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Background
Tuberculosis (TB) caused by Mycobacterium tuberculosis (M. tuberculosis) is a serious problem worldwide, with about 10.4 and 14 million incident and prevalent cases respectively (Karim et al., 2009; WHO, 2009; WHO, 2016a). TB continues to be a global threat and has shown to be difficult to control in regions with high prevalence to human immune deficiency virus (HIV) infection (Louw et al., 2011). Sub-saharan Africa is currently experiencing dual epidemics of both TB and HIV. TB disease in South Africa is hyperendemic, with incidence rates of up to 948 cases per 100 000 population (Bekker and Wood, 2010). Other parts of southern Africa have more than 50% of TB cases coinfected with HIV (WHO, 2014).
History of tuberculosis
Tuberculosis (TB) is an ancient disease that has plagued humankind throughout history and continues to cause great epidemics. History suggests that TB appeared about 70 000 years ago and that it remained sporadic up to the 18th century (Daniel, 2006; Banuls et al., 2015). Indeed, the discovery of M. tuberculosis by Robert Koch (1843–1910) came into a framework of previous popular and scientific knowledge regarding TB who announced the discovery on March 24, 1882 (Anquetin et al., 2006; Lawn and Zumla, 2011). Koch’s work on the anthrax bacillus grown in pure culture paved the way for understanding the fundamental and underlying etiology of infectious diseases, including TB (Weyer et al., 2011). It then became epidemic during the industrial revolution but was soon reduced by the introduction of the Bacillus Calmette–Guérin (BCG) vaccine in 1921 and the use of antimicrobial drugs, such as streptomycin (STR) (1943), isoniazid (INH) (1952) and rifampicin (RIF) (1963) (Banuls et al., 2015). TB incidence increased again in the 1980s, due to the deterioration of health conditions in large cities (Banuls et al., 2015). About one-quarter of the world’s population, or approximately 1.7 billion individuals, is infected with TB. Of these, almost 10 million people have active TB and 1.8 million die from this disease each year (WHO, 2016). Substantial geographical and age variation occur (Houben et al., 2016). Human populations act as the natural reservoir of the pathogen (Salgame et al., 2015). More than 90 % of TB cases occur in developing countries and the regions most affected by this disease are Africa, South-East Asia and East Europe (Banuls et al., 2015).
Background
Molecular detection of first and second-line drug-resistance in tuberculosis (TB) is important for early detection and treatment. Second-line drugs of ofloxacin (OFX), amikacin (AMK), kanamycin (KAN) and capreomycin (CAP) are crucial for treatment and prevention of extensively drug-resistant (XDR) TB. Molecular tests such as GenoType® MTBDRsl are important and should be evaluated in high burden regions of Africa. The study assessed the performance of GenoType® MTBDRsl version 1 (Hain Lifescience, GmbH, Nehren,Germany) against MGIT DST and used DNA sequencing (Sanger) to resolve discordance.
Methods
Six hundred and eighty-nine culture positive specimens were tested using GenoType® MTBDRsl. The diagnostic performance was compared to the gold standard MGIT DST method. Sanger DNA sequencing was performed on gyrA, gyrB, rrs, eis, tlyA, Rv1258cRv1634 and Rv0194. We further investigated the impact of new rrs gene mutations on minimum inhibitory concentrations (MIC) using microtiter plate (AlamarBlue®).
Methods
Genomic DNA was extracted from two XDR-TB strains andthe whole genome of Mycobacterium tuberculosis sequenced on the Illumina HiSeq platform at the Broad Institute(Cambridge, MA, USA). Reads from two strains were assembled and annotated to the H37Rv genome. Functional effects of amino acid mutations were predicted using the protein variation effect analyser (PROVEAN) tool.
Results
Transcriptomic comparison of XDR to MDR and to susceptible TB strains To identify genes overexpressed in XDR strains, we performed transcriptomic comparison in relation to MDR strains and susceptible strains. Pairwise comparisons of reads per kb per million mapped reads (RPKM) between the three groups were performed in order to identify overexpression categories: (i) Genes upregulated in XDR relative to MDR and susceptible TB strains; and (ii) Genes unique to both XDR and MDR TB strains, relative to the susceptible group. Using a 1.5-fold overexpression, we identified 1257 (30.7%) genes unique to XDR-TB and 577 (14.1%) genes unique to MDR-TB strains (Figure 11). A further 1887 (46.1%) genes were common among all strains (Table 17). Among the genes unique to XDR-TB, genes involved in efflux, lipid metabolism and non-coding RNA were identified with a 1.5-fold overexpression (Table 18).
Chapter 1: 1.1 Background
1.2 Hypothesis
1.3 Aim
1.4 Objectives
1.5 References
Chapter 2: Literature review
2.1 History of tuberculosis
2.2 The tubercle bacillus
2.2.1 Cell wall and envelope
2.2.2 Phylogeny
2.2.3 Mycobacterium tuberculosis genome and transcriptome
Chapter 3: Performance evaluation of GenoType® MTBDRsl version 1 enhanced by DNA sequencing for detection of Mycobacterium tuberculosis second-line drug resistance in Southern Africa
3.1 Abstract
3.2 Introduction
3.3 Materials and Methods
3.3.1 Bacterial cultures and specimen origin
3.3.2 Drug susceptibility testing
3.3.3 GenoType® MTBDRsl assay
3.3.4 Polymerase chain amplification and DNA sequencing
3.3.5 Spoligotyping
Chapter 4: Draft genome sequences of two extensively drug-resistant strains of Mycobacterium tuberculosis
4.1 Abstract
4.2 Introduction
4.3 Materials and methods
4.3.1 Strains and drug susceptibility testing
4.3.2 Genomic DNA extraction from mycobacteria
4.3.3 Genome sequencing, analysis and annotation
4.4 Results
4.4.1 Genomic data
4.4.2 Drug resistance markers
Chapter 5: RNA sequencing and inhibition of efflux pump genes involved in second-line drug resistance in Mycobacterium tuberculosis
5.1 Abstract
5.2 Background
5.3 Methods
5.3.2 Strains phenotype and genotype
5.3.3 Strain growth
5.3.4 Minimum inhibitory concentrations
5.3.5 RNA preparation and sequencing
5.3.6 RNA sequencing data analysis
5.3.7 Protein-protein interaction network
5.4 Results
Chapter 6: The role of efflux pumps Rv0194, Rv1258c and Rv1634 on antimicrobial susceptibilities and virulence in Mycobacterium tuberculosis
6.1 Abstract
6.3 Materials and methods
6.3.1 DNA amplification and bioinformatics features of Rv1258c, Rv1634 and Rv0194 genes
6.3.2 Bacterial strains, plasmids, and growth conditions
Chapter 7: Concluding remarks
7.1 Summary
7.2 Future Research
7.3 References
Appendix A: Detailed methods
A1 Spoligotyping
A2 RNA extraction
A3 RNA purification
