Horng-Yunn Dou

Molecular epidemiology and evolutionary genetics of Mycobacterium tuberculosis in Taipei

BackgroundThe 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.MethodsA 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.ResultsOf 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.ConclusionOur 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.

Dissemination of multidrug-resistant Acinetobacter baumannii carrying BlaOxA-23 from hospitals in central Taiwan

BACKGROUND Imipenem-resistant Acinetobacter baumannii (IRAB) poses a great threat to healthcare systems. Production of carbapenem-hydrolyzing class D β-lactamases (CHDLs) is the major mechanism for imipenem resistance. In this study, we found a high prevalence of IRAB carrying a gene encoding CHDL, bla(OxA-23), in central Taiwan and elucidated the molecular characteristics and possible mechanisms of the spread of these isolates. METHODS During 2007, we collected 291 nonrepetitive A baumannii isolates from 10 teaching hospitals in Taiwan. The antimicrobial susceptibility of the isolates was determined by agar dilution or Etest. The genes encoding carbapenemase and related structure were detected by polymerase chain reaction mapping and sequencing, and the clonal relationship of the isolates was analyzed by pulsed-field gel electrophoresis. Plasmid localization of bla(OxA-23) was determined by extraction of plasmid with commercial kit and Southern blot analysis. RESULTS Among 142 IRAB isolates, 30 harbored the bla(OxA-23). The prevalence of IRAB with bla(OxA-23) was highest in central Taiwan compared to other areas [24.8% (27/109) vs. 1.6% (3/182); p < 0.001]. These IRAB with bla(OxA-23) were also resistant to other antimicrobial agents, except colistin. The PCR methods showed the presence of bla(OxA-51) in all isolates. We could exclude clonal spreading due to the diversity of the pulsotype. The bla(OxA-23) gene was detected in the plasmids of 6 isolates. Tn2006 was present in 22 (73.3%) isolates, and Tn2008, in 6 other isolates (26.7%). Two strains had bla(oxa-23)-ΔATPase but lacked upstream ISAba1. CONCLUSION The high prevalence of bla(OxA-23)-harboring IRAB in central Taiwan might be attributed to the transposition event of Tn2006.

Genetic diversity of the Mycobacterium tuberculosis Beijing family based on SNP and VNTR typing profiles in Asian countries.

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.

Associations of Mycobacterium tuberculosis genotypes with different ethnic and migratory populations in Taiwan.

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.

High prevalence of latent tuberculosis infection in dialysis patients using the interferon-{gamma} release assay and tuberculin skin test

BACKGROUND AND OBJECTIVES Patients in ESRD on hemodialysis with latent tuberculosis (TB) infection have 10 to 25 times the risk of reactivation into active disease compared with healthy adults. This study investigates the prevalence of latent TB infection in dialysis patients from a country with an intermediate burden of TB and its associated risk factors using the QuantiFERON-TB Gold in-tube test (QGIT) and the tuberculin skin test (TST). DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS This was a prospective, cross-sectional study performed at a medical center in Taiwan on dialysis patients. Each patient underwent QGIT, two-step TST using 2 tuberculin units (TU) of PPD RT-23, a chest x-ray to exclude active TB, and an interview to determine TB risk factors. RESULTS Ninety-three of 190 eligible patients were enrolled: 35 men and 58 women. 64.8% were vaccinated with the Bacille-Calmette-Guérin (BCG) vaccination. Overall, 34.4% were positive by QGIT and 10.8% were indeterminate. Using a 10-mm TST cutoff, 53.9% were positive. There was poor correlation between TST and QGIT at any TST cutoff criteria. There was a significant increasing trend of QGIT positivity with age in those younger than 70 years, and, conversely, a decreasing trend of TST reactivity with age. Significant risk factors for QGIT positivity included age and past TB disease. CONCLUSIONS This study shows a high prevalence of latent TB infection in dialysis patients in a country with an intermediate burden of TB. QGIT in dialysis patients correlated better than TST with the risk of TB infection and past TB disease.

The pattern of cytokine production in vitro induced by ancient and modern Beijing Mycobacterium tuberculosis strains.

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.

Recombinant BCG coexpressing Ag85B, CFP10, and interleukin-12 induces multifunctional Th1 and memory T cells in mice

Lin C‐W, Su I‐J, Chang J‐R, Chen Y‐Y, Lu J‐J, Dou H‐Y. Recombinant BCG coexpressing Ag85B, CFP10, and interleukin‐12 induces multifunctional Th1 and memory T cells in mice. APMIS 2012; 120: 72–82.

Clonal Expansion of Both Modern and Ancient Genotypes of Mycobacterium tuberculosis in Southern Taiwan

We present the first comprehensive analysis of Mycobacterium tuberculosis isolates circulating in the Kaohsiung region of southern Taiwan. The major spoligotypes found in the 224 isolates studied were Beijing lineages (n = 97; 43.3%), EAI lineages (n = 72; 32.1%) and Haarlem lineages (n = 18; 8.0%). By 24 MIRU-VNTR typing, 174 patterns were identified, including 24 clusters of 74 isolates and 150 unique patterns. The combination of spoligotyping and 12-MIRU-VNTR revealed that 129 (57.6%) of the 224 isolates were clustered in 18 genotypes. Moreover, 63.6% (7/11) of infected persons younger than 30 years had a Beijing strain, which could suggest recent spread among younger persons by this family of TB strains in Kaohsiung. Among the 94 Beijing family (SIT1, SIT250 and SIT1674) isolates further analyzed for SNPs by mass spectrometry, the most frequent strain found was ST10 (n = 49; 52%), followed by ST22 (n = 17; 18%) and ST19 (n = 11; 12%). Among the EAI-Manila family isolates analyzed by region deletion-based subtyping, the most frequent strain found was RD type 1 (n = 63; 87.5%), followed by RD type 2 (n = 9; 12.5%). In our previous study, the proportion of modern Beijing strains (52.5%) in northern Taiwan was significantly higher than the proportion of EAI strains (11%). In contrast, in the present study, EAI strains comprised up to 32% of Beijing strains in southern Taiwan. In conclusion, both ‘modern’ (Beijing) and ‘ancient’ (EAI) M. tuberculosis strains are prevalent in the Kaohsiung region, perhaps suggesting that both strains are somehow more adapted to southern Taiwan. It will be interesting to investigate the dynamics of the lineage composition by different selection pressures.

Molecular epidemiology of Mycobacterium tuberculosis in aboriginal peoples of Taiwan, 2006–2011

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.

Prevalence of Mycobacterium tuberculosis in Taiwan: A Model for Strain Evolution Linked to Population Migration

The global evolution and spread of Mycobacterium tuberculosis (MTB), one of the most successful bacterial pathogens, remain a mystery. Advances in molecular technology in the past decade now make it possible to understand MTB strain evolution and transmission in the context of human population migration. Taiwan is a relatively isolated island, serving as a mixing vessel over the past four centuries as colonization by different waves of ethnic groups occurred. By using mycobacterial tandem repeat sequences as genetic markers, the prevalence of MTB strains in Taiwan revealed an interesting association with historical migrations of different ethnic populations, thus providing a good model to explore the global evolution and spread of MTB.