Browsing by Author "Evans, Joanna"

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    Detection of mixed Mycobacterium tuberculosis infections in South African TB Patients
    (2009) Stead, Michael Craig; Evans, Joanna; Grewal, Harleen
    Recently, the widely accepted theory that TB disease resulted from one infecting M. tuberculosis strain leading to heightened immune protection against subsequent infections, has been revised. Epidemiological studies and the advances in molecular genotyping techniques have highlighted the rapidly frequent isolation of several different M. tuberculosis strain lineages in single disease episodes, often with differing drug susceptibilities. This has important implications on drug susceptibility testing and the treatment of patients. It has also highlighted the relative contributions of exogenous reinfection and endogenous reactivation in TB disease progression. However, our understanding of the nature and frequency of mixed infections is lacking. This study investigated the frequency and detection of mixed TB infections in the Delft region of the Western Cape, as part of a larger clinical trial on the effects of multi-nutrient supplementation and standard treatment on the TB bacteriological response. Newly diagnosed, adult TB patients (n=154) produced a single weekly sputum sample over an 8-week period. Genomic DNA was extracted from colonies grown from MGIT cultures on LJ slopes. Spoligotyping was used as an initial screen to detect mixed infections as well as to assess the epidemiology of M. tuberculosis in these serial isolates (n=686). In addition, clonal relatedness of the isolates was assessed by MIRU-VNTR analysis. Thereafter PCR assays to detect infections of W-Beijing and non-W-Beijing isolates, as well as to differentiate mixed non-W-Beijing isolates were carried out. Phenotypic and genotypic drug susceptibility was carried out. Spoligotyping indicated that W-Beijing isolates constituted a large proportion (47.8%) of circulating M. tuberculosis in this region, with other strains detected including LAM (17.1%), T (14.7%), X (6.4%), H (7.9%), S (4.3%), and F33 (2.1%) strains. Using both spoligotyping and PCR assays, mixed infections were detected 21 (16.3%) of 129 patients screened. Phenotypic and genotypic DST confirmed that all isolates identified in patients as harbouring mixed strains by spoligotyping were fully susceptible to both RIF and INH. MIRU-VNTR 2 analysis for genetic relatedness identified 1 clonal cluster in the mixed samples identified by spoligotyping, consisting of the T1, T4, W-Beijing and W-Beijing + X3 isolates. The frequency of mixed infections, particularly in high disease burdened areas, is high, and warrants further attention. This finding has great implications with regards to the interpretation of epidemiological and DST data, and the subsequent treatment of patients.
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    Investigation of the Genetic Basis of Antibiotic Resistance in Mycobacterium tuberculosis
    (2010) Evans, Joanna; Segal, Heidi
    The emergence of antibiotic resistant strains of Mycobacterium tuberculosis, coupled with the time it takes to perform phenotypic drug susceptibility testing of this organism, makes the treatment of tuberculosis increasingly difficult. Several genotypic assays for the rapid detection of drug resistance in M. tuberculosis have been developed, but the sensitivity with which these assays identify resistance differs geographically. Additionally, the identification of phenotypically resistant isolates with no identifiable genotypic marker suggests that other factors, such as differential gene expression, may play a role in the development of drug resistance in M. tuberculosis. This investigation aims to both develop and evaluate rapid genotypic assays for the detection of resistance to both first- and second-line drugs in M. tuberculosis, and to investigate the role of alternative sigma factors in the progression to multidrug resistant M. tuberculosis. The sensitivity of the GenoType® MTBDRplus [HAIN Life science] assay for the detection of rifampicin and isoniazid resistance was evaluated in clinical M. tuberculosis isolates with varying phenotypic susceptibility profiles from Cape Town, South Africa. Additionally, the use of multiplex allele-specific-PCR assays for the detection of two commonly identified isoniazid resistance determinants was evaluated in parallel. The GenoType® MTBDRplus [HAIN Lifescience] assay identified rifampicin and isoniazid resistance in clinical M. tuberculosis isolates with sensitivities of 93.5% and 82.1%, respectively, and similar results were obtained using multiplex allele-specific-PCR assays for the detection of isoniazid resistance. Novel multiplex allele-specific-PCR assays for the detection of resistance to ofloxacin and kanamycin/amikacin in M. tuberculosis were designed, and their ability to correctly identify resistance to these second-line drugs was evaluated. Multiplex allele-specific-PCR assays for the detection of GyrA D94G and rrs A1401G correctly identified ofloxacin and kanamycin/amikacin resistance in M. tuberculosis in 64.3% and 80.0% of phenotypically resistant isolates, respectively. Whilst the development of genotypic assays for the rapid detection of drug resistance in M. tuberculosis show promise, variation in the geographical distribution of specific resistance determinants necessitates that phenotypic drug susceptibility testing be performed in parallel. However, screening for GyrA D94G and A90V together with rrs A1401G would identify up to 88.0% of XDR-TB in this region prior to obtaining phenotypic drug susceptibility results, making these assays extremely useful as a rapid genotypic tool for the detection of second-line drug resistance in this setting. The role of alternative sigma factor expression in the progression to drug resistance in M. tuberculosis was investigated in rifampicin mono-resistant M. tuberculosis H37Rv isogenic mutants using real-time quantitative PCR assays. Investigation of rifampicin mono-resistant M. tuberculosis H37Rv isogenic mutants indicated an association between specific RpoB mutations and an enhanced ability to grow in the presence of isoniazid. Furthermore, mutants that were able to grow in the presence of isoniazid displayed upregulation of sigE, which encodes a sigma factor involved in the maintenance of cell wall structure. A role for differential gene expression induced by the use of alternative sigma factors in the development of drug resistance in M. tuberculosis was demonstrated in rifampicin mono-resistant M. tuberculosis H37Rv isogenic mutants, and further confirmed in clinical isolates of M. tuberculosis.
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    Investigation of the genetic basis of antibiotic resistance in Mycobacterium tuberculosis
    (2010) Evans, Joanna; Segal, Heidi
    The emergence of antibiotic resistant strains of Mycobacterium tuberculosis, coupled with the time it takes to perform phenotypic drug susceptibility testing of this organism, makes the treatment of tuberculosis increasingly difficult. Several genotypic assays for the rapid detection of drug resistance in M. tuberculosis have been developed, but the sensitivity with which these assays identify resistance differs geographically. Additionally, the identification of phenotypically resistant isolates with no identifiable genotypic marker suggests that other factors, such as differential gene expression, may play a role in the development of drug resistance in M. tuberculosis. This investigation aims to both develop and evaluate rapid genotypic assays for the detection of resistance to both first- and second-line drugs in M. tuberculosis, and to investigate the role of alternative sigma factors in the progression to multidrug resistant M. tuberculosis.
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    The prevalence of IS6100 and its association with hypermutability in cystic fibrosis isolates of pseudomonas aeruginosa from local hospitals
    (2006) Evans, Joanna; Segal, Heidi
    Includes bibliographical references (leaves 92-115 ).