Genetic characterization of drug resistant clinical isolates of Mycobacterium tuberculosis circulating within the Copperbelt province and Northern regions of Zambia.

Thesis (PhD)--Stellenbosch University, 2019.ENGLISH ABSTRACT: The emergence and spread of drug resistant (DR) tuberculosis (TB) strains in the form of multidrug resistant (MDR)- and extensively drug resistant (XDR)-TB is a major threat to the global fight against TB. Treatment for these forms of TB is prolonged, up to 24 months, and involves the use of a combination of highly toxic, less potent anti TB drugs. In 2015 alone, the World Health Organisation (WHO) estimated 580,000 new MDR-TB cases across the world. Nine African countries are listed as high MDR-TB burden countries by the WHO. A review of published research revealed that diverse genotypes are associated with DR TB in Africa, and demonstrated that DR TB strains are associated with community and nosocomial outbreaks. Furthermore, the role of migration in the transmission of DR TB strains has been demonstrated in certain parts of Africa. Of concern is the under-use of molecular epidemiological tools, resulting in gaps in knowledge of the transmission dynamics of DR TB on the continent. This study aims to address some of these gaps by describing the molecular epidemiology of DR TB in regions of the Copperbelt province and Northern regions of Zambia. We used molecular strain typing tools of whole genome sequencing (WGS), Sanger (targeted gene) sequencing, insertion sequence 6110-restriction fragment length polymorphism (IS6110-RFLP) and spoligotyping to describe the genotypes of DR Mycobacterium tuberculosis (M.tb) strains circulating within parts of Zambia. We demonstrated that a variety of genotypes are associated with DR TB in Zambia. The predominant genotype was lineage 4, with majority of strains belonging to Latin American Mediterranean (LAM). Other lineages belonged to 2 and 3. The genotyping analysis showed clustering of strains among patients being from different regions of the country thereby suggesting that DR TB is possibly widespread across the country. In addition, this analysis also identified household transmission of MDR-TB between two household contacts, placing emphasis on the need for routine tracing of MDR-TB patient contacts in Zambia. Further analysis of WGS and Sanger sequencing data identified 8 pre-XDR-TB cases. These belonged to lineage 4.6.1 (Uganda lineage), lineage 2.2 (Beijing genotype) and lineage 4.3 (LAM), giving a preliminary first insight into the genotypes associated with pre-XDR-TB in Zambia. Alarmingly, transmission of these pre-XDR-TB strains was demonstrated, with clustered strains sharing identical drug resistance-conferring mutations and low nucleotide variance differences. This finding emphasises the need for more comprehensive drug susceptibility testing, as failing to identify second line resistance may place the patient at risk of acquisition of additional resistance when treated with a standardised MDR-TB regimen. Nosocomial transmission of DR TB has not been described in Zambia, despite the high risk of transmission in health care facilities. Assessment of the knowledge, attitudes and practices of health care workers at MDR-TB health care facilities in Ndola district revealed knowledge gaps and administrative deficiencies which could be placing these critical personnel at risk of acquiring DR TB at the work place. Findings highlighted continuous infection prevention and control trainings and provision of adequate personal protective equipment (PPE) as key areas of improvement. The current study provides a first insight into the genetics of DR TB strains circulating in Zambia. These findings address knowledge gaps and contribute to our understanding of DR TB in Africa. To address the DR TB epidemic in Zambia, the TB control program need to expand the Xpert test-and-treat diagnostic strategy to all people entering healthcare facilities with symptoms of TB. More comprehensive drug susceptibility testing needs to be done to ensure patients are adequately treated. Following diagnosis of DR TB patients need to be counselled to initiate treatment and families and close contacts should be screened for TB.AFRIKAANS OPSOMMING: Die opkoms en verspreiding van middelweerstandige (DR) tuberkulose (TB), spesifiek die multi weerstandige (MDR-TB) en uiters weerstandige (XDR-TB) vorme van TB is 'n groot bedreiging vir die globale stryd teen TB. Behandeling vir hierdie vorms van DR TB word verleng, tot 24 maande, en behels die gebruik van 'n kombinasie van hoogs toksiese en swakker anti-TB-middels. In 2015 het die Wêreldgesondheidsorganisasie (WGO) beraamd daar is 580,000 nuwe MDR / rifampisien weerstande (RR) -TB gevalle regoor die wêreld. Nege Afrika-lande word deur die WGO as hoë MDR-TB lande gelys. 'n Literatuuroorsig het aan die lig gebring dat diverse genotipes met DR TB op die vasteland geassosieer word, en getoon dat DR TB-stamme geassosieer word met gemeenskaps- en hospitaal uitbrake. Verder is die rol van migrasie in die oordrag van DR TB-stamme in spesifieke dele van Afrika gedemonstreer. Daar is kommerwekkend min molekulêre epidemiologiese studies, met ‘n gevolglike gebrek in kennis oor die transmissie dinamika van DR TB op die vasteland. Die doel van hierdie studie is om sommige van hierdie leemtes aan te spreek deur die transmissie dinamika van DR TB in dele van Zambië te beskryf Ons het genotipiese onderskeidings tegnieke, spoligotipering, IS6110-restriksie fragmentlengte polimorfisme (IS6110-RFLP), heel genoomvolgorde bepaling (WGS) en Sanger volgordebepaling gebruik om die genotipes van DR Mycobacterium tuberculosis te beskryf wat in dele van Zambië sirkuleer. Ons het gewys dat 'n wye verskeidenheid genotipes geassosieer word met DR TB in Zambië. Drie van die vernaamste stamme is gevind (linie 2, 3 en 4) met die oorheersende genotipes wat behoort in linie 4 (Latyns-Amerikaanse Mediterreens (LAM)). Groepering van stamme onder pasiënte uit verskillende streke van die land is getoon, wat daarop dui dat DR TB moontlik wydverspreid oor die land voorkom. Hierdie analise het ook huishoudelike oordrag van MDR-TB geïdentifiseer, wat klem lê op die behoefte aan roetine opsporing van MDR-TB-pasiënt kontakte in Zambië. Verdere analise van WGS en Sanger volgorde bepalingsdata het 8 pre-XDR-TB gevalle geïdentifiseer, wat aan linie 4.6.1 (T1 genotipe), linie 2.2 (Beijing genotipe) en linie 4.3 (LAM) behoort. Dit is die eerste beskrywing van genotipes wat verband hou met pre-XDR-TB in Zambië. Oordrag van hierdie stamme is gedemonstreer, deurdat groepe dieselfde weerstandsmutasies het, asook beperkte variasie in die heelgenoomdata toon. Hierdie kommerwekkende bevinding beklemtoon die behoefte aan meer omvattende middelweerstandigheidstoetse, aangesien versuim om tweede-linie weerstand te diagnoseer, die pasiënt se risiko verhoog om addisionele weerstand op te bou, indien ‘n gestandaardiseerde MDR-TB-regimen gebruik word. Hospitaaloordrag van DR TB is nog nie voorheen in Zambië beskryf nie, ten spyte van die hoë risiko van oordrag in gesondheidsorgfasiliteite. Assessering van die kennis, houdings en praktyke van gesondheidswerkers by MDR-TB gesondheidsorgfasiliteite in Ndola-distrik, het gebrekkige kennis en administratiewe tekortkominge onthul, wat hierdie kritieke personeel in gevaar sou stel om DR TB in die werkplek op te doen. Ons bevindings beklemtoon die belang van deurlopende infeksie voorkomings- en beheerpraktyke en die voorsiening van voldoende persoonlike beskermingstoerusting (PPE) as sleutelareas van verbetering. Hierdie studie is die eerste beskrywing van die genetika van DR TB-stamme in omloop in Zambië. Hierdie bevindings vul ons kennis aan en dra by tot ons begrip van DR TB in Afrika. Om die DR TB -epidemie in Zambië aan te spreek, moet die TB-beheerprogram die Xpert toets-en-behandel strategie uitbrei sodat alle mense met TB simptome bereik word. Meer omvattende middelweerstandigheidstoetsing moet gedoen word om te verseker dat pasiënte effektief behandel word. Na die diagnose van DR TB by pasiënte moet beraadslaag word om behandeling te begin en gesinne en naby kontakte moet vir TB gesif word