The majority of isolates were of single infection (71%), and the prevalence of mutant alleles of Pfcrt76 decreased from 64.5% in 2002 to 16% in 2004; likewise, mutant Pfmdr1-86 alleles decreased from 46.6% to 2.7%. Overall, there was a decline of mutant isolates by a factor of 17 and 4 for Pfmdr1 and Pfcrt, respectively. In contrast, isolates with wild-type alleles increased significantly from < 20% in 2002 to 67.6% for Pfcrt76 and 83.5% for Pfmdr1-86 in 2004. This observation suggest a biologic trend of decrease of CQ mutants and a subsequent increase of CQ susceptible parasites in circulation after the discontinued use of CQ in 2001 as a first-line drug in Tanzania. High prevalence of susceptible P. falciparum found in circulation not only supports other reports of a decline of mutant parasites after a reduction of drug selection pressure but suggests that the fitness cost is high in mutant parasites. Typing parasite isolates from infected mosquitoes, an alternative means of data collection, has the potential to increase the spatial and temporal coverage, and this approach is practical in highly endemic regions of Africa.
Forest malaria is a complex but common phenomenon occurring in southeast Asia. We studied its epidemiology through a prospective community-based study in central Vietnam. A total of 585 individuals were followed for two years by active case detection and biannual cross-sectional surveys. The prevalence of antibodies to Plasmodium falciparum was constantly about 20% across surveys and the incidence rate of clinical episodes of P. falciparum malaria was 0.11/person-year. Multivariate analysis showed that regular forest activity was the main risk factor for clinical malaria and malaria infections. Untreated bed nets had a significant protective effect (60%), except for people regularly sleeping in the forest. The population-attributable fraction for regular forest activity was estimated to be 53%. Our results confirm the major role played by forest activity on the malaria burden in this area and provide the basis for targeting control activities to forest workers. New interventions based on insecticide-treated materials need to be urgently evaluated.
Vaccination of mice with a recombinant protein, Ov-ASP-1, the Onchocerca volvulus homologue of the activation associated secreted gene family stimulated very high titres of both IgG1 and IgG2a without adjuvant. rOv-ASP-1 was also immuno-reactive with IgG isotypes from both O. volvulus-infected (INF) and putatively immune (PI) humans, with higher IgG4 in the former group. The protein also stimulated IFN-gamma secretion by PBMC from INF and PI and IL-5 only in INF. Using a mouse diffusion chamber model, vaccination with rOv-ASP-1 resulted in partial but significant protection against challenge with infective third-stage larvae (L3) but only when formulated with Freund's complete adjuvant (FCA) or alum. Protection was Th1-dependent (highly elevated IgG2a) with FCA and contingent on a strongly Th2-skewed (IgG1) response with alum. IgE responses to rOv-ASP-1 with or without adjuvant were weak or absent. When immunization using rOv-ASP-1 in adjuvant failed to induce adequate Th1 (FCA) or Th2 (alum) responses, protection efficacy was compromised. The recombinant protein appears to stimulate a mixed Th1/Th2 response but the outcome in terms of protective immunity is the result of a subtle interplay of its intrinsic and adjuvant-augmented properties. Ov-ASP-1 is potentially secreted based on its localization in the secretory granules of L3.
Sterile protective immunity against challenge with Plasmodium spp. sporozoites can be induced in multiple model systems and humans by immunization with radiation-attenuated Plasmodium spp. sporozoites. The infected hepatocyte has been established as the primary target of this protection, but the underlying mechanisms have not been completely defined. Abs, CD8+ T cells, CD4+ T cells, cytokines (including IFN-γ and IL-12), and NO have all been implicated as critical effectors. Here, we have investigated the mechanisms of protective immunity induced by immunization with different vaccine delivery systems (irradiated sporozoites, plasmid DNA, synthetic peptide/adjuvant, and multiple Ag peptide) in genetically distinct inbred strains, genetically modified mice, and outbred mice. We establish that there is a marked diversity of T cell-dependent immune responses that mediate sterile protective immunity against liver-stage malaria. Furthermore, we demonstrate that distinct mechanisms of protection are induced in different strains of inbred mice by a single method of immunization, and in the same strain by different methods of immunization. These data underscore the complexity of the murine host response to a parasitic infection and suggest that an outbred human population may behave similarly. Data nevertheless suggest that a pre-erythrocytic-stage vaccine should be designed to induce CD8+ T cell- and IFN-γ-mediated immune responses and that IFN-γ responses may represent an in vitro correlate of pre-erythrocytic-stage protective immunity.
