New and improved methods for the diagnosis of susceptibility to tuberculosis drugs

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dc.contributor.advisorTheron, Granten_ZA
dc.contributor.advisorDe Vos, Margarethaen_ZA
dc.contributor.authorDerendinger, Brigittaen_ZA
dc.contributor.otherStellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.en_ZA
dc.date.accessioned2023-01-25T08:52:10Zen_ZA
dc.date.accessioned2023-05-18T06:56:50Zen_ZA
dc.date.available2023-01-25T08:52:10Zen_ZA
dc.date.available2023-05-18T06:56:50Zen_ZA
dc.date.issued2023-01en_ZA
dc.descriptionThesis (PhD)--Stellenbosch University, 2023.en_ZA
dc.description.abstractENGLISH 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.af_ZA
dc.description.abstractAFRIKAANS OPSOMMING: Middelbestande-tuberkulose (DR-TB) is ‘n wêreldwye bedreiging en dit is belangrik om pasiënte te diagnoseer en met toepaslike behandeling in die kortste moontlike tyd te begin. Optimalisering van bestaande vinnige molekulêre toetse en implementering van nuwe medisyne-vatbaarheidstoetse (DST) om pasiënte op behandeling te monitor, is belangrik om die oordrag van DR-TB te beperk. Eerstens (hoofstuk 2), Mycobacterium tuberculosis (Mtb)-DNA herwinbaar vanaf gebruikte Xpert MTB/RIF (Xpert) toetse ‒ “cartridge extract” (CE) ‒ wat andersins weggegooi sou word, kan gebruik word vir tweedelyn molekulêre-DST. Geen bykomende DNA-ekstraksie of monstersuiwering was nodig nie. Ons het Xpert semikwantitatiewe kategorie "low" gedefinieer om te verseker dat geen MTBDRsl-toetse op CE vermors word wat waarskynlik ongeldige resultate sou gee nie. Dit het die behoefte verlig om 'n tweede sputummonster te neem, wat tot gevolg diagnostiese vertragings kon verminder en pasiënte vinniger op behandeling kon plaas. Hierdie benadering word ontwikkel tot 'n “cartridge device” en sal deur TB-programme geëvalueer word. Tweedens en derdens (hoofstuk 3 en 4), MTBDRplus en MTBDRsl (vinnige molekulêre WHO-aanbeveelde toetse), het suboptimale sensitiwiteit en hoë onbepaalde koerse gerapporteerde, veral in smeer-negatiewe monsters. Ons het hipotese dat die “ramp rate” (spoed van temperatuurverandering tussen PCR-siklusse) toetsprestasie kan beïnvloed. Ons het getoon dat die regstelling van “ramp rate” op die “thermocycler” (vervaardiger-aanbevole “ramp rate” ≤2.2°C/s) waarskynlik die opbrengs van vinnige diagnose vir eerste- en tweedelyn DST, gedoen met MTBDRplus en MTBDRsl, onderskeidelik, verbeter het, veral in paucibacillêre monsters. Ons het 'n opname gedoen wat getoon het dat suboptimale “ramp rate” is 'n algemene probleem, maar is maklik om reg te stel. Die manuskrip het die WHOkursusopleidingsmateriaal geingelig: https://openwho.org/courses/multi-drug-resistant-tb). Laastens (hoofstuk 5), bedaquiline (BDQ), 'n lewensreddende TB-middel, is vinnig aan die toeneem, maar grootliks in die afwesigheid van DST. In 'n groep van programmatiese pasiënte wat steeds kultuur-positief is na ≥4 maande van 'n BDQ-gebaseerde behandeling, het meer as die helfte “isolates” gehad wat BDQ-weerstand gekry (meestal verkryging, sommige oordrag). Verskeie Rv0678 en pepQ variante was geassosieer met fenotipiese weerstand, baie voorheen onbeskryf. Pasiënte met basislyn fluoroquinolone-weerstand, clofazimine blootstelling en ≤4 effektiewe middels was meer geneig om BDQ-weerstandig te wees. Hierdie tesis het tot vier eerste-outeur manuskripte gelei (drie gepubliseer, een ingedien). Daarbenewens twee 2de-outeur manuskripte en sewe manuskripte as middel mede-outeur. Hierdie nege totaal word kortliks in hoofstuk 6 bespreek en kan in die bylaes gevind word (en as bykomende publikasies ingesluit). Die kandidaat het aangebied by drie internasionale en twee nasionale eweknie-geëvalueerde konferensies. Samevattend toon hierdie werk dat tweedelyn-middelweerstand tyd-tot-behandeling-aanvang verminder kan word deur tweedelyn-DST op CE van gebruikte toetse te doen. Die aantal smeer-negatiewe pasiënte by wie DST moontlik is, sal aansienlik verbeter na “ramp rate”- korreksie vir MTBDRplus en MTBDRsl. Laastens toon ons die bestaan van 'n potensieel aansteeklike-poel van BDQ-weerstandige “isolates” wat onder programmatiese toestande geskep is, sowel as die uitdagings en risiko's wat verband hou met die begin van pasiënte met komplekse TB-behandelingsgeskiedenis op 'n regime wat 'n nuwe geneesmiddel bevat sonder gereeld beskikbare DST. Ons bevindinge gee ook inligting oor hoe en by wie nuwe TB-middels geprioritiseer word vir gebruik.en_ZA
dc.description.versionDoctoralen_ZA
dc.format.extent240 pages : illustrateden_ZA
dc.identifier.urihttp://hdl.handle.net/10019.1/126942en_ZA
dc.language.isoen_ZAen_ZA
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.subject.lcshTuberculosis -- Susceptibilityen_ZA
dc.subject.lcshDrug resistanceen_ZA
dc.subject.lcshMycobacterium tuberculosisen_ZA
dc.subject.lcshSimulated patientsen_ZA
dc.titleNew and improved methods for the diagnosis of susceptibility to tuberculosis drugsen_ZA
dc.typeThesisen_ZA
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