Browsing by Author "Nagel, Simone"
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- ItemDeciphering the mechanisms of Isoniazid reisistance to mycobacterium tuberculosis(Stellenbosch : Stellenbosch University, 2018-12) Nagel, Simone; Streicher, Elizabeth; Klopper, Marisa; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.ENGLISH ABSTRACT: Isoniazid (INH) serves as the backbone of combined anti-tuberculosis therapy. However, the effectivity of this drug has been compromised due to increasing resistance to it in Mycobacterium tuberculosis (M. tuberculosis). This resistance arises through spontaneous mutations in certain genes/genomic regions of M. tuberculosis. The current drug resistance testing algorithm for INH consists of a genotypic MTBDRplus line-probe assay (LPA), which reports resistance to INH based on mutations in katG gene and inhA promoter, and a phenotypic drug susceptibility test (DST), used to confirm INH susceptibility in rifampicin resistant cases on LPA. However, the algorithm is problematic as there are discrepancies between mutations causing INH resistance detected on LPA and phenotypic DST. This may result in incorrect diagnoses and the prescription of incorrect drug treatment regimens. The current study investigates 398 clinical isolates obtained from NHLS Port Elizabeth with discrepant status between the LPA and the phenotypic DST. The isolates were investigated by means of a series of standardized methods such as Sanger sequencing, Whole Genome Sequencing and spoligotyping. This was done to determine if the reasons for the discrepancies between the LPA and phenotypic DST were due to novel mechanisms of resistance to INH. Genotyping via spoligotyping indicated a prevalence of the Beijing genotype in this cohort, as well as mixed infections and novel spoligotype patterns that don’t resemble any known lineage or family markers. Sanger sequencing revealed canonical mutations that were missed by the LPA as well as mutations occurring elsewhere in the katG gene, indicating there are blind spots in the LPA that may have detrimental effects on patient response to treatment. Per the whole genome sequencing results, novel mechanisms were associated with spontaneous non-canonical single nucleotide polymorphisms in M. tuberculosis. These polymorphisms occurred in genetic regions outside the LPA region of hybridisation and conferred varying levels of resistance to INH. The clinical isolates in this dataset consisted of 29.2% low-level resisistant (minimum inhibitory concentration between 0.1-1.0 µg/L to INH) and 61% highlevel resistant (minimum inhibitory concentration of > 1µg/ml to INH) isolates.The degree of resistance to INH is relevant, as it informs prescription of treatment regimens. It is recommended that cases of low-level resistance to INH be treated with the short course Bangladesh regimen consisting of high dose INH, whereas high-level resistance to INH requires the removal of this antibiotic from the regimen.Homologous recombineering was also done to functionally confirm the role these novel mechanisms have on INH resistance, and the subsequent level of resistance caused. The results obtained in this study will ultimately be used to improve current diagnostic methods for detection of INH resistance.