Molecular characterization of mycobacterium tuberculosis complex isolates in Mozambique

Abstract: Mozambique is one of the high burden tuberculosis (TB) and human immunodeficiency virus (HIV) countries with a prevalence of HIV infection in adults of 11.5% and an estimated TB prevalence of 559 per 100 000 population. Fifty six percent of the TB patients in Mozambique are estimated to be HIV positive. TB control strategies might significantly be affected by differences in virulence, epidemiologic characteristics and epidemiology of particular strains of the Mycobacterium tuberculosis complex. Molecular epidemiology studies allow the identification of circulating strain types, understanding of transmission dynamics, as well as investigations of the evolution of the M. tuberculosis complex. The studies included in this thesis described the molecular epidemiology of M. tuberculosis complex in Mozambique, identified predominant genotypes responsible for TB transmission and prevalence and investigated the association between predominant spoligotypes and HIV sero-status. The prevalence and transmission of the Beijing genotype in Mozambique was also evaluated. With the aim to explore the public health risk for bovine TB, isolates from two sites were investigated, Maputo (tuberculous lymphadenitis or TBLN cases) and Govuro district (TBLN and pulmonary cases), the last site, Govuro, with known high prevalence of bovine TB in cattle (39.6%). Furthermore, a phylogenetic phylogeographic snapshot of worldwide M. tuberculosis complex diversity was created based on the classification of the Multiple-locus variable-number tandem repeat analysis (MLVA). For the first time, the genetic diversity of circulating M. tuberculosis complex strains in Mozambique was described. It was found that the TB epidemic in Mozambique was caused by a wide diversity of spoligotypes with predominance of the Latin-American Mediterranean (LAM, n=165 or 37%); East African-Indian (EAI, n=132 or 29.7%); the evolutionary recent T clade (n=52 or 11.6%) and the globally-emerging Beijing clone (n=31 or 7%). The predominant lineages were also common in neighboring countries, indicating TB transmission by migration from one country to another. The Beijing lineage, distributed worldwide and responsible for large epidemics was found to be particularly common in the Southern region of Mozambique, especially in Maputo City (17%) and associated with HIV infection (p=0.023). By combined use of region of difference (RD) analysis and spacer oligonucleotide typing (spoligotyping), a distinct group of four isolates had deletion of RD150, a signature of the “sublineage 7” recently emerging in South Africa. The same group was very similar to the South African “sublineage 7” by Restriction Fragment Length Polymorphism (RFLP) and Mycobacterial Interspersed Repetitive Units–Variable-Number Tandem Repeat (MIRU-VNTR), suggesting that this sublineage could have been recently introduced in Mozambique from South Africa. No M. bovis was found in TBLN cases from Maputo. It was demonstrated that TBLN in Maputo was caused by a variety of M. tuberculosis genotypes, similar to the ones causing pulmonary TB, suggesting that in Maputo, cases of TBLN arise from the same source as pulmonary TB, rather than from an external zoonotic source. For the first time, evidence of the occurrence of M. bovis in humans in Mozambique was revealed. In a study presently being conducted in the district of Govuro, among six M. tuberculosis complex isolates, one was M. bovis. Nevertheless, further research is needed on cases of abdominal TB and other forms of extrapulmonary TB, in Govuro and in other pastoral areas, where the prevalence of bovine TB in cattle is known to be high, in order to have a better answer about the public health importance of this zoonotic disease in Mozambique.