Molecular characterization and drug susceptibility of isolates from MDR-TB patients in the Eastern Cape and North West provinces of South Africa

Thesis (PhD)--Stellenbosch University, 2016.ENGLISH SUMMARY: South Africa is among the countries facing rising numbers of Mycobacterium tuberculosis (Mtb) drug resistant strains. In 2000, DOTS-Plus strategy was introduced nationally to combat drug resistant tuberculosis (TB). This necessitated the introduction of drug susceptibility testing for second-line drugs (SLDs) in order to detect and treat cases in a timely and effective manner. However, this was only routinely implemented following the description of extensively drug resistant (XDR-TB, defined as MDR-TB plus resistance to a fluoroquinolone and a second-line injectable, in South Africa in 2006. The impact of implementing a standardized MDR-TB therapy policy in South Africa on individual treatment outcomes and acquisition of additional drug resistance has not been widely documented. Improved knowledge of factors that lead to acquisition of second-line drug resistance will help better predict who is most at risk of drug resistance and contribute to the development of new tools and strategies to combat MDR-TB. To fill this gap, we sought to determine the prevalence of SLD resistance among MDR-TB patients in the DOTS-Plus cohort and its impact on treatment outcomes for these patients in two provinces in South Africa; Eastern Cape (EC) and North West (NW) province. The results show that treatment success was strongly influenced by the setting where the patients were treated. Default and death accounted for 58.1% (193/333) of all unfavourable outcomes in provinces. The EC province had the lowest (13.4%, 51/381) cure rate and the highest default rate of 38.3%; compared to a default rate of 6.39% in NW. This study also describes the resistance patterns against second line drugs among newly diagnosed MDR-TB patients in the NW and EC province using Genotype MTBDRsl assay (version 1) and targeted sequencing of genes known to confer resistance, and how these patients acquired resistance during treatment. These finding have important implications for infection control, because undiagnosed highly resistant strains could have been transmitted to contacts during treatment. The concordance between Genotype MTBDRsl and sequencing was 82% for all gyrA gene and 67% for the rrs gene. Resistance to all drugs (including ethambutol) tested at baseline was 15.8% (47/298) and resistance to both ofloxacin and kanamycin was 1.3% (4/298). Heteroresistance associated with the gyrA and embB gene was also observed. Furthermore, the study discusses the implementation of the DOTS-Plus policy with regards to whether it significantly contributed to the emergence of XDR-TB in individual patients. Implications for implementation of standardized MDR-TB treatment in the absence of knowledge of baseline resistance are also discussed. Analysis of 48 MDR-TB patients, with initial and last available isolates, showed that 45,8% gained resistance to second line drugs during treatment which suggests that the combination of in-hospital treatment with a standardized MDR-TB treatment regimen increased the risk to the patient gaining XDR during treatment. This thesis has contributed to our understanding of drug resistance in TB, and implications of implementing standardized MDR-TB treatment in South Africa. We propose an algorithm for rapidly diagnosing patients that are at risk of extensively drug resistant tuberculosis (XDR-TB) using a combination of the methods endorsed by the World Health Organization (WHO).AFRIKAANSE OPSOMMING: Suid-Afrika is ‘n land met stygende getalle middelweerstandige stamme van Mycobacterium tuberculosis. In 2000 is die DOTS-plus strategie oor die hele land ingestel om middelweerstandige tuberkulose (TB) te beveg. Dit het die bekendstelling van middelvatbaarheidstoetsing vir tweedeliniemiddels genoodsaak om gevalle tydig en effektief op te spoor en te behandel. Dit is egter eers in 2006, na die beskrywing van uitgebreide middelweerstandige (XDR)-TB, omskryf as MDR-TB plus weerstand teen fluorokinoloon en ’n tweedelinie inspuitbare middel as roetine geïmplementeer in Suid-Afrika. Die impak van die implementering van 'n gestandaardiseerde MDR-TB-terapiebeleid op individuele behandelingsuitkomste en die opdoen van addisionele middelweerstandigheid in SA is nie goed gedokumenteer nie. Verbeterde kennis van die faktore wat tot die opdoen van tweedeliniemiddelweerstandigheid lei, sal lei tot beter voorspellings oor wie die hoogste risiko loop vir middelweerstandigheid, en ook bydra tot die ontwikkeling van nuwe middels en strategieë om MDR-TB te beveg. Ten einde hierdie gaping te vul, het ons probeer vasstel wat die voorkomssyfer van tweedeliniemiddelweerstandigheid onder MDR-TB-pasiënte in die DOTS-plus-studiegroep is en die uitwerking daarvan op behandelingsuitkomste vir hierdie pasiënte in twee provinsies in Suid-Afrika, naamlik die Oos-Kaap en Noordwes. Die resultate toon dat behandelingsukses sterk beïnvloed word deur die plek waar die pasiënte behandel is. 58.1% (193/333) van alle ongunstige uitkomste in die provinsies is te wyte aan versuiming van behandeling en sterfte. Die Oos-Kaap het die laagste genesingskoers (13.4%, 51/381) en die hoogste versuimingskoers (38.3%) gehad, in vergelyking met 'n versuimingskoers van 6.39% in Noordwes. Hierdie studie beskryf ook die weerstandspatrone teen tweedeliniemiddels onder nuut-gediagnoseerde MDR-TB-pasiënte in Noordwes en die Oos-Kaap met genotipe-MTBDRsl-toetsing (weergawe 1) en geteikende DNS volgordebapaling van gene bekend vir die oordra van weerstandigheid, en hoe hierdie pasiënte weerstand gedurende die behandeling opgebou het. Hierdie bevindinge het belangrike implikasies vir infeksiebeheer omdat ongediagnoseerde, hoogs weerstandige stamme na kontakte gedurende behandeling oorgedra kan word. Die ooreenstemming tussen die genotipe-MTBDRsl en DNS volgorde was 82% vir al die gyrA-gene en 67% vir die rrs-geen. Weerstandigheid teen alle middels (insluitende etambutol) wat op aanvangsvlak getoets is, was 15.8% (47/298) en weerstandigheid teen sowel ofloksasien as kanamisien was 1.3% (4/298). Heteroweerstandigheid wat met sowel die gyrA-geen as die embB-geen geassosieer word, is ook waargeneem. Die studie bespreek verder die implementering van die DOTS-plus beleid en of dit betekenisvol aanleiding gee tot die verskyning van XDR-TB in individuele pasiënte. Die implikasies vir die implementering van gestandaardiseerde MDR-TB-behandeling met gebrek aan enige kennis oor aanvangsweerstandigheid, word ook bespreek. Die ontleding van 48 MDR-TB pasiënte, met ’n aanvanklike en ’n laaste kultuur toon dat 45,8% weerstand teen tweedeliniemiddels gedurende behandeling opgebou het. Dit dui daarop dat die kombinasie van behandeling in 'n hospitaal met ’n gestandaardiseerde behandelingsplan die risiko vir die pasiënt verhoog om XDR gedurende behandeling op te doen. Hierdie tesis dra by tot ons kennis oor en begrip van middelweerstandigheid in TB en die implikasies van die implementering van gestandaardiseerde MDR-TB-behandeling in Suid-Afrika. Ons doen 'n algoritme aan die hand om pasiënte wat gevaar loop om uitgebreide middelweerstandige tuberkulose (XDR-TB) op te doen, vinnig te diagnoseer met 'n kombinasie van metodes wat deur die Wêreld-gesondheidsorganisasie (WGO) goedgekeur is