Molecular epidemiology and deletion analysis of mycobacterium tuberculosis strains associated with pulmonary and extrapulmonary tuberculosis
Date of Award
Doctor of Philosophy in Health Science (PhD)
Biological and Biomedical Sciences
Pakistan ranks 6th amongst the list of 22 high tuberculosis (TB) burden countries with an estimated incidence rate of 231 person and prevalence rate of 364 person per 100,000 population respectively (WHO, 2011). TB remains one of the leading causes of dealth due to infectious diseases in Pakistan and is responsible for 5% of the country's total disease burden (WHO, 2011). Molecular epidemiological studies have shown a predominance of the Central Asian Strainl (CAS1) with Beijing strains constituting 3% of Mycobacterium tuberculosis isolates in Pakistan. M tuberculosis strains display high degree of genomic deletions called large sequence polymorphisms (LSPs) or region of differences (RDs) causing genetic variation. However, the role of LSPs in pathogenicity amongst Al tuberculosis strains is still not clear. The overall aims of this study were to explore the frequency of Region of Differences (RDs) genomic deletions amongst strain prevalent in Pakistan including CAS1, CAS subfamily and Beijing spoligotypes. The presence of commonly reported RD deletions, RD1, RD750, RD207, RD149, RD152, RD105, RD 150, RD142 and RD181 was investigated using a PCR based method. The biological relevance of RD149 and concurrent RD149-RD152 deletions on CAS1 strains was explored using growth in broth as well as growth and cytokine induction in THP-1 cell line model. RD genomic deletion analysis was performed on 235 M tuberculosis (185 pulmonary; 50 extrapulmonary) strains, including 171 CAS strains (133 CAS1 (ST26), 38 CAS subfamily), 8 Beijing isolates, 47 isolates belonging to other previously defined ("Other") clusters, and 9 previously undefined Unique isolates. All the 133 CAST isolates studied had RD750 deletions. RDI49 and RD152 deletions were observed in 39.8% (n=53) and 21.8 % (n=29) respectively, whereas concurrent RD149-RD152 deletions were observed in 18.79% (n=25) of CAS 1 strains. All the 38 CAS subfamily isolates were characterized by a RD750 deletion; 21.05% (n=8) had RD149 deletions and 21.05% (n=8) had RD152 deletions. No deletions in RDI, RD207, RD105, RD150, RD142 and RD181 regions were observed in CAS1 and CAS subfamily isolates. All Beijing isolates (n=8) showed RD207, RD149, RD152, RD105 and RD150 deletions. RD142 and RD181 deletions however, were observed in 62.5% (n=5) and 87.5% (ip--7) of Beijing strains respectively. Of the "Other" cluster strains, 29.8% (n=14) had RD149 deletions, followed by 2.13% (n=1) with RD152 deletions. No deletions of studied RDs were observed in the Unique strains (n=9). CASI strains showed more frequent RD149 deletions as compared with the CAS subfamily (p=0.036) and more frequent RD152 deletions compared with "Other" cluster (p=0.0003). RD149 and RD152 deletions were more frequent in Beijing isolates compared with CAST strains (p<0.001). Concurrent RD149 and RD152 deletions were more frequent in CAS] compared with "Other" clusters (p<0.001) and in Beijing strains compared with CAST (p<0.001). Given the fact that CAS1 is the prevalent spoligotype in this region, biological significance of RD149 and concurrent RD149 and RDI52 deletions in these strains was investigated. Growth of CAS1 strains with deletions was slower in broth (RD149, p=0.024 and RD149-RD152, p=0.025) than that of strains without deletions. CAST strains with RD149 deletions further showed reduced intracellular growth (1)=0.013) as compared with CAS 1 strains without deletions and also as compared to H37Rv (p=0.007) and CAS1 strains with concurrent RD149-RD152 deletion (1)=0.029). All CAS1 strains induced higher levels of TNFa and ILI 0 secretion in THP-1 cells than H37Rv. In addition, CAS I strains with RD149 deletions induced more TNFa secretion than CAS1 strains without deletions (p=0.013). CAS1 RD149 deletion strains from extrapulmonary sources showed more rapid intracellular growth (p=0.015) and lower levels of TNFa (p=0.01), CCL2 (p=0.003) and IL6 (p=0.008) secretion as compared with strains from pulmonary sources. These data suggest that the presence of RD149 deletion in CAS1 strains is associated with reduced growth and increased TNFct induction in host cells possibly, reducing strain virulence. The differences observed for extrapulmonary strains may indicate an adaptation that increases their potential for dissemination and tropism outside the lung. Overall, RD149 deletions result in genetic diversity within strains and could have an important effect on interactions with host cells with important clinical consequences.
Kanji, A. A. (2012). Molecular epidemiology and deletion analysis of mycobacterium tuberculosis strains associated with pulmonary and extrapulmonary tuberculosis (Unpublished doctoral thesis). Aga Khan University, Karachi, Pakistan.