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Background: The Beijing lineage of Mycobacterium tuberculosis (MTB) is the most predominant MTB strain in Asian countries and is spreading worldwide, however, the East AfricanâIndian (EAI) lineage is also particularly prevalent in many tropical Asian countries. The evolutionary relationships among MTB EAI isolates from Taiwan and those of tropical Asian countries remain unknown. Methods: The EAI strains collected from patients in Taiwan were analyzed using spacer oligonucleotide typing and mycobacterial interspersed repetitive unitâvariable number of tandem repeats (MIRU-VNTR) typing, and compared with published profiles from Cambodia and Singapore to investigate potential epidemiological linkages. Results: Among the three countries, the EAI lineage was most prevalent in Cambodia (60%; Singapore, 25.62%; and Taiwan, 21.85%), having also the highest rates of multidrug resistance and lowest rates of clustering of MTB isolates. We describe a convenient method using seven selected MIRU-VNTR loci for first-line typing to discriminate Beijing and EAI lineages. A potential epidemiological linkage in these tropical Asian countries is also discussed based on a minimum-spanning tree constructed using 24 MIRU-VNTR loci of MTB EAI strains. Conclusion: This study identified evolutionary relationships among MTB EAI isolates from Taiwan and those of two other tropical Asian countries, Cambodia and Singapore. Keywords: East AfricanâIndian family, Mycobacterium tuberculosis, tropical Asian countries

Background The tuberculosis (TB) pandemic remains a leading cause of human morbidity and mortality, despite widespread use of the only licensed anti-TB vaccine, bacille Calmette-Guerin (BCG). The protective efficacy of BCG in preventing pulmonary TB is highly variable; therefore, an effective new vaccine is urgently required. Methods In the present study, we assessed the ability of novel recombinant BCG vaccine (rBCG) against Mycobacterium tuberculosis by using modern immunological methods. Results Enzyme-linked immunospot assays demonstrated that the rBCG vaccine, which coexpresses two mycobacterial antigens (Ag85B and CFP10) and human interleukin (IL)-12 (rBCG2) elicits greater interferon-γ (IFN-γ) release in the mouse lung and spleen, compared to the parental BCG. In addition, rBCG2 triggers a Th1-polarized response. Our results also showed that rBCG2 vaccination significantly limits M. tuberculosis H37Rv multiplication in macrophages. The rBCG2 vaccine surprisingly induces significantly higher tumor necrosis factor-α (TNF-α) production by peripheral blood mononuclear cells that were exposed to a nonmycobacterial stimulus, compared to the parental BCG. Conclusion In this study, we demonstrated that the novel rBCG2 vaccine may be a promising candidate vaccine against M. tuberculosis infection.

In total, 303 randomly selected clinical Mycobacterium tuberculosis (MTB) isolates from 303 patients (collected January to December 2012) in central Taiwan were examined. The major lineages found were Beijing (N = 114, 37.62%), Haarlem (N = 76, 25.08%) and East African–Indian (EAI) (N = 42, 13.86%). Notably, younger persons (≤30 years old) were 6.58 times more likely to be infected with a Beijing genotype compared to older persons (>70 years) (p

Summary Previous research revealed a 6-fold higher incidence of tuberculosis (TB) amongst aborigines compared to Han Chinese in Taiwan. To investigate the reasons for this disparity, we genotyped Mycobacterium tuberculosis (MTB) strains obtained from members of different aboriginal tribes in different geographical regions of Taiwan by using molecular methods. In total, 177 isolates of MTB collected from patients at four hospitals in Taiwan from January 2006 to December 2011 were analysed by spoligotyping, mycobacterial interspersed repetitive unit-variable number tandem-repeat (MIRU-VNTR) typing. The most prevalent strains in the eastern and central regions of Taiwan were Beijing (45.7% in eastern) and Haarlem (39.1% in eastern, 37.1% in central) lineages, whereas in southern regions the most prevalent strains were EAI (47.7%) and Haarlem (20.5%) lineages. The high prevalence of EAI in southern Taiwan aborigines may be closely associated with Austronesian culture. This study provides a first overview of the M. tuberculosis strains circulating in aboriginal populations in Taiwan. The high prevalences of certain MTB lineages within aboriginal sub-populations suggest that transmission of MTB may have been restricted to close contacts.

BACKGROUND We present the first comprehensive analysis of Mycobacterium tuberculosis (MTB) isolates circulating in southern Taiwan. In this 9-year population-based study, the TB situation in the Kaohsiung region was characterized by genotypic analysis of 421 MTB isolates. METHODS All 421 isolates of MTB were analyzed by spoligotyping and MIRU-VNTR typing. Drug-resistance patterns were also analyzed. RESULTS The percentage of EAI (East African-Indian) strains increased across sampling years (2000-2008) in southern Taiwan, whereas the proportion of Beijing lineages remained unchanged. Clustering was more frequent with EAI genotype infections (odds ratio = 3.6, p

Mycobacterium tuberculosis (MTB) continues to be a leading cause of human deaths due to an infectious agent. Current efforts are focused on making better TB vaccines. We describe the generation and immunological characterization of recombinant BCG (rBCG). This rBCG was generated by incorporating an expression plasmid encoding two mycobacterial antigens (Ag85B and CFP10) and human interleukin (IL)-12 into a BCG strain. Immunogenicity studies in mice showed that rBCG coexpressing Ag85B, CFP10, and IL-12 (rBCG::Ag85B-CFP10-IL-12) induces a robust immune response in mice. The rBCG vaccine promotes a T-cell response against MTB that is characterized by a high proportion of polyfunctional and memory T cells in spleen and lung. Our results showed strong immunogenicity and mycobacterial growth inhibition of rBCG::Ag85B-CFP10 plus IL-12 than that of BCG vaccine.

We compared mycobacterial interspersed repetitive unit (MIRU)-variable number tandem repeat (VNTR) typing to traditional spoligotyping for discriminating Mycobacterium tuberculosis (MTB) strains. Our 17-loci MIRU-VNTR typing method was found to be superior to spoligotyping for non-Beijing family strains. To extend the method we also established PCR-based rapid genotyping protocols for Beijing, East-African-Indian and U lineages.

The distribution of human Mycobacterium tuberculosis (MTB) genotypes is reportedly associated with geography, ethnicity and population migrations. Three groups of 208 patients with tuberculosis in Taiwan were sampled to test this observation: (1) 41 aborigines of Austronesian ethnicity, who have been inhabiting in Taiwan for more than 500 years; (2) 58 veterans of Han Chinese origin, who moved as the first generation from Mainland China to Taiwan 55-60 years ago; and (3) 109 patients representing the general Taiwanese population of Han Chinese whose ancestors migrated to Taiwan around 200-400 years ago. A total of 208 MTB isolates, one per patient, were analyzed by spoligotyping and mycobacterial interspersed repetitive unit (MIRU) typing. Beijing ancient strains and Haarlem strains predominated among aborigines, while Beijing modern strains were common among veterans and the general population. All Beijing strains were further analyzed by typing the NTF loci and RD deletion. Results suggest a chronological trend among Beijing isolates from the three groups: isolates from the aborigines had signatures compatible with ancient lineages, and those from veterans and the general population were more contemporary. Our data indicate that the distribution of MTB genotypes/strains in Taiwan is associated with different populations whose migratory activities occurred between 55 and 500 years ago. These results suggest that transmission of MTB may have been relatively restricted to close contacts.

Tuberculosis incidence among aborigines is significantly higher than for Han Chinese in Taiwan, but the extent to which Mycobacterium tuberculosis (MTB) strain characteristics contribute to this difference is not well understood. MTB isolates from aborigines and Han Chinese living in eastern and southern Taiwan, the major regions of aborigines, were analyzed by spoligotyping and 24-loci MIRU-VNTR. In eastern Taiwan, 60% of aboriginal patients were ≤20 years old, significantly younger than the non-aboriginal patients there; aborigines were more likely to have clustered MTB isolates than Han Chinese (odds ratio (OR) = 5.98, p

It is unclear to what extent the host-responses elicited by Beijing versus non-Beijing strains of Mycobacterium tuberculosis (MTB) contribute to the predominance of modern Beijing strains in Taiwan and some other Asian countries. The purpose of this study was to compare the expression profiles of virulence-related genes in human monocyte-derived macrophages infected in vitro with Beijing (ancient and modern strains) and non-Beijing strains (EAI strains) of MTB that are epidemic in Taiwan. We found that modern Beijing strains induced lower levels of pro-inflammatory cytokines, whereas EAI strains induced higher levels. Notably, the most prevalent modern Beijing sub-lineage, possessing intact RD150 and RD142 chromosomal regions, induced very low levels of pro-inflammatory cytokines, especially interleukin-1β. Moreover, in an intracellular growth assay, the survival of the same modern Beijing strain in human monocyte-derived macrophages was significantly higher than that of an ancient Beijing strain and an EAI strain. Taken together, these results may explain why modern Beijing strains of MTB predominate in Taiwan.

The Mycobacterium tuberculosis (MTB) Beijing strain is highly virulent, drug resistant, and endemic over Asia. To explore the genetic diversity of this family in several different regions of eastern Asia, 338 Beijing strains collected in Taiwan (Republic of China) were analyzed by mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing and compared with published MIRU-VNTR profiles and by the Hunter-Gaston diversity index (HGDI) of Beijing strains from Japan and South Korea. The results revealed that VNTR2163b (HGDI>0.6) and five other loci (VNTR424, VNTR4052, VNTR1955, VNTR4156 and VNTR 2996; HGDI>0.3) could be used to discriminate the Beijing strains in a given geographic region. Analysis based on the number of VNTR repeats showed three VNTRs (VNTR424, 3192, and 1955) to be phylogenetically informative loci. In addition, to determine the geographic variation of sequence types in MTB populations, we also compared sequence type (ST) data of our strains with published ST profiles of Beijing strains from Japan and Thailand. ST10, ST22, and ST19 were found to be prevalent in Taiwan (82%) and Thailand (92%). Furthermore, classification of Beijing sublineages as ancient or modern in Taiwan was found to depend on the repeat number of VNTR424. Finally, phylogenetic relationships of MTB isolates in Taiwan, South Korea, and Japan were revealed by a minimum spanning tree based on MIRU-VNTR genotyping. In this topology, the MIRU-VNTR genotypes of the respective clusters were tightly correlated to other genotypic characters. These results are consistent with the hypothesis that clonal evolution of these MTB lineages has occurred.

Background The control of tuberculosis in densely populated cities is complicated by close human-to-human contacts and potential transmission of pathogens from multiple sources. We conducted a molecular epidemiologic analysis of 356 Mycobacterium tuberculosis (MTB) isolates from patients presenting pulmonary tuberculosis in metropolitan Taipei. Classical antibiogram studies and genetic characterization, using mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing and spoligotyping, were applied after culture. Methods A total of 356 isolates were genotyped by standard spoligotyping and the strains were compared with in the international spoligotyping database (SpolDB4). All isolates were also categorized using the 15 loci MIRU-VNTR typing method and combin with NTF locus and RD deletion analyses. Results Of 356 isolates spoligotyped, 290 (81.4%) displayed known spoligotypes and 66 were not identified in the database. Major spoligotypes found were Beijing lineages (52.5%), followed by Haarlem lineages (13.5%) and EAI plus EAI-like lineages (11%). When MIRU-VNTR was employed, 140 patterns were identified, including 36 clusters by 252 isolates and 104 unique patterns, and the largest cluster comprised 95 isolates from the Beijing family. The combination of spoligotyping and MIRU-VNTR revealed that 236 (67%) of the 356 isolates were clustered in 43 genotypes. Strains of the Beijing family was more likely to be of modern strain and a higher percentage of multiple drug resistance than other families combined (P = 0.08). Patients infected with Beijing strains were younger than those with other strains (mean 58.7 vs. 64.2, p = 0.02). Moreover, 85.3% of infected persons younger than 25 years had Beijing modern strain, suggesting a possible recent spread in the young population by this family of TB strain in Taipei. Conclusion Our data on MTB genotype in Taipei suggest that MTB infection has not been optimally controlled. Control efforts should be reinforced in view of the high prevalence of the Beijing strain in young population and association with drug resistance.