Molecular characterization of the drug resistant tuberculosis epidemic in the Eastern Cape, South Africa

Klopper, Marisa (2015-03)

Thesis (PhD)--Stellenbosch University, 2015

Thesis

ENGLISH ABSTRACT : South Africa has a high burden of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB), with the Eastern Cape (EC) being one of the worst affected provinces in the country. This study provides the first in-depth analysis of the molecular epidemiology of drug-resistant TB in the EC. A convenience sample of drug-sensitive and drug-resistant isolates was collected over one year by the National Health Laboratory Services in Port Elizabeth. These isolates were characterized by various molecular techniques. Our results were compared to data from three additional provinces, to contextualise the population structure of MDR-TB strains. Each province had a distinct population structure. The population structure of XDR-TB cases in the Western Cape was significantly influenced by strains originating from the EC. A high degree of clustering of drug resistance mutation patterns was detected in each setting, suggestive of transmission. Clustering was particularly pronounced in the EC, with 93% of pre-XDR and XDR-TB isolates belonging to the Atypical Beijing genotype. We showed that this genotype was programmatically selected through a weakened MDR-TB regimen that failed to recognise inhA defined ethionamide resistance. This weakened regimen has facilitated transmission and is the underlying cause of mortality. We propose that existing molecular assays which detect inhA mutations should be used to identify patients at risk of XDR-TB and to adjust treatment. Through spoligotyping, restriction fragment length polymorphism typing and mutation analysis we demonstrated that the EC Atypical Beijing isolates evolved from a common progenitor, giving rise to two sub-groups, each with unique features, including mutations that confer resistance to up to 11 anti-TB drugs. This finding was supported by whole genome sequencing (WGS) and RNA sequencing demonstrating close relatedness and suggests the emergence and spread of totally drug-resistant TB in the EC. We showed that isolates harbouring the rrs A1401G mutation displayed a decreased susceptibility to capreomycin, thereby questioning the utility of this drug in the treatment of XDR-TB when amikacin resistance was already noted. Importantly, strains harbouring the rpoB516 mutation were shown to be susceptible to rifabutin, despite low-level resistance to rifampicin (RIF). Therefore the use of rifabutin in the EC may improve therapeutic success and limit transmission of XDR-TB. WGS was used to investigate molecular features that may confer a selective advantage to the EC Atypical Beijing genotype strains. These analyses revealed that all represented Atypical Beijing genotype strains – including those diagnosed as pan-susceptible – harboured a mutation in ethA, conferring phenotypic ethionamide resistance. This surprising finding may explain the apparent increased ability of the Atypical Beijing genotype strains to develop higher drug-resistance in the context of an ethionamide-containing MDR-TB treatment regimen. It is unclear why some strains additionally acquire inhA promoter mutations. This requires further investigation. A large number of genes were shown by RNAseq to be differentially regulated, however, their influence on the physiological properties of the bacillus remain to be determined. Together these findings have challenged the use of standardised MDR-TB treatment without comprehensive DST. This view is now widely recognised but has not influenced the South African TB guidelines (2014) which promote treatment of RIF resistance without relevant knowledge of drug resistance. We propose that the effective treatment of highly resistant TB can only be achieved with the development of new drugs, new drug combinations and comprehensive rapid DST.

AFRIKAANSE OPSOMMING : Suid-Afrika het ‘n hoë voorkoms van multi-middelweerstandige (MDR) en uiters middelweerstandige (XDR) tuberkulose (TB), veral in die Oos-Kaap. Hierdie studie bied die eerste in-diepte analise van die molekulêre epidemiologie van middelweerstandige TB in die Oos-Kaap. ‘n Gerieflikheidsteekproef wat oor een jaar geneem is en bestaan het uit middelsensitiewe sowel as middelweerstandige isolate is van die National Health Laboratory Services in Port Elizabeth ontvang. Hierdie isolate is deur verskeie molekulêre metodes gekarakteriseer. Ons resultate is vergelyk met data van drie addisionele provinsies om die populasiesamestelling van MDR-TB stamme in konteks te plaas. Elke provinsie het ‘n unieke populasiesamestelling getoon. Die populasiesamestelling van XDR-TB gevalle in die Wes-Kaap is beduidend deur stamme van die Oos-Kaap beïnvloed. ‘n Hoë mate van groepering van weerstandigheidspatrone is in elke provinsie gevind, wat dui op transmissie. Groepering was besonder duidelik in die Oos-Kaap, waar 93% van pre-XDR en XDR-TB isolate van die Atipiese Beijing genotipe was. Ons het getoon dat hierdie genotipe programmaties geselekteer is deur ‘n suboptimale MDR-TB behandelingsregime wat nie inhA-gedefiniëerde ethionamied weerstandigheid in ag neem nie. Hierdie ondoeltreffende behandelingsregime het transmissie gefasiliteer en is die onderliggende oorsaak van mortaliteit. Ons stel voor dat bestaande molekulêre toetse gebruik word wat inhA mutasies opspoor om XDR-TB risiko-pasiënte te identifiseer en hul behandeling dienooreenkomstig aan te pas. Ons het gedemonstreer dat twee sub-groepe van Oos-Kaap Atipiese Beijing isolate ontwikkel het uit ‘n gemene voorsaat, elk met unieke eienskappe, insluitend mutasies wat weerstandigheid teen tot 11 middels veroorsaak. Hierdie bevinding word gerugsteun deur heelgenoom volgordebepaling en ribonukleïensuur volgordebepaling en dui op die ontluiking en verspreiding van algeheel middelweerstandige TB in die Oos-Kaap. Ons het getoon dat isolate wat die rrs A1401G mutasie het, verminderde vatbaarheid vir capreomisien het, en dit bevraagteken die bruikbaarheid van hierdie middel in die behandeling van XDR-TB waar amikasienweerstandigheid teenwoordig is. Van belang is dat stamme wat die rpoB516 mutasie het, vatbaar is vir rifabutien, ten spyte van weerstandigheid teen rifampisien. Die gebruik van rifabutien kan dus die uitkomste van XDR-TB pasiënte in die Oos-Kaap verbeter, en ook transmissie beperk. Heelgenoom volgordebepaling is gebruik om molekulêre eienskappe te ondersoek wat moontlik ‘n selektiewe voordeel kan bied aan die Oos-Kaapse Atipiese Beijing genotipe stamme. Ons het getoon dat alle verteenwoordigde Atipiese Beijing genotipe stamme – insluitend dié wat as algeheel middelsensitief gediagnoseer is – ‘n ethA mutasie het wat ethionamied weerstandigheid veroorsaak. Dit mag die oënskynlike verhoogde vermoë van die Atipiese Beijing genotipe stamme om hoër weerstandigheid te ontwikkel verklaar. Verder het ribonukleïensuur volgordebepaling getoon dat ‘n groot aantal gene verskillend gereguleer is. Hierdie verskille moet verder ondersoek word om die invloed daarvan op die fisiologiese eienskappe van die bacillus te verklaar. Hierdie bevindinge betwis die gebruik van gestandardiseerde MDR-TB behandeling in die afwesigheid van omvattende middelsensitiwiteitstoetse. Hierdie siening word tans algemeen aanvaar, en tog het dit nie die Suid-Afrikaanse TB-riglyne (2014), wat behandeling van rifampisienweerstandigheid sonder die relevante kennis van middelweerstandigheid voorstaan, beïnvloed nie. Ons stel voor dat die effektiewe behandeling van hoogs weerstandige TB net bereik kan word deur die ontwikkeling van nuwe middels, nuwe kombinasies van middels en vinnige, omvattende middelsensitiwiteitstoetse.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/96615
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