This thesis is concerned with the detection of viruses in blood and blood products, with the general aim of improving the safety of blood donations and factor concentrates for clinical use. The prevention of virus transmission by blood and blood products is currently based on the screening of blood donations for virus specific antibody or antigen and by incorporation of virus inactivation procedures in the manufacturing process. In this study virus detection was by reverse transcription (RT) of virus RNA (where appropriate) followed by amplification of cDNA of virus DNA by the polymerase chain reaction (PCR). The viruses studied were hepatitis C virus (HCV), parvovirus B19 and hepatitis A virus (HAV). For HCV, the relationship between detection of anti-HCV by recombinant immunoblot assay (RIBA-2) and viraemia was examined and a good correlation (84.4%) between a positive result in the RIBA-2 and detection of HCV by PCR was observed. However 5.4% of donations that were RIBA-2 indeterminate were PCR positive, demonstrating that PCR can be useful in the confirmation of ambiguous serological results. An assay system was developed that identifies different HCV genotypes by restriction fragment length polymorphisms (RFLP) in the 5' non-coding region of the genome. In a worldwide study of the distribution of HCV genotypes in blood donors, two new genotypes were discovered. Distinct geographical distributions of HCV genotypes were observed; HCV types 1, 2 and 3 were widely distributed while genotypes 4, 5 and 6 were detected exclusively in donors from one country only.

The objectives of the study were (1) to assess total and specific antibodies in sera and saliva of secretors and non-secretors with reference to carriage of meningococci and smoking: (2) to assess the ability of saliva from secretors and non-secretors to inhibit bacterial binding to epithelial cells; (3) to assess bactericidal activity of sera from secretors and non-secretors. As a preliminary step, a sensitive precise and accurate enzyme linked immunosorbent assay (ELISA) was developed to assess the total and specific levels of IgA, IgM and IgG in serum and saliva. In a parallel trial, antibody levels obtained with the ELISA and SRID assay revealed that the ELISA was significantly more sensitive. Total serum and salivary IgG, IgA and IgM and levels of these isotypes specific for Neisseria lactamica and five isolates of meningococci were determined by ELISA from material obtained from 357 pupils and staff of a secondary school in which an outbreak of meningitis occurred. There were no differences in total or specific levels of serum IgG, IgA or IgM or salivary IgG or IgA of secretors compared with non-secretors. Non-secretors had significantly lower levels of total salivary IgM and lower levels of IgM specific for N.lactamica and five meningococcal isolates. No correlation between levels of serum and salivary IgM suggested that this IgM was produced locally and had not leaked from the serum. Although carriers had higher levels of antibodies than non-carriers, the effect of secretor status on antibody levels was still significant. Smoking had no effect on levels of total antibodies as those for neisseria. A flow cytometry assay was developed to assess the effect of salivary antibodies on the attachment of meningococci to epithelial cells.

Background: Cutaneous Leishmaniasis caused by the Leishmania mexicana complex is associated with unpleasant or disfiguring lesions, for which there only limited treatment options. The life cycle of L. mexicana consists of two stages which involve different immune evasion mechanisms: promastigote and amastigote. Understanding parasitic interactions with host cells and developing a protective vaccine could improve the management and treatment of the disease. Aims: • To construct and compare the immunogenicity of 3 Leishmania genes in 3 different plasmids. • Study the effect of long term in vitro culture on the virulency of L. mexicana and its interaction with host cells. • To analyse the influence of Leishmania infection on MHC class I expression by susceptible human cell lines (U937 macrophages, U937 and MonoMac-6 monocytes), and Toll-like receptor, cytokines and chemokines gene expression profiles. Methodology: Three Leishmania genes (L. mexicana GP63, L. donovani centrin1 and L. donovani centrin3) were cloned into three plasmids (pcRT7/CT-TOPO; VR1012; and pcDNA3.1/Hygro(-)). The immunogenicity of the prepared DNA constructs and their empty counterparts was assessed in Balb/c mice using gene gun immunisation method. An in vivo model of attenuated L. mexicana was produced by growing the parasite in vitro for up to passage 20, and testing the infectivity of these parasites in vivo and in vitro. The influence of infecting target cells with virulent and avirulent L. mexicana at different growth stages on MHC class I expression was determined by flow cytometry. Gene expression profiles were determined by qPCR analysis of extracted mRNA. Results: All tested Leishmania DNA constructs were highly immunogenic compared to the controls, as assessed using ELISPOT and cell proliferation assays. A novel survival assay developed in this study illustrated that macrophages derived from immunised mice were resistant to Leishmania infection. The parasite at passage 1 was highly infectious (virulent), but this progressively decreased to be completely avirulent at passage 20. This was associated with a significant down regulation of virulence-associated genes (GP63, LPG2, CPC, CPB2, CPB2.8, CHT1, LACK and LDCEN3) at passage 20, and was also accompanied by morphological changes. The avirulent parasite was unable to transform to the pathogenic amastigote stage in infected target cells. The gene expression profile of toll-like receptors (TLR-1, TLR-2, TLR-4, and TLR-9), cytokines (IL-1, IL-6, IL-10, IL-12β, TNF-α, and TGF-β), and chemokines (CCL-1, CCL-2, CCL-3, CCL-4, CCL-5, and CCL-22) in target cells was were induced and inhibited according to the virulence status of the parasite. Similar and significant down regulation of MHC class 1 was induced by infection of target cells for 24 hours with both virulent and avirulent L. mexicana parasite, however after 48 hours of infection only cells infected with avirulent but not virulent parasite have significantly restored their MHC class I expression. Conclusions: Since no differences in the immunogenicity of the three plasmids encoding the same Leishmania gene were observed, immunogenicity is not dependent on the plasmid type. The failure of the avirulent L. mexicana parasite to infect Balb/c mice, and its inability to produce the pathogenic amastigote stage in vitro suggests that it might have potential as a vaccine candidate. This was also supported by the up regulation of Th2 mediators following the infection with virulent compared avirulent parasites. The level of MHC class I down regulation was dependent on parasite growth stage, virulency and infection dose.

No description available.

No description available.

Current malaria vector control strategies in Africa target indoor resting and biting mosquitoes and rely heavily on a small number of insecticides. These interventions have lead to the selection of insecticide resistance, behavioural adaptations of the vectors and leave naturally exophilic species nearly untouched. Gravid Anopheles gambiae s.l. female searching for an oviposition site would be a novel target for vector control. However, little is known about the oviposition-site selection behaviour (criteria) of this mosquito. The major aim of the presented research was to investigate if gravid An. gambiae s.l make informed choices when selecting an oviposition site and to identify physical, chemical and biological parameters associated with these choices under standardized experimental and natural field conditions. Standardized field tests and dual-choice oviposition bioassays were used to evaluate responses to soil and rabbit food pellets infusions and habitat water and also to test if bacteria and the volatile chemicals that bacteria produce are relevant to habitat selection. A case–control approach was used to study natural aquatic habitats on Rusinga Island in Lake Victoria during the long rainy season in 2012 to compare the characteristics of habitats colonized (cases) and not colonized (controls) by early instar Anopheles larvae. Factors evaluated included biological characteristics of the sites, zooplankton, invertebrate fauna, physical parameters, nutrients, bacteria communities and volatile chemicals released from the water. Multivariate analyses were used to investigate associations between oviposition site characteristics and habitat selection by Anopheles. The experimental work illustrated that wild and caged An. gambiae s.l. females discriminate between potential aquatic habitats for oviposition and gravid An. gambiae s.l. female select suitable habitats using preferred and avoided chemical cues from water bodies. It furthermore emphasizes that natural infusions can be used to manipulate the oviposition behaviour of An. gambiae s.l. In the field no evidence was found that bacteria from natural habitat water were involved in habitat selection. Although chemical cues were highly diverse analysis suggests that cases and control habitats differ in the headspace volatile profile of the water. High turbidity >200 nephelometric turbidity units (NTU) was the only environmental factor strongly associated with cases. Other risk factors were higher grass coverage (positive association), and the abundance of creeping water bugs of the family Naucoridae and fish (negative associations). This study demonstrates that gravid An. gambiae females choose suitable habitats for oviposition using a complex system of chemical and visual cues from water bodies. Habitats preferred by An. gambiae exhibited distinct and measurable characteristics that can be potentially exploited to attract and kill gravid females to improve malaria vector monitoring and control.

The study of late-stage human African trypanosomiasis (HAT) within mouse models is lengthy and complex, with the removal of brain tissue being required to monitor parasitic burden. This results in the inability to track real-time infections within the central nervous system (CNS). With nearly 70 million people at risk of HAT infection in Africa every year, research into new drug therapies which are capable of crossing the blood brain barrier and efficacious towards the parasite are imperative. However, progress has been slow and difficult, partly due to the limitations with the current drug relapse mouse model for African trypanosomiasis (T. b. brucei GVR35), which requires a follow-up time of 180-days. In this study, we report the generation of highly bioluminescent parasites and their use in an in vivo imaging model of late-stage African trypanosomiasis. Bloodstream forms of the chronic model strain GVR35 were transfected with a “red-shifted” luciferase, which produced detectable signal in CNS at 21-days p.i mimicking that of the wild type line. This model enabled the tracking of a single animal through the entire chronic infection, with the detection of parasites occurring earlier than blood film microscopy. The model was further employed to assess the effects of known anti-trypanosomal drugs on bioluminescence, and to demonstrate how the reduction in bioluminescent signal combined with qPCR can determine a dose-dependent effect after treatment. The non-invasive in vivo imaging model will reduce the time and numbers of mice required to assess preclinical efficacy of new anti-trypanosomal drugs. This study shows the development and optimisation of a new, efficient method to evaluate novel anti-trypanosomal drugs in vivo with the added advantage of reducing the current drug relapse model from 180-days to 90-days.

Surgical site infections (SSI) are a prevalent health care-associated infection (HAl). Prior to the mid-19th century, surgical sites commonly developed postoperative wound complications. It was in the 1860's, after Joseph Lister introduced carbolic acid and the principles of antisepsis that postoperative wound infection significantly decreased. Today, patient preoperative skin preparation with an antiseptic agent prior to surgery is a standard of practice. Povidone-iodine and chlorhexidine gluconate are currently the most commonly used antimicrobial agents used to prep the patient's skin. In this current study, the epidemiology, diagnosis, surveillance and prevention of SSI with chlorhexidine were investigated. The antimicrobial activity of chlorhexidine was assessed. In in-vitro and in-vivo studies the antimicrobial efficacy of 2% (w/v) chlorhexidine gluconate (CHG) in 70% isopropyl alcohol (IPA) and 10% povidoneiodine (PVP-I) in the presence of 0.9% normal saline or blood were examined. The 2% CHG in 70% IPA solutions antimicrobial activity was not diminished in the presence of 0.9% normal saline or blood. In comparison, the traditional patient preoperative skin preparation, 10% PVP-I antimicrobial activity was not diminished in the presence of 0.9% normal saline, but was diminished in the presence of blood. In an in-vivo human volunteer study the potential for reduction of the antimicrobial efficacy of aqueous patient preoperative skin preparations compromised by mechanical removal of wet product from the application site (blot) was assessed. In this evaluation, 2% CHG and 10% povidone-iodine (PVP-I) were blotted from the patient's skin after application to the test site. The blotting, or mechanical removal, of the wet antiseptic from the application site did not produce a significant difference in product efficacy. In a clinical trial to compare 2% CHG in 70% IPA and PVP-! scrub and paint patient preoperative skin preparation for the prevention of SSI, there were 849 patients randomly assigned to the study groups (409 in the chlorhexidine-alcohol and 440 in the povidone-iodine group) in the intention-to-treat analysis. The overall surgical site infection was significantly lower in the 2% CHG in 70% IPA group than in the PVP-I group (9.5% versus 16.1 %, p=0.004; relative risk, 0.59 with 95% confidence interval of 0.41 to 0.85). Preoperative cleansing of the patient's skin with chlorhexidine-alcohol is superior to povidone-iodine in preventing surgical site infection after clean-contaminated surgery.

Healthcare associated infections may arise from many sources, including patient?s own skin flora and the clinical environment, and inflict a significant burden within the health service. Adequate and effective skin antisepsis and surface disinfection are therefore essential factors in infection control. Current EPIC guidelines recommend 2 % chlorhexidine (CHG) in 70 % isopropyl alcohol (IPA) for skin antisepsis however poor penetration has been reported. Eucalyptus oil (EO) is a known permeation enhancer, producing synergistic antimicrobial activity when combined with CHG. In this current study, the antimicrobial efficacy of EO and its main constituent 1,8-cineole were assessed against a panel of clinically relevant microorganisms, alone and in combination with CHG. The superior antimicrobial efficacy of EO compared with 1,8-cineole, and synergistic effects with CHG against planktonic and biofilm cultures, confirmed its suitability for use in subsequent studies within this thesis. Impregnation of EO, CHG and IPA onto prototype hard surface disinfectant wipes demonstrated significantly improved efficacy compared with CHG/IPA wipes, with clear reductions in the time required to eliminate biofilms. Optimisation of the EO/CHG/IPA formulation resulted in the development of Euclean® wipes, with simulated-use and time kill studies confirming their ability to remove microbial surface contamination, prevent cross contamination and eliminate biofilms within 10 minutes. The employment of isothermal calorimetry provided additional information on the type and rate of antimicrobial activity possessed by Euclean® wipes. A clinical audit of the Euclean® wipes at Birmingham Children?s Hospital, Birmingham, U.K. revealed divided staff opinion, with the highest cited advantage and disadvantage concerning the odour. Finally, skin penetration and cell toxicity studies of EO/CHG biopatches and Euclean® solution developed during this study, revealed no permeation into human skin following biopatch application, and no significant toxicity. These current studies enhance the knowledge regarding EO and its potential applications.

Sepsis is an overwhelming inflammatory response to infection that can progress to septic shock, characterised by refractory hypotension and insufficient organ perfusion. It is predominantly associated with nosocomial infections, and tends to occur in extremes of age, where it is associated with very high mortality rates. Sepsis and sepsis-associated multi-organ failure represent a tremendous healthcare burden and a significant cause of morbidity and mortality worldwide. Incidence has continued to increase over several decades, and septic shock is now the leading cause of death in intensive care units, claiming an estimated 20,000 lives per day worldwide. Despite decades of endeavour, research has not produced any specific pharmacological treatments shown definitively to improve survival in septic patients. Cardiovascular collapse plays a key role in mortality, and correspondingly, pre-clinical and clinical assessment of the impact of interventions on disease progression has been based upon systemic haemodynamics and achieving target blood pressures. In recent years, however, it has become increasingly clear that systemic stabilisation does not necessarily prevent the onset of organ failure, and may actually be achieved at the expense of visceral perfusion. Sepsis is now increasingly understood to be a ‘disease of the microcirculation’, though pre-clinical models rarely assess this parameter, perhaps accounting for the lack of research translatability. There is thus a critical need to develop more clinically relevant pre-clinical models, which focus on clinically prognostic endpoints, in order to identify novel drug targets for the treatment of sepsis. This thesis will describe the development of a novel approach to monitoring cardiovascular dysfunction in two widely used pre-clinical models of sepsis: lipolysaccharide (LPS)-induced endotoxaemia, and cecal ligation and puncture (CLP). This approach comprises a multi-parameter monitoring system in which all levels of the cardiovascular system – global haemodynamics, cardiac function, microcirculatory blood flow and haematological markers – can be evaluated in a single animal. Using a novel application of laser speckle contrast imaging technology, in combination with gold standard haemodynamic monitoring techniques, we demonstrate that mesenteric blood flow is severely and time-dependently decreased following the induction of sepsis, despite stabilisation of arterial pressure, heart rate and cardiac output. Decreased mesenteric perfusion is shown to correlate with the development of metabolic acidosis and organ dysfunction, suggesting that global haemodynamic indices are insufficiently predictive of syndrome progression. Mesenteric blood flow, on the other hand, appears to be a sensitive and clinically relevant prognostic marker of disease severity, correlating with haematological markers of organ dysfunction, and sensitive to standard clinical interventions known to improve survival in patients. This thesis will go on to describe a mechanistic investigation of sepsis-induced microcirculatory flow impairment, using vasodilatory and anti-coagulant agents. Finally, it will describe the use of this model, in addition to in vitro models, for assessing the potential role of two ion channels – transient receptor potential vanilloid (TRPV) 1 and 4 – in modulating vascular dysfunction during sepsis.