Masters Degrees (African Centre for HIV/AIDS Management)

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Browsing Masters Degrees (African Centre for HIV/AIDS Management) by browse.metadata.advisor "Grobbelaar, Melanie"

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    Transmission of bedaquiline-resistant tuberculosis in the Western Cape Province, South Africa
    (Stellenbosch : Stellenbosch University, 2022-12) Steinhobel, Amy Debra; Grobbelaar, Melanie; Stellenbosch University. Faculty of Economic and Management Sciences. African Centre for HIV/AIDS Management.
    ENGLISH SUMMARY: Bedaquiline (BDQ) is the first novel drug to be approved for the treatment of multi-drug resistant TB (MDR-TB) in 40 years. Reduced susceptibility to BDQ commonly occurs through variants in the genes Rv0678, atpE and pepQ. The aim of this study was to determine the prevalence of BDQ genotypic resistance in the Western Cape, using a high throughput screening method to investigate the acquisition of variants in the three candidate genes and associated phenotypic resistance, as well as potential transmission network of BDQ-resistance. A total of 326 patient isolates with rifampicin-resistant TB (RR-TB) at baseline were selected between January 2018 to February 2019 using the STATA software for sample processing from the longitudinal drug-resistant strain bank at the Division of Molecular Biology and Human Genetics at Stellenbosch University (SU). Crude DNA was used for library preparation and targeted deep sequencing (TDS) of the BDQ-resistance candidate genes (Rv0678, atpE and pepQ) using a protocol designed by the translational genomics research institute (TGen). The first TDS run was performed at TGen in Flagstaff, USA on an Illumina MiSeq platform and 146 patient isolates were sequenced. The expertise was then transferred to SU and the protocol was optimised. The second TDS run was done at the South African Medical Research Council (SAMRC) on an Illumina MiniSeq platform and 180 patient isolates were sequenced. Data obtained from collective TDS runs were analysed using the TB-specific Amplicon Sequencing Analysis Pipeline and yielded 15 isolates with variants suitable for further phenotypic drug susceptibility testing (pDST) using the BACTEC MGIT 960 system. Of the variants detected; six were Rv0678 variants, eight were pepQ variants and one was an atpE variant. Two of the isolates with Rv0678 indels at high frequencies (>97%) were BDQ-resistant, suggesting a phenotypic BDQ-resistance frequency of 0.61% (2/362). Spoligotyping revealed that 9/13 isolates belonged to lineage 2 (Beijing genotype) and three isolates belonged to lineage 4 (one T1 strain, one LAM3 strain and one LAM1-LAM4 strain). A subset of six isolates, from the first TDS run, was selected for minimum inhibitory concentration (MIC) testing using the CRyPTIC UKMYC6 plates and for whole-genome sequencing (WGS) on the MiniSeq platform. The UKMYC6 plate data and the WGS data for the subset identified the drug-resistant profiles, which ranged from rifampicin mono-resistant to extensively drug-resistant TB. The two isolates presented with identical sub-lineages (2.2.2) and the phylogeny indicated the close relatedness of the patient isolates. The findings from this study suggest that it is possible to use NGS to screen hundreds of patient isolates to identify BDQ resistance-associated variants (RAVs) in the community. However, this approach is dependent on the availability of a comprehensive catalogue of BDQ RAVs with the accompanying pDST and MIC data to support the genotype-phenotype. In the future NGS surveillance methods could be used in clinical decision-making for designing effective treatment regimens containing BDQ, as well as to investigate transmission within the community. Additionally, this could help to preserve this novel antibiotic to ensure its longstanding use in successful drug-resistant TB therapy in the future.