This thesis considers the application of PCR to the detection and characterisation of human immunodeficiency virus (HIV) nucleic acid and hepatitis B virus (HBV) DNA in clinical samples. Primers were selected from the <I>po1 </I>and <I>env </I>sequences for the detection of HIV-1 DNA, and amplifications of the envelope gene were used to demonstrate sequence variability. HIV-1 DNA was detected in all 32 patients (59 of the 61 PBMC samples tested). Viral DNA sequences were detected in nuclear extracts tested, 71% of cytoplasmic extracts, and in 50% of purified monocytic cells. Primers were selected to differentiate between HIV-1 DNA structural forms. Covalently closed circular unintegrated viral DNA (CUVD) with 1 or 2 LTR sequences was detected by amplifying across the junction site, and linear unintegrated viral DNA (LUVD) was detected using a ligation mediated PCR (L-PCR). CUVD was detected in 65% of patient samples and was associated with disease progression (0.02>P>0.01). The detection of LUVD was negative for all patient samples. HBV DNA was detected in patient serum samples using 4 specific primers in a nested PCR. The primers were selected to hybridize to the S-gene sequence of HBV using the nucleotide consensus sequence for 5 HBsAg subtypes (<I>adrcg, adw, adyw, ayw,</I> and <I>awy<SUB>2</SUB></I>). The presence of HBeAg in serum is indicative of infectivity. The detection of HBV DNA by PCR was compared with HBe status in 115 patient samples. All HBeAg positive and 25% of HBeAg negative samples were positive for HBV DNA. To determine the source of the viral DNA in these samples, the DNA extraction method was applied to free virus, and IgG and IgM complexed virus to establish the association of viral DNA with free virus and HBeAg.

The foundation of this project was the development of an indirect serotyping ELISA for the identification of HCV genotypes 1-3. This thesis is concerned with the further development of the serotyping ELISA. Initially the assay was extended by the incorporation of antigen for recently discovered genotypes 4, 5 and 6. This version of the assay was able to detect type-specific antibody in 87% sera from blood donors and patients with chronic HCV infection from various geographical areas. The specificity of this assay was >97% when compared to genotyping by a PCR method. This serotyping ELISA was applied to a number of different population studies, including the U.S.A., Norway, Pakistan, Egypt and Hong Kong, and in doing so has contributed to current knowledge of genotype distributions worldwide. In an attempt to improve the sensitivity of the assay, peptides corresponding to an additional immunogenic region in NS4 were included and assessed for their contribution to the sensitivity and specificity of the assay. The performance of the serotyping assay was also compared to other PCR-based methods of genotyping. An investigation into samples producing discrepant results in two distinct cohorts was performed by sequence analysis of the 5' NCR, core and NS4 regions. The frequency of discrepant results was higher when testing samples from haemophiliacs than from patients with chronic HCV who had no history of multiple exposure to the virus. Sequence analysis revealed that mis-typing by the ELISA may have resulted from amino acid changes within NS4 from the non-haemophiliac study group, whereas little antigenic variation was identified in discrepant haemophiliac samples. Discrepant results in this multiply-exposed cohort might be explained by the detection of antibody to genotypes from previous or multiple infections. This work has enabled the specific identification of HCV serotypes causing infections worldwide by the development of a highly specific serotyping ELISA. Evidence concerning the possible biological differences between HCV genotypes suggests that serotyping may become an important method in clinical practice for the selection of patients for antivirus treatment.

There is increasing evidence to suggest that the normal non-pathogenic commensal flora, of the healthy individual, may act as a reservoir of antibiotic resistant determinants. In order to investigate this potential gene pool further, three commensal faecal flora surveys examining three separate populations have been completed. Firstly, 100 faecal specimens, which had been submitted to clinical diagnostic laboratories from general practitioners in Edinburgh and found not to contain any pathogens, were examined for the presence of antibiotic resistance amongst the lactose-fermenting, aerobic faecal bacteria. A further 100 specimens were obtained from healthy members of the community in Edinburgh and were investigated as before. In developing countries, the carriage of antibiotic resistance amongst pathogens has been reported as being significantly higher than in the developed countries. Consequently, to make such a comparison about non pathogenic commensals, the third survey was carried out in the black communities of South Africa; 361 faecal specimens were obtained and examined for the presence of antibiotic resistant faecal flora. The ampicillin resistant bacteria, from each survey, were purified and investigated further. Conjugation experiments identified the transferability of the resistance determinants. In those strains able to transfer the antibiotic resistance genes, plasmid preparations and restriction profiles were prepared. From such work, the presence of single or epidemic plasmids within the given community could be identified. The causative B-lactamase was determined by analytic isoelectric focusing. The epidemiology of the B-lactamases mediating resistance within this population was thus determined. A follow up survey was carried out in Edinburgh approximately two years after the initial survey. Faecal specimens were obtained from five people who had participated in the 'healthy' commensal flora survey. Previously their specimens were found to harbour transferable ampicillin resistant determinants; the responsible plasmids were isolated and identified. Whether or not ampicillin resistant bacteria had been maintained was assessed prior to investigation of the resistance mechanism employed; persistence of a single plasmid type was sought.

The time of spread of HIV-1 to non-lymphoid tissue was investigated by sequence comparisons of variants infecting a range of tissues from three individuals with AIDS in the p 17<SUB>gag</SUB> gene, and flanking regions of V1/V2. Phylogenetic analysis revealed several lineages in each individual that contained sequences from lymphoid and nonlymphoid tissue, such as brain, in both regions. This observation contrasts strongly with the previously described organ-specific sequences in the V3 region in this study population and other investigations. By estimating mean synonymous pairwise distances in the p 17<SUB>gag</SUB> region, we were able to calculate the time of divergence of variants infecting lymphoid and non-lymphoid tissues, such as brain. In lymphoid tissue the mean diversity of <I>gag</I> sequences implied an approximate population age of 2.65 to 5.6 years, while those infecting brain were significantly more variable, suggesting an even earlier time of diversification (4.1 to 6.2 years). In two of the three individuals, these times of divergence indicate that infection of the brain may have occurred as an early event in the progression to disease, preceding the onset of AIDS by several years. This is the first report where it has been possible to estimate times of diversification in different tissues in vivo and is of importance in understanding the dynamics of the spread of HIV-1 into non-lymphoid tissue, and its possible adaptation for replication in different cell types. In summary, both analysis of the P17<SUB>gag</SUB> and V1/V2 regions revealed high levels of heterogeneity of HIV-1 in tissue such as brain producing multiple lineages upon phylogenetic analysis. We have therefore found no evidence for specifically neurotropic variants of HIV-1 and question the idea that spread into the central nervous system or other non-lymphoid tissues requires specific adaptation.

A survey of antibiotic resistance on Gram negative aerobic bacilli, which had been isolated from blood cultures during the period 1980-1991 from Edinburgh Royal Infirmary, was performed. All viable cultures were investigated for susceptibility to an extensive range of antibiotics including the newer carbapenems imipenem and meropenem. One hundred and sixty seven Gram negative, oxidase negative, aerobic bacilli, that were found to be resistant to cefuroxime (≥8.0μg/ml), were investigated further. The predominant species present were: enterobacter, serratia and acinetobacter. Isoelectric focusing (IEF) and conjugation studies were performed. None of the strains that transferred ampicillin resistance to <I>Escherichia coli</I> J62<SUB>-2</SUB> co-transferred resistance to any of the later generation cephalosporins or to imipenem. It was apparent that inducible chromosomal β-lactamases were the main cause of cefuroxime resistance in these strains. Amongst the cefuroxime resistant strains an isolate of <I>Acinetobacter baumannii</I> was found to be resistant to all β-lactams including imipenem and meropenem. Isoelectric focusing revealed the presence of a chromosomal cephalosporinase and a novel β-lactamase of pI 6.65. Despite the fact that the original clinical isolate could be cured of its resistance to carbapenems and penicillins by growing in the presence of ethidium bromide with the concurrent loss of the enzyme of pI 6.65, no resistance plasmid was transferred. Plasmids were isolated but the physical loss of a plasmid in the cured strain could not be demonstrated. The enzyme hydrolysed penicillin, ampicillin and cephaloridine slowly during enzyme assay but inactivation of carbapenems could only be demonstrated by microbiological means. The enzyme was thus designated ARI 1 (Acinetobacter resistant to imipenem). The molecule size of the ARI 1 enzyme was 23 kDa. It was inhibited by BRL 42715 indicating that it was a serine active site β-lactamase. It was not inhibited by EDTA.

No description available.

Of the plethora of parasitic diseases that afflict mankind, malaria remains the most significant with 100-300 million cases reported annually and 600,000 fatalities. Treatment and control measures have been hampered by the emergence of drug resistance to most antimalarial therapies. Early signs of drug resistance to the current frontline option, the artemisinins, make it imperative that novel drug candidates are discovered. One possible short-term solution is drug repositioning, via screening existing FDA-approved (Food and Drug Administration agency) drug libraries for antimalarial activity. Towards this goal, two, fast, simple, and reliable in vitro SYBR Green-based drug susceptibility assays were optimised. The first, the SYBR Green microplate method offered a medium throughput option that was used to screen two FDA-approved libraries (Z score = 0.68 +0.06), LOPAC and ENZO (~700 compounds). Approximately 60 hits, defined as > 50 % inhibition at 2.5 µM, were identified by the preliminary screen. The SYBR Green flow cytometer method, capable of providing direct parasitaemia estimates and stage-specific information, was used for second-phase characterisation of the hits. From these, antiamoebic compound emetine dihydrochloride hydrate was identified as a potent inhibitor of the multidrug resistant Plasmodium falciparum, strain K1, with an IC50 of 47 nM (95 % confidence interval 44.92-49.17). Further characterisation of the compound involved analysis of the parasite killing profile, to determine the parasite reduction ratio (PRR) and parasite clearance time (PCT) as well drug interaction analysis with existing antimalarials. Emetine was shown to have a similar killing profile to atovaquone inferring a similar mitochondrial mode of action, corroborated by fluorescence staining with the JC-1 mitochondrial probe. Taken together, emetine’s pharmacokinetic matching and synergy with atovaquone provide an exciting drug combination for further investigation. The relatively high hit rate presented in the study, and in vitro workflow outlined for emetine, also showed drug repositioning to be a promising option for antimalarial drug discovery.

Cystic Echinococcosis is a zoonotic infection of humans caused by the metacestode(larval) stages of the cestode Echinococcus granulosus (family Taeniidae). Diagnosis of the infection often involves immunodiagnostic approaches using cyst fluid antigens and these have also been used in serological follow up of patients after surgical treatment or chemotherapy. However the usefulness of other metacestode antigenic extracts for these purposes has not been fully investigated. The laminated layer is a polysaccharide/protein complex that surrounds the outside of the hydatid cyst and is a structure unique to the genus Echinococcus. In the current study a crude extract of this layer was prepared by sonication and tested for reactivity against sera from hydatid patients from Turkana, Kenya. This extract reacted both in ELISA and in Immunoblotting, primarily recognising antigenic bands around 55 kDa and 12 - 36 kDa. This latter region appeared to be more specific in terms of total IgG and IgG1 and IgG 4 subclass responses. The glycoproteins in this region also bound particular lectins such as soyabean Aglutinin and a lectin affinity purification column was produced to try and isolate the more specific glycoproteins. However this part of the project was not successful and no purified fraction was produced. The crude laminated layer extract was then compared with hydatid fluid to look for differences in antibody profile in treated and untreated Turkana patients over time in an attempt to identify possible markers of disease progression/regression. Sera were obtained from 10 albendazole treated patients over time courses ranging from 9 months to several years. Similar samples were also obtained from 4 patients who had refused treatment. Samples were analysed by ELISA against against Hydatid cyst fluid(HCF) and Laminated layer(LL). Results of the time courses showed that antibody levels fluctuated in both treated and untreated patients and that some of these changes were associated with changes in cyst morphology. In some cases the laminated layer showed similar recognition patterns to HCF but in others there were peaks of activity against one antigen which was not evident against the other.

Phosphodiesterases are key enzymes in cyclic-nucleotide signalling pathways, regulating the levels of cAMP and cGMP in the cell. Cyclic nucleotides play an important role in regulating progression of the complex parasite life cycle. There are four Plasmodium PDEs, PDEα-δ. PDEβ has proved refractory to deletion and is predicted to be essential in asexual blood stages. PDEs α, γ and δ have been successfully disrupted in previous studies, which revealed stage-specific roles for PDEγ and PDEδ in the mosquito. PDEβ is a promising drug target due to the proven ability to create specific inhibitors for individual human PDEs (e.g. Viagra inhibits human PDE5), the established safety record of current inhibitors and the predicted essential nature of PDEβ in P. falciparum blood stages. The lack of useful reagents; knockout strains, tagged lines and recombinant proteins, has severely limited progress in this field. Conditional genetic disruption (required to study essential genes) is notoriously difficult in P. falciparum. This project has attempted to generate a conditional knock-out using the destabilisation domain system and the novel DiCre system, which involves recombinase-mediated genomic excision of the target DNA upon introduction of a drug. A PDEβ haemagglutanin (HA) tagged line has been successfully generated and used to investigate the cellular biology of PDEβ, including, the subcellular localisation and cyclic nucleotide specificity of PDEβ, which until now has remained speculative. A small library of PDE inhibitors generated by Pfizer has been evaluated using a parasite growth inhibition assay and a PDE assay, with compounds active at sub-micromolar concentrations against the parasite and the protein. These assays have used wild type parasites and also a PDEα KO line which has no obvious phenotype in blood stage parasites.

The genus Alphavirus is one of two within the family Togaviridae and comprises approximately 30 species, including several pathogens of humans and other animals that are of medical, veterinary and economic importance. Most alphaviruses are maintained in nature by a biological transmission cycle between susceptible vertebrate hosts and blood-feeding arthropods such as mosquitoes. In common with many Old World alphavirus pathogens Chikungunya virus (CHIKV) is the aetiological agent of a syndrome characterized by fever, skin rash and acute or chronic poly-arthralgia or arthritis. Whereas previously CHIKV outbreaks were sporadic and self-limiting, in 2004 it re-emerged in coastal Kenya and there followed a series of outbreaks that have continued until the present day, resulting in many millions of cases. Inroads into understanding the pathogenesis of CHIKV disease were until recently hampered through the lack of a convenient small animal model capable of exhibiting symptoms similar to those observed in humans in response to viral challenge. By contrast, studies with other alphaviruses, most notably Sindbis and Semliki Forest viruses (SINV and SFV), have provided insights into many aspects of the infectious process including several key determinants of virulence. In the present study a reverse genetics system was developed to investigate the pathogenesis of CHIKV disease, the virus replication cycle and its interactions with susceptible hosts. This tool was used to investigate the hypothesis that a CHIKV encoded virulence factor is located within the cleavage domain of the non-structural polyprotein precursor of the viral replicase. Results from this study indicate that a unique amino acid within this conserved site is instrumental in contributing the inhibition of the type 1 IFN response in infected hosts. It was also shown that the type 1 IFN response was induced at an earlier stage in mice challenged with virus containing an engineered mutation at this site and that joint-swelling was less severe than with wild-type virus.