Application of spoligotyping in the understanding of the dynamics of Mycobacterium tuberculosis strains in high incidence communities
Thesis (PhD (Biomedical Sciences. Molecular Biology and Human Genetics))--University of Stellenbosch, 2007.
Tuberculosis (TB) is a global health problem and demands rigorous control management efforts. A dramatic increase in the acquisition and spread of drug resistant TB globally has been observed in recent years. A grim picture has emerged for the control program with the discovery of extreme drug-resistant TB, which is virtually untreatable and is of immense concern for the future of TB control. In the last decade strain-specific genetic markers have been identified to examine the molecular epidemiology and spread of TB, including IS6110 DNA-fingerprinting and spoligotyping. Although spoligotyping has less discriminatory power than the gold standard, IS6110 DNA-fingerprinting, it is simpler, faster and less expensive, as it is PCR-based. Spoligotyping has been applied to enhance our understanding of the dynamics of drug susceptible and drug resistant strains of Mycobacterium tuberculosis in high incidence communities, by studying 3 aspects of the TB epidemic: molecular epidemiology of drug resistant TB, recurrent TB and the evolution of M. tuberculosis. By using spoligotyping and other genotypic and phenotypic analysis of drug-resistant M. tuberculosis isolates from the Western Cape Province of South Africa showed that drug resistance is widespread and recently transmitted. An emerging drug resistant M. tuberculosis outbreak has been identified, termed DRF150, which has specific genotypic characteristics and is resistant to 5 first-line drugs in 45% of the cases. Inappropriate chemotherapy; poor adherence to treatment and prolonged periods of infectiousness due to the delay in susceptibility testing has led to the development and spread of this drug resistant genotype. The study demonstrates the ability of the spoligotyping technique to accurately determine the pathogenic mechanism of recurrent disease by spoligotyping, making it useful in large-scale intervention studies. Application of spoligotyping and a newly developed PCR-method showed that the occurrence of multiple infections was higher than what was previously assumed and also more frequent in retreatment cases than new cases. These findings have important implications for the understanding of protective immunity, and the development and testing of new vaccines and drugs. Various different molecular markers including spoligotyping has been used to reconstruct the evolutionary history of isolates with less than 6 copies of IS6110 element (termed Low Copy Clade (LCC)), which were previously poor defined. It was also shown that LCC is widely disseminated and play an important role in the global tuberculosis epidemic. Reconstruction of the evolutionary relationship of M. tuberculosis Principal Genetic Group 2 strains, identified previously unknown genetic relationships between strain families and laid the foundation to establish correlations between genotype and phenotype. Spoligotyping signatures, created by evolution of the Direct Repeat region in M. tuberculosis, were identified, which will enable the analysis of the strain population structure in different settings and will also enable the rapid identification of strain families that acquire drug-resistance or escape protective immunity in drug and vaccine trials. This study contributed to our understanding of the molecular epidemiology of drug resistant TB, recurrent TB and the evolution of M. tuberculosis in high incidence communities.