Molecular characterization of Mycobacterial strains from clinical specimens of patients with Tuberculosis at the Dr George Mukhari Tertiary Laboratory in South Africa

Tuberculosis (TB) is a complicated old disease ranking above human immunodeficiency virus (HIV) and acquired immune deficiency syndrome (AIDS) with the largest disease burden, especially in poor resource settings. Each year, more than 10 million people develop TB, while nearly two million die. South Africa has the world’s eighth highest TB incidence (520/100 000 population), and fifth highest multidrug resistant TB (MDR-TB) incidence, with over 19 000 cases estimated, and an HIV prevalence of 18% nationwide. Efforts to end TB in South Africa are significantly compromised by an unabated drug-resistant TB epidemic, discordant results between different methods, mixed Mtb infections and nontuberculous mycobacterial infections. In response to these challenges, the world health organisation (WHO) recommended standard routine diagnostic algorithms that have been implemented, with standardised treatment algorithms which are aimed at reducing the spread of TB drug resistance, timely initiating effective anti-TB treatment, and improving treatment outcomes.
The aim of this thesis was to characterize diagnostic challenges by genotyping mycobacterial strains from clinical specimens of patients with TB at the Dr George Mukhari Tertiary Laboratory, South Africa.
In chapter 3, we investigated whether the rifampicin resistance conferring rpoB Ile491Phe was missed as “INH monoresistant” by the current standard diagnostic algorithms in South Africa, and found the rpoB Ile491Phe mutation associated with poor RIF based treatment outcome and decreased bedaquiline sensitivity, in 15% INH monoresistant TB isolates using a targeted polymerase chain reaction (PCR) assay and deep sequencing, which isolates were later reclassified as MDR-TB. Moreover, whole genome sequencing (WGS) revealed the concurrent silent spread in South Africa of an MDR-TB strain lineage extending the Swaziland outbreak, and an independently emerged second Ile491Phe-bearing lineage.
Chapter 4 focused on the frequency of discordant RIF Mtbc isolates from TB patients between the genotypic and phenotypic DST methods, and evaluated the clinical outcomes of the affected patients. We found that the Mtbc isolates with discordant genotypic and phenotypic results harboured 26.5% of disputed and 59.0% of undisputed rpoB mutations, as well as a combination of mutations within/outside the RRDR region at a rate ot 14.5%. We further found that discordant RIF Mtbc results caused by disputed rpoB mutations in our settings are associated with unfavourable treatment outcomes.
In Chapter 5, we reviewed publications under mixed Mtb infections that were detected using different methods and factors that are associated with mixed infections, reported worldwide. We found that the prevalence of mixed Mtb infections worldwide, was different in different geographical settings of the WHO TB regions, and also depended on which specimen types and molecular methods were used for analysis.
In chapter 6, we investigated the frequency of mixed Mtbc infections and heteroresistance, and correlated these with patient clinical outcomes. We found mixed Mtbc infections in 6 (7.2%) isolates and clonal heteroresistance in 3 (3.6%) Mtbc isolates, of the unknown lineages. All Mtbc isolates were from patients who were co-infected with HIV with low CD4 cell count. Differences in DST among isolates from the same patient were observed in ten cases, of which six were from patients with mixed infection. Our findings showed a low prevalence of mixed Mtbc infections and clonal heteroresistance in our study setting.
In chapter 7, we describe the prevalence and patients’ characteristics of different nontuberculous mycobacteria (NTMs). M. intracellulare, M. avium, M. fortuitum, M scrofulaceum, and M. kansasii respectively, were the most prevalent and clinically significant mycobacteria species in our settings. The uncommon mycobacteria species being M celatum, M. smegmatis, M shimoidei, M. asiaticum and M. mucogenicum. The GenoType Mycobacterium CM and AS assay enable the rapid identification of a broad range of potentially clinically significant Mycobacterium species.
The results of this thesis will not only guide with future researches to better understand and resolve the core issues with TB, but will also help with the knowledge in terms of which optimal molecular diagnostic tests can be implemented to standard routine algorithms, to accurately detect all mutations that are associated with drug resistant TB and discordant results between methods; mixed infections and NTM infections.