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South Africa (SA) is burdened with one of the highest tuberculosis (TB) infection rates worldwide. The dual epidemic of HIV and MDR- and extensively drug-resistant (XDR) -TB outbreaks prompted the World Health Organisation to call for a new rapid molecular diagnostics tool(s). To curb the spread of drug-resistant TB, SA introduced genotypic Hain MTBDRplus line probe assays (Hain LPA) for the routine rapid diagnosis of MDR-TB. This study aimed to determine the frequency, geographic distribution and genetic basis for phenotypic and genotypic drug susceptibility testing (DST) discordant findings. The cultures used in this study were isolated during the period June 2007- July 2008 from Western Cape, Gauteng, KwaZulu-Natal, and Northern Cape. A total of 118 comparable MGIT and Hain LPA DST were obtained with 41 isolates verified to be discordant. The predominant families were the LAM, T, X and, S. Hain LPA failed to identify INH resistance (R) in 46.3% (19/41) of isolates with MIC‟s supporting the phenotypic resistance. Genotypic RIF-R was shown in 31.7% (13/41) of isolates which was not expressed phenotypically but interestingly the MIC‟s favour the LPA resistance. Sequencing analysis of the rpoB gene region identified new mutations; Leu458Pro, Leu436Pro, and Asp441Gly, associated with missing wildtypes. For INH, the katG and inhA gene regions were sequenced. Mutations at codon Gly213Val, Pro232Lys, Lys254Asn, and Ser259Asn were detected in katG and a variety found in inhA. These mutations have not been previously reported neither are they incorporated on the Hain LPA strips. Detailed knowledge of the frequency distribution of resistance-linked mutations and associated MICs in different regions of SA, could facilitate understanding of the limitations of current molecular tests and inform testing algorithms. These findings impact on the use of new molecular diagnostics as well as epidemiological monitoring of drug-resistant strains.

Multidrug resistance (MDR) is one of the foremost causes of treatment failure in childhood malignancies. MDR is a multifactorial process, but classic resistance to cytotoxic drugs has most often been associated with over-expression of one or more MDR transporter proteins in malignant cells, conferring on them the ability to extrude an extraordinarily diverse array of endo- and xenobiotics out of the cell. The best characterized multidrug transporters, P-glycoprotein (Pgp) and the Multidrug Resistance-associated Protein (MRP), belong to the ATP-binding cassette (ABC) gene superfamily, and have been previously implicated in the development of drug resistance in the clinical context. The work described herein examined the various aspects of the MDR genotype and phenotype in the childhood malignancies acute lymphoblastic leukaemia (ALL) and neuroblastoma. The first series of studies tested the hypothesis that morphine, a potential Pgp substrate, might influence the efficacy and/or toxicity of clinically used chemotherapy agents which are substrates for Pgp. The results, however, provided no evidence in a variety of human tumour cell lines of morphine influencing response to selected chemotherapeutic drugs. This finding is particularly important as morphine remains to be the opioid of choice for the treatment of cancer pain in the clinic. The second series of studies examined the effect of single nucleotide polymorphisms (SNPs) in the MDR1 gene, encoding Pgp, and in the MRP1 gene, on patient outcome in childhood ALL or neuroblastoma, with a view to identifying novel prognostic markers for these malignancies. It was found that two of the examined SNPs in the MRP1 gene were associated with improved outcome in neuroblastoma, which had not previously been demonstrated in this disease. Moreover, each of the relevant MRP1 SNPs were associated with lower MRP1 gene expression in both patient samples and tumour cell lines, supporting previous studies indicating that low MRP1 expression in neuroblastoma is strongly associated with improved patient outcome. Importantly, the results of this study suggest a role for selected MRP1 polymorphisms in predicting clinical response in neuroblastoma. Finally, a series of studies were undertaken, using both in vitro and in vivo model systems, to test the efficacy of putative small molecule inhibitors of the MRP1 gene and its transcriptional regulator, the MYCN oncogene, in neuroblastoma. These studies demonstrated for the first time the efficacy of a novel compound, 4H10, at reversing multidrug resistance either in cultured neuroblastoma cells, or in the MYCN transgenic mice, which develop neuroblastoma that closely mirror the human disease. The results indicate that inhibition of MRP1 function has potential clinical importance in the treatment of neuroblastoma, and therefore warrant further research in this area. In contrast, the results failed to provide evidence of the in vivo efficacy of the novel putative small molecule MYCN inhibitors analysed in these studies. Collectively, the findings of these studies contribute to a better understanding of the mechanisms of clinical drug resistance, and may help in the development of new approaches for risk assessment and treatment of aggressive childhood malignancies and thereby improve the long-term outlook of children diagnosed with these debilitating diseases.

Multidrug resistance (MDR) is one of the foremost causes of treatment failure in childhood malignancies. MDR is a multifactorial process, but classic resistance to cytotoxic drugs has most often been associated with over-expression of one or more MDR transporter proteins in malignant cells, conferring on them the ability to extrude an extraordinarily diverse array of endo- and xenobiotics out of the cell. The best characterized multidrug transporters, P-glycoprotein (Pgp) and the Multidrug Resistance-associated Protein (MRP), belong to the ATP-binding cassette (ABC) gene superfamily, and have been previously implicated in the development of drug resistance in the clinical context. The work described herein examined the various aspects of the MDR genotype and phenotype in the childhood malignancies acute lymphoblastic leukaemia (ALL) and neuroblastoma. The first series of studies tested the hypothesis that morphine, a potential Pgp substrate, might influence the efficacy and/or toxicity of clinically used chemotherapy agents which are substrates for Pgp. The results, however, provided no evidence in a variety of human tumour cell lines of morphine influencing response to selected chemotherapeutic drugs. This finding is particularly important as morphine remains to be the opioid of choice for the treatment of cancer pain in the clinic. The second series of studies examined the effect of single nucleotide polymorphisms (SNPs) in the MDR1 gene, encoding Pgp, and in the MRP1 gene, on patient outcome in childhood ALL or neuroblastoma, with a view to identifying novel prognostic markers for these malignancies. It was found that two of the examined SNPs in the MRP1 gene were associated with improved outcome in neuroblastoma, which had not previously been demonstrated in this disease. Moreover, each of the relevant MRP1 SNPs were associated with lower MRP1 gene expression in both patient samples and tumour cell lines, supporting previous studies indicating that low MRP1 expression in neuroblastoma is strongly associated with improved patient outcome. Importantly, the results of this study suggest a role for selected MRP1 polymorphisms in predicting clinical response in neuroblastoma. Finally, a series of studies were undertaken, using both in vitro and in vivo model systems, to test the efficacy of putative small molecule inhibitors of the MRP1 gene and its transcriptional regulator, the MYCN oncogene, in neuroblastoma. These studies demonstrated for the first time the efficacy of a novel compound, 4H10, at reversing multidrug resistance either in cultured neuroblastoma cells, or in the MYCN transgenic mice, which develop neuroblastoma that closely mirror the human disease. The results indicate that inhibition of MRP1 function has potential clinical importance in the treatment of neuroblastoma, and therefore warrant further research in this area. In contrast, the results failed to provide evidence of the in vivo efficacy of the novel putative small molecule MYCN inhibitors analysed in these studies. Collectively, the findings of these studies contribute to a better understanding of the mechanisms of clinical drug resistance, and may help in the development of new approaches for risk assessment and treatment of aggressive childhood malignancies and thereby improve the long-term outlook of children diagnosed with these debilitating diseases.

Objective The consistent emerging of multidrug-resistant tuberculosis (MDR-TB) cases are increasingly becoming a major threat and challenge in global TB control, especially in some resource-limited settings like India, China, South Africa. Currently there is no widely acknowledged treatment strategy for MDR-TB. Effectiveness and of current DOTS-Plus strategy is remaining controversial. This systematic review aims to investigate treatment outcomes for MDR-TB under DOTS-Plus and potential factors associated with poor outcome (death, default and failure). Methodology The literatures were searched in Pubmed, Medline, the Cochrane library, Essential Evidence Plus, EMBASE and CNKI. Some manual search articles were also added and 164 literatures in total were founded related to treatment outcomes for multidrug resistant patients under DOTS-Plus. After basically screening and carefully full-text reading, nine studies meeting the inclusion criteria were included. A total of 3358 participants from 8 high MDR-TB countries were investigated. Result Baseline characters were varied across these nine studies, including HIV prevalence (0-1.6%), MDR-TB prevalence (0-4.7%), previous treatment history (without TB treatment, with TB treatment but not under directly observed therapy, short courses (DOTS) and with TB treatment under DOTS), and male/female ratio (54%-86.5%). All studies reported a successful outcome rate (cure and complete) higher than 60 percent, and three of the studies reported higher than 70 percent, which are comparatively high in MDR-TB treatment. Factors associated with poor outcomes that reported by these studies were including alcohol use/ abuse, homelessness, unemployment, imprisonment, BMI, cavitary and bilateral disease, missing doses, and resistant to some second-line drugs. Conclusion In sum, the overall treatment outcomes from these nine studies under DOTS-Plus were acceptable, and most of them were satisfactory. Nevertheless, in consideration of potential bias arising from these cohort analyses, conclusions should be drawn carefully. Several major challenges restrict low- and middle- income countries from implementing DOTS-Plus, which put high command on TB infrastructure, policy commitment, human resources and financial support. Further effort could be put on systematical review and meta-analysis on cost-effectiveness of DOTS-Plus programs. In China, policy makers should pay attention to arrive at national and provincial guidelines of MDR-TB treatment under DOTS-Plus. / published_or_final_version / Public Health / Master / Master of Public Health

Intrinsic antimicrobial resistance of the opportunistic human pathogen Pseudomonas aeruginosa has mainly been attributed to the presence of several chromosomally-encoded multidrug efflux systems. The MexAB-OprM system exports the largest range of structurally unrelated antimicrobial agents and its expression is modulated by multiple regulatory controls. To develop a better understanding of mexAB-oprM overexpression in nalC mutants, which characteristically produce the effector protein PA3719 that binds and disrupts MexR transcriptional repression of mexAB-oprM, the PA3719-MexR interaction domains were investigated. Using a bacterial two-hybrid system, the C-terminus of PA3719 was found to be sufficient to mediate MexR-binding, and the binding region was found to be distinct from the MexR DNA-binding motif. The two-hybrid system was also used in an attempt to understand the role of PA3720, a protein of unknown function that is also overexpressed in nalC mutants. Results from this study confirm that PA3720 does not function to bind and alleviate NalC transcriptional repression of the PA3720-PA3719 operon. This study also attempted to identify the signals involved in overexpressing PA3720-PA3719, in the hopes to elucidate the natural function of MexAB-OprM. Random transposon mutagenesis using a PA3720-PA3719 promoter-lacZ fusion containing P. aeruginosa strain was conducted, but failed to clearly identify any disrupted genes associated with PA3720-PA3719 overexpression. Using the same PA3720-PA3719 promoter-lacZ fusion, expression of these genes was assessed as a function of growth in both wildtype and nalC mutant P. aeruginosa strains. Interestingly, PA3720-PA3719 expression was found to be growth-regulated, with an increased amount of expression occurring in late log/early stationary phase, even in the absence of nalC. This suggests that another regulator(s) is/are involved in modulating PA3720-PA3719 levels in late log/early stationary phase. Since PA3719 ultimately influences mexAB-oprM expression, its involvement in mediating growth-phase mexAB-oprM expression was assessed by examining mexA expression in both wildtype and PA3719 deletion P. aeruginosa strains. PA3719 was found to be involved in some, but not all, of the growth phase control of mexAB-oprM. These results suggest that mexAB-oprM growth-phase regulation is complex, as both MexR-dependent and MexR-independent regulatory pathways seem to exist. Overall, this study has produced a better understanding of mexAB-oprM regulation in nalC mutant P. aeruginosa strains. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2007-09-25 19:00:42.929

Multidrug resistance protein 1 (MRP1) is an integral membrane protein belonging to the ATP-binding cassette (ABC) superfamily that utilizes ATP binding and hydrolysis to transport various endogenous substrates and/or xenobiotics across membranes against a concentration gradient. The overall goal of my research was to examine the nucleotide and protein interactions of MRP1 using various biochemical methods. In the first study, Cu2+(Ph)3 which promotes cross-linking of two nearby Cys residues and limited proteolysis were used to study conformational changes of MRP1 at different stages of ATP binding and hydrolysis at the nucleotide binding domains (NBDs). The limited trypsin digestion patterns indicated that some Cys residues of MRP1 could be cross-linked in the nucleotide-free state and that the Cys cross-linked MRP1 was more susceptible to trypsinolysis. Furthermore, binding of ATP, AMP-PNP, and trapping of ADP by MRP1 prevented the cross-linking events from occurring, but binding of ATPγS did not. However, the ATPγS-bound MRP1, like nucleotide-free MRP1, showed enhanced sensitivity towards trypsinolysis. These studies show that the two ATP analogs, AMP-PNP and ATPγS, interact with MRP1 in different ways. In the second study, the interaction of MRP1 with other cellular proteins was examined. An in vivo chemical cross-linking approach combined with affinity purification and MS analysis was initially used to identify protein partners directly interacting with MRP1. When this approach proved unsuccessful, a second approach involving immunoaffinity purification of MRP1-containing complexes followed by MS analysis was adopted. Six potential candidate interacting protein partners of MRP1 were identified via this approach and two of them, FUS and drebrin, were further characterized by co-immunoprecipitation and colocalization experiments. FUS seems unlikely to be an important binding partner of MRP1 since confocal and subcellular fractionation studies showed it to be exclusively localized in the nucleus. On the other hand, drebrin depletion by siRNA knock-down resulted in a moderate decrease in MRP1 overall expression levels although the membrane localization of MRP1 remained unchanged. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2008-12-08 17:44:52.767