Doctoral Degrees (Molecular Biology and Human Genetics)

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Browsing Doctoral Degrees (Molecular Biology and Human Genetics) by browse.metadata.advisor "De Vos, Margaretha"

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    New and improved methods for the diagnosis of susceptibility to tuberculosis drugs
    (Stellenbosch : Stellenbosch University, 2023-01) Derendinger, Brigitta; Theron, Grant; De Vos, Margaretha; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: Drug-resistant tuberculosis (DR-TB) is a global threat. Diagnosing patients with DR-TB and initiating them onto appropriate treatment, in the shortest possible time, is of utmost importance. The optimisation of existing rapid molecular assays and the implementation of drug susceptibility testing (DST) for new drugs to monitor patients on treatment is crucial in curbing transmission. Firstly (chapter 2), showed that Mycobacterium tuberculosis (Mtb) DNA recoverable from used Xpert MTB/RIF (Xpert) cartridges ‒ cartridge extract (CE) – that would otherwise be discarded, can be used for downstream second-line molecular DST. No additional DNA extraction or sample purification was needed. We defined a threshold of Xpert semiquantitative category “low” to ensure that no MTBDRsl assays are wasted on CE likely to give invalid results. This alleviated the need for collection of a second sputum specimen, thereby reducing diagnostic delays, and enabling patients to be placed on treatment sooner. This approach is being developed into a cartridge extraction device and will be evaluated by TB programmes. Secondly and thirdly (chapter 3 and 4), MTBDRplus and MTBDRsl focussed on WHOendorsed rapid molecular assays that have reported suboptimal sensitivities and high indeterminate rates especially in smear-negative specimens. We hypothesised that ramp rate (speed of temperature change between PCR cycles) could impact assay performance. We showed that correcting thermocycler ramp rate (manufacturer-recommended ramp rate ≤2.2°C/s) likely improved the yield of rapid diagnoses for first-and second-line DST, done with MTBDRplus and MTBDRsl respectively, especially in paucibacillary specimens. Our survey showed that suboptimal ramp rate is a common problem but is easily fixable. This manuscript informed WHO course training material (https://openwho.org/courses/multi-drug-resistant-tb). Finally (chapter 5), bedaquiline (BDQ), a lifesaving TB drug, is undergoing rapid scale-up but largely in the absence of DST. In a group of programmatic patients still culture-positive after ≥4 months of BDQ-based treatment, more than half had isolates that gained BDQ resistance (mostly acquisition, some transmission). Several Rv0678 and pepQ variants were associated with phenotypic resistance, many previously undescribed. Patients with baseline fluoroquinolone-resistance, clofazimine exposure, and ≤4 effective drugs were more likely to be BDQ-resistant. This thesis has resulted in four first author manuscripts (three published, one submitted). Additionally, two 2nd author manuscripts and seven manuscripts as a middle co-author. These nine total are briefly discussed in chapter 6 and can be found in the appendices (and included as ancillary publications). The candidate presented three times at international and twice at national peer-reviewed conferences. In summary, this work shows that second-line DR time-to-treatment initiation could be reduced by doing second-line DST on CE from used cartridges. The number of smear-negative patients in whom DST is possible will improve substantially after ramp rate correction for MTBDRplus and MTBDRsl. Finally, we show the existence of a potentially infectious pool of BDQ resistant strains created under programmatic conditions, as well as the challenges and risks associated with starting patients with complex TB-treatment histories on a regimen containing a novel drug without routinely available DST. Our findings also inform on how and in whom new TB drugs are prioritised for use.