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In settings with high tuberculosis (TB) endemicity, various genotypes of the Mycobacterium tuberculosis complex (MTBC) often differ in prevalence. However, the factors leading to these differences remain poorly understood. Here we studied the MTBC population in Dar es Salaam, Tanzania over a six-year period, using 1,082 unique patient-derived MTBC whole-genome sequences (WGS) and associated clinical data. We show that the TB epidemic in Dar es Salaam is dominated by multiple genotypes introduced to Tanzania from different parts of the world during the last 300 years. The most common MTBC genotypes deriving from these introductions exhibited differences in transmission rates and in the duration of the infectious period, but little differences in overall fitness, as measured by the effective reproductive number. Moreover, measures of disease severity and bacterial load indicated no differences in virulence between these genotypes during active TB. Instead, the combination of an early introduction and a high transmission rate accounted for the high prevalence of L3.1.1, the most dominant MTBC genotype in our setting. Yet, a longer co-existence with the host population did not always result in a higher transmission rate, suggesting that distinct life-history traits have evolved in the different MTBC genotypes. Taken together, our results point to bacterial factors as important determinants of the TB epidemic in Dar es Salaam.Author summaryTuberculosis (TB) is the deadliest human infectious disease caused by one single agent, Mycobacterium tuberculosis (Mtb). The origins of Mtb have been traced to East Africa millennia ago, where it likely became adapted to infect and transmit in humans. Here we show that in Dar es Salaam, Tanzania, an East African setting with a very high burden of TB, infections are caused by distinct Mtb genotypes introduced in recent evolutionary times from different parts of the world. These genotypes differed in traits important to Mtb transmission in the Dar es Salaam host population; while some Mtb genotypes transmitted more efficiently during a certain period of time, others elicited that patients would be infectious for longer periods. These traits evolved independently in the different Mtb genotypes and could not be explained by the time of co-existence between the host population and the pathogen. This suggests that bacterial factors are important determinants of the TB epidemic. More generally, we demonstrate that distinct pathogenic life history characteristics can co-exist in one host population.

A major challenge in the use of DNA vaccines is efficient DNA delivery in vivo. Establishing a safe and efficient electric transfer method is the key to developing rapid DNA vaccines against emerging infectious diseases. To overcome the complexity of designing new electric transfer machines for DNA delivery, a clinically approved electric transfer machine could be considered as an alternative. Here, we report an electroacupuncture machine-based method for DNA vaccine delivery after intramuscular injection of the COVID-19 DNA vaccine. The S gene of SARS-CoV-2 in the pVAX1 plasmid (pSARS2-S) was used as an antigen in this study. We optimized the clinically used electroacupuncture machine settings for efficient induction of the neutralizing antibody titer after intramuscular injection of pSARS2-S in mice. We found that pSARS2-S immunization at 40 Vpp for 3–5 s could induce high neutralizing antibody titers and Th1-biased immune responses. IFN-γ/TNF-α-secreting CD4+ and CD8+ T cells were also observed in the DNA vaccination group but not in the recombinant protein vaccination group. T-cell epitope mapping shows that the major reactive epitopes were located in the N-terminal domain (a.a. 261–285) and receptor-binding domain (a.a. 352–363). Importantly, pSARS2-S immunization in hamsters could induce protective immunity against SARS-CoV-2 challenge in vivo. In the preclinical toxicology study, blood biochemistry, hematology, and DNA persistence analysis reveal that the DNA delivery method is safe. Furthermore, the raised antisera could also cross-neutralize different variants of concern. These findings suggest that DNA vaccination using an electroacupuncture machine is feasible for use in humans in the future.

A series of recombinant human type 5 adenoviruses that express the full-length or membrane-truncated spike protein (S) of SARS-CoV-2 (AdCoV2-S or AdCoV2-SdTM, respectively) was tested the efficacy against SARS-CoV-2 via intranasal (i.n.) or subcutaneous (s.c.) immunization in a rodent model. Mucosal delivery of adenovirus (Ad) vaccines could induce anti-SARS-CoV-2 IgG and IgA in the serum and in the mucosal, respectively as indicated by vaginal wash (vw) and bronchoalveolar lavage fluid (BALF). Serum anti-SARS-CoV-2 IgG but not IgA in the vw and BALF was induced by AdCoV2-S s.c.. Administration of AdCoV2-S i.n. was able to induce higher anti-SARS-CoV-2 binding and neutralizing antibody levels than s.c. injection. AdCoV2-SdTM i.n. induced a lower antibody responses than AdCoV2-S i.n.. Induced anti-S antibody responses by AdCoV2-S via i.n. or s.c. were not influenced by the pre-existing serum anti-Ad antibody. Novelty, S-specific IgG1 which represented Th2-mediated humoral response was dominantly induced in Ad i.n.-immunized serum in contrast to more IgG2a which represented Th1-mediated cellular response found in Ad s.c.-immunized serum. The activation of S-specific IFN-ɣ and IL-4 in splenic Th1 and Th2 cells, respectively, was observed in the AdCoV2-S i.n. and s.c. groups, indicating the Th1 and Th2 immunity were activated. AdCoV2-S and AdCoV2-SdTM significantly prevented body weight loss and reduced pulmonary viral loads in hamsters. A reduction in inflammation in the lungs was observed in AdCoV-S via i.n. or s.c.-immunized hamsters following a SARS-CoV-2 challenge. It correlated to Th1 cytokine but no inflammatory cytokines secretions found in AdCoV-S i.n. -immunized BALF. These results indicate that intranasal delivery of AdCoV2-S vaccines is safe and potent at preventing SARS-CoV-2 infections.

Shiu-Ju Yang,1 Ting-Chun Wei,1 Chih-Hao Hsu,1 Sin-Ni Ho,1 Chi-Yun Lai,2 Shiu-Feng Huang,2,3 Yih-Yuan Chen,4 Shih-Jen Liu,1 Guann-Yi Yu,1 Horng-Yunn Dou1,5 1National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan, Miaoli, 35053, Taiwan; 2Pathology Core Laboratory, National Health Research Institutes, Zhunan, Miaoli, 35053, Taiwan; 3National Institute of Molecular and Genomic Medicine, National Health Research Institutes, Zhunan, Miaoli, 35053, Taiwan; 4Department of Biochemical Science and Technology, National Chiayi University, Chia-Yi, 60070, Taiwan; 5Department of Biological Science & Technology, National Yang Ming Chiao Tung University, Hsinchu, TaiwanCorrespondence: Horng-Yunn DouNational Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, 35, Keyan Road, Zhunan Town, Miaoli County, 35053, TaiwanTel +886-37-206-166 ext 35529Fax +886-37-583-009Email hydou@nhri.edu.twBackground: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus which caused a global respiratory disease pandemic beginning in December 2019. Understanding the pathogenesis of infection and the immune responses in a SARS-CoV-2-infected animal model is urgently needed for vaccine development.Methods: Syrian hamsters (Mesocricetus auratus) were intranasally inoculated with 105, 5× 105, and 106 TCID50 of SARS-CoV-2 per animal and studied for up to 14 days. Body weight, viral load and real-time PCR amplification of the SARS-CoV-2 N gene were measured. On days 3, 6 and 9, lung, blood, liver, pancreas, heart, kidney, and bone marrow were harvested and processed for pathology, viral load, and cytokine expression.Results: Body weight loss, increased viral load, immune cell infiltration, upregulated cytokine expression, viral RNA, SARS-CoV-2 nucleoprotein, and mucus were detected in the lungs, particularly on day 3 post-infection. Extremely high expression of the pro-inflammatory cytokines MIP-1 and RANTES was detected in lung tissue, as was high expression of IL-1β, IL-6, IL-12, and PD-L1. The glutamic oxalacetic transaminase/glutamic pyruvic transaminase (GOT/GPT) ratio in blood was significantly increased at 6 days post-infection, and plasma amylase and lipase levels were also elevated in infected hamsters.Conclusion: Our results provide new information on immunological cytokines and biological parameters related to the pathogenesis and immune response profile in the Syrian hamster model of SARS-CoV-2 infection.Keywords: SARS-CoV-2, hamster, immune response, pathogenesis

The development of efficient vaccines against COVID-19 is an emergent need for global public health. The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major target for the COVID-19 vaccine. To quickly respond to the outbreak of the SARS-CoV-2 pandemic, a nucleic acid-based vaccine is a novel option, beyond the traditional inactivated virus vaccine or recombinant protein vaccine. Here, we report a DNA vaccine containing the spike gene for delivery via electroporation. The spike genes of SARS-CoV and SARS-CoV-2 were codon optimized for mammalian cell expression and then cloned into mammalian cell expression vectors, called pSARS-S and pSARS2-S, respectively. Spike protein expression was confirmed by immunoblotting after transient expression in HEK293T cells. After immunization, sera were collected for antigen-specific antibody and neutralizing antibody titer analyses. We found that both pSARS-S and pSARS2-S immunization induced similar levels of antibodies against S2 of SARS-CoV-2. In contrast, only pSARS2-S immunization induced antibodies against the receptor-binding domain of SARS-CoV-2. We further found that pSARS2-S immunization, but not pSARS-S immunization, could induce very high titers of neutralizing antibodies against SARS-CoV-2. We further analyzed SARS-CoV-2 S protein-specific T cell responses and found that the immune responses were biased toward Th1. Importantly, pSARS2-S immunization in hamsters could induce protective immunity against SARS-CoV-2 challenge in vivo. These data suggest that DNA vaccination could be a promising approach for protecting against COVID-19., Author summary SARS-CoV-2 continues to threaten global health, and development of SARS-CoV-2 vaccine is an urgent priority to halt COVID-19 epidemic. Here, we developed DNA vaccines containing the spike gene from SARS-CoV-2 and delivered via electroporation. We observed that the DNA vaccine induced a long-lasting humoral response against SARS-CoV-2, and the elicited serum antibodies efficiently blocked the binding of SARS-CoV-2 RBD to its entry receptor (ACE2). Importantly, the DNA vaccine induced strong Th1-biased immune responses against SARS-CoV-2, which could provide protective effect with low side effects. Finally, we demonstrated that SARS-CoV-2 DNA vaccine conferred protective efficacy against SARS-CoV-2 infection in Syrian hamsters. Our findings suggested that DNA vaccination could be a useful approach for controlling COVID-19 pandemic in near future.

Analysis of single nucleotide substitutions (SNS) in nucleic acids is the basis for the diagnosis of drug-resistant pathogens as well as human genetic disorders among a broad range of other applications. However, SNS are often inaccessible for the analysis by hybridization probe due to the location in double stranded regions of folded single stranded DNA or RNA analytes. Here we introduce an electrochemical sensor that is able to analyze SNS in folded nucleic acids. The electrochemical sensor takes advantage of a universal electrode-bound hairpin (UTH). It relies on the recognition of targeted nucleic acids by analyte-specific adaptor strands R and L that also hybridize with UTH. Strand L can bind several methylene blue (MeB)-probes thus placing MeB groups close to the electrode surface, which enables cathodic charge transfer. We demonstrated that the same UTH-functionalized electrode and MeB-probe can be used to analyze DNA analytes with different sequences. The sensor is highly selective toward SNS even at room temperature and can be regenerated for next round by rinse with water. The ability of the sensor to analyze SNS within secondary structure folded DNA was demonstrated. The sensing system is capable of detecting SNS in bacterial DNA, useful to differentiate drug-resistant from drug-susceptible mycobacterium tuberculosis. The proposed platform uniquely combines high SNS selectivity with ability to analyze potentially any DNA or RNA sequence including those folded in stable structures, therefore, creates a basis for a cost efficient electrochemical sensing of nucleic acids applicable both in medical diagnostics and environmental monitoring of microorganisms.

Pulmonary tuberculosis (TB) is a difficult-to-eliminate disease. Although the Bacille Calmette-Guerin (BCG) vaccine against Mycobacterium tuberculosis (MTB) has been available for decades, its efficacy is variable and has lessened over time. Furthermore, the BCG vaccine no longer protects against newly emerged Beijing strains which are responsible for many current infections in adults. Development of a novel vaccine is urgently needed. In this study, we first tested the efficacy of our recombinant BCG vaccines rBCG1 and rBCG2, compared to parental BCG, against MTB strain H37Ra in mice. Both the bacterial load and the level of lymphocyte infiltration decreased dramatically in the three groups treated with vaccine, especially rBCG1 and rBCG2. Furthermore, the Th1 and Th17 responses increased and macrophage numbers rose in the vaccination groups. Th1-mediated production of cytokines TNF-α, IFN-γ, and MCP-1 as well as M1-polarized cells all increased in lung tissue of the rBCG1 and rBCG2 groups. Clodronate-induced depletion of macrophages reduced the level of protection. Based on these results, we conclude that rBCG vaccines induce a significant increase in the number of M1 macrophages, which augments their potential as TB vaccine candidates.

24 páginas, 3 figuras, 1 tabla. The sequence data generated by this study has been deposited on SRA (https://www.ncbi.nlm.nih.gov/sra) under the accession number PRJNA670836. Extended data is available here: https://github.com/fmenardo/MTBC_L1_L3. DOI: https://doi.org/10.5281/zenodo.4609804 (Menardo, 2021)., Background: Lineage 1 (L1) and 3 (L3) are two lineages of the Mycobacterium tuberculosis complex (MTBC) causing tuberculosis (TB) in humans. L1 and L3 are prevalent around the rim of the Indian Ocean, the region that accounts for most of the world's new TB cases. Despite their relevance for this region, L1 and L3 remain understudied. Methods: We analyzed 2,938 L1 and 2,030 L3 whole genome sequences originating from 69 countries. We reconstructed the evolutionary history of these two lineages and identified genes under positive selection. Results: We found a strongly asymmetric pattern of migration from South Asia toward neighboring regions, highlighting the historical role of South Asia in the dispersion of L1 and L3. Moreover, we found that several genes were under positive selection, including genes involved in virulence and resistance to antibiotics. For L1 we identified signatures of local adaptation at the esxH locus, a gene coding for a secreted effector that targets the human endosomal sorting complex, and is included in several vaccine candidates. Conclusions: Our study highlights the importance of genetic diversity in the MTBC, and sheds new light on two of the most important MTBC lineages affecting humans., This work was supported by the Swiss National Science Foundation (grants 310030_188888, CRSII5_177163, IZRJZ3_164171 and IZLSZ3_170834) and the European Research Council (309540 EVODRTB and 883582-ECOEVODRTB).

We developed a series of recombinant human type 5 adenoviruses that express the full-length or membrane-truncated spike protein (S) of SARS-CoV-2 (AdCoV2-S or AdCoV2-SdTM, respectively). We tested the immunoprotective efficacy against SARS-CoV-2 via intranasal (i.n.) or subcutaneous (s.c.) immunization in a rodent model following two-dose immunizations. Mucosal delivery of adenovirus (Ad) vaccines could induce anti-SARS-CoV-2 IgG and IgA in the serum and in the mucosal, respectively as indicated by vaginal wash (vw). Serum anti-SARS-CoV-2 IgG but not IgA was induced in the vw by s.c. injection of AdCoV2-S. Intranasal administration of AdCoV2-S was able to induce higher anti-SARS-CoV-2 antibody levels than s.c. injection. Immunization with AdCoV2-SdTM induced a lower antibody response than AdCoV2-S. In addition, the degree of neutralization of clinically isolated SARS-CoV-2 in the serum correlated with the above anti-SARS-CoV-2 responses; the most potent neutralizing activity was observed in the AdCoV2-S i.n. group, and less viral neutralizing activity was observed in response to AdCoV2-S s.c. and AdCoV2dTM i.n. Novelty, S-specific IgG1 which represented Th2-mediated humoral response was dominantly induced in Ad i.n.-immunized serum in contrast to more IgG2a which represented Th1-mediated cellular response found in Ad s.c.-immunized serum. The activation of S-specific IFN-ɣ and IL-4 in Th1 and Th2 cells, respectively, was observed in the AdCoV2s i.n. and s.c. groups, indicating the Th1/Th2-balenced immunity was activated. During the protection study, two doses of i.n. AdCoV2-S or i.n. AdCoV2-SdTM significantly prevented body weight loss and reduced pulmonary viral loads in hamsters. A significant reduction in inflammation in the lungs was observed in AdCoV-S-immunized hamsters following a SARS-CoV-2 challenge. It correlated to Th1 cytokine but no inflammatory cytokines secretions found in i.n. AdCoV-immunized respiratory tract. These results indicate that intranasal delivery of AdCoV2-S vaccines is safe and potent at preventing SARS-CoV-2 infections.

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.

ObjectiveTo investigate the association between diabetes and latent tuberculosis infections (LTBI) in high TB incidence areas.DesignCommunity-based comparison study.SettingOutpatient diabetes clinics at 4 hospitals and 13 health centres in urban and rural townships. A community-based screening programme was used to recruit non-diabetic participants.ParticipantsA total of 2948 patients with diabetes aged older than 40 years were recruited, and 453 non-diabetic participants from the community were enrolled.Primary and secondary outcome measuresThe interferon-gamma release assay (IGRA) and the tuberculin skin test were used to detect LTBI. The IGRA result was used as a surrogate of LTBI in logistic regression analysis.ResultsDiabetes was significantly associated with LTBI (adjusted OR (aOR)=1.59; 95% CI 1.11 to 2.28) and age correlated positively with LTBI. Many subjects with diabetes also had additional risk factors (current smokers (aOR=1.28; 95% CI 0.95 to 1.71), comorbid chronic kidney disease (aOR=1.26; 95% CI 1.03 to 1.55) and history of TB (aOR=2.08; 95% CI 1.19 to 3.63)). The presence of BCG scar was protective (aOR=0.66; 95% CI 0.51 to 0.85). Duration of diabetes and poor glycaemic control were unrelated to the risk of LTBI.ConclusionThere was a moderately increased risk of LTBI in patients with diabetes from this high TB incidence area. This finding suggests LTBI screening for the diabetics be combined with other risk factors and comorbidities of TB to better identify high-risk groups and improve the efficacy of targeted screening for LTBI.

Mycobacterium tuberculosis (TB) infection poses a huge challenge worldwide. It is challenging for rapid identification of TB due to its low growth rate, and therefore it could be widely spread. For this reason, a novel combination of propidium monoazide (PMA) and real-time polymerase chain reaction (RT-PCR) was reported in this work for fast detection of live TB. Experimental results showed that the PMA-RT-PCR protocol can detect live TB with a detection limit as low as 14 colony formation units (CFU)/reaction by measuring the threshold cycle number (Ct value) in a calibration curve. Furthermore, PMA could be used successfully to distinguish dead TB such that no PCR products could be generated. More importantly, all the procedure could be finished within 2 hours. This is the first time that an integrated microfluidic system was demonstrated to use molecular diagnostics for fast detection of TB and could be potentially used for prognosis monitoring of TB treatment.

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

Background The emergence of imipenem-nonsusceptible (INS) Acinetobacter baumannii complex has had a great impact on healthcare systems worldwide. Understanding the risk factors related to INS infection is useful for infection control. The risk factors for INS A. baumannii have been well documented; however, the risk factors related to INS Acinetobacter nosocomialis infection lack documentation. The purpose of this study was to identify the risk factors associated with INS A. nosocomialis bacteremia. Methods This retrospective 9-year study included 329 adults with A. nosocomialis bacteremia in a tertiary medical center in Taiwan. Acinetobacter nosocomialis was identified using a multiplex polymerase chain reaction method and sequence analysis of a 16S–23S intergenic spacer. Results Among 329 patients with A. nosocomialis bacteremia, 67 had INS isolates (20.4%). Patients with INS isolates tended to have a more severe form of the diseases [with ICU admission and a higher APACHE (Acute Physiology and Chronic Health Evaluation) II score], specific underlying diseases (associated with chronic lung diseases and end-stage renal diseases, but less commonly alcoholism and chemotherapy), multiple invasive procedures, pneumonia as a primary focus of infection, and prior antimicrobial use (sulbactam, antipseudomonal penicillins, aminoglycosides, and carbapenems). Multivariable analysis showed that ICU admission, chronic lung diseases, arterial line catheterization, total parenteral nutrition, and prior use of carbapenems were independent risk factors; prior use of carbapenems was found to be the most influential (odds ratio 6.36, 95% confidence interval 2.00–20.21; p = 0.002). Conclusion To our knowledge, this is the first study describing the risk factors associated with INS A. nosocomialis bacteremia. Regulated antibiotic control policy, especially for carbapenem, and infection control measures targeting patients hospitalized in ICU, with chronic lung diseases and multiple invasive procedures, may be helpful in reducing INS A. nosocomialis infection.

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/Purpose Tuberculosis (TB) is endemic in Taiwan and usually affects the lung, spinal TB accounting for 1–3% of all TB infections. The manifestations of spinal TB are different from those of pulmonary TB. The purpose of this study was to define the epidemiological molecular types of mycobacterial strains causing spinal TB. Methods We retrospectively reviewed the medical charts of adult patients diagnosed with spinal TB from January 1998 to December 2007. Patients with positive culture results and/or pathological findings characteristic of TB were enrolled in this study. Spoligotyping was performed to type the Mycobacterium tuberculosis isolates. Results A total of 38 patients with spinal TB were identified. Their mean age was 68 years, and their median duration of symptoms was 60 days (range 3–720 days). The lumbar and thoracic spine accounted for 76% of the sites involved. Thirteen specimens (from seven male and six female patients) were available for typing. Spoligotyping of these 13 specimens revealed three Beijing (23%) and 10 non-Beijing types (77%). The non-Beijing types included two EAI2 Manilla (15%), two H3 (15%), two unclassified (15%), and one each of BOVIS1, U, T2, and orphan type. No significant predominant strain was found in this study, and no drug-resistant Beijing strains were identified. Conclusion TB spondylitis was found to occur in older patients. Spoligotyping results showed that most of the TB spondylitis cases were caused by non-Beijing type Mycobacterium tuberculosis .

To better understand the transmission and evolution of Mycobacterium tuberculosis (MTB) in Taiwan, six different MTB isolates (representatives of the Beijing ancient sublineage, Beijing modern sublineage, Haarlem, East-African Indian, T1, and Latin-American Mediterranean (LAM)) were characterized and their genomes were sequenced. Discriminating among large sequence polymorphisms (LSPs) that occur once versus those that occur repeatedly in a genomic region may help to elucidate the biological roles of LSPs and to identify the useful phylogenetic relationships. In contrast to our previous LSP-based phylogeny, the sequencing data allowed us to determine actual genetic distances and to define precisely the phylogenetic relationships between the main lineages of the MTB complex. Comparative genomics analyses revealed more nonsynonymous substitutions than synonymous changes in the coding sequences. Furthermore, MTB isolate M7, a LAM-3 clinical strain isolated from a patient of Taiwanese aboriginal origin, is closely related to F11 (LAM), an epidemic tuberculosis strain isolated in the Western Cape of South Africa. The PE/PPE protein family showed a higher dn/ds ratio compared to that for all protein-coding genes. Finally, we found Haarlem-3 and LAM-3 isolates to be circulating in the aboriginal community in Taiwan, suggesting that they may have originated with post-Columbus Europeans. Taken together, our results 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.

Mycobacterium tuberculosis strain W06, analyzed by molecular methods, was classified as a modern Beijing M. tuberculosis strain, the most predominant strain in Taiwan. To our knowledge, this is the first draft genome announcement of a Beijing M. tuberculosis strain in Taiwan.

Most Mycobacterium tuberculosis rifampin-resistant strains have been associated with mutations in an 81-bp rifampin resistance-determining region (RRDR) in the gene rpoB . However, if this region alone were targeted, rifampin-resistant strains with mutations outside the RRDR would not be detected. In this study, among 51 rifampin-resistant clinical isolates analyzed by sequencing 1,681-bp-long DNA fragments containing the RRDR, 47 isolates contained mutations within the RRDR, three isolates contained mutations both within and outside the RRDR, and only one isolate had a single missense mutation (Arg548His) located outside the RRDR. A drug susceptibility test of recombinant Mycobacterium smegmatis and M. tuberculosis isolates carrying mutated rpoB (Arg548His) showed an increased MIC for rifampin compared to that of the control strains. Modeling of the Arg548His mutant RpoB-DNA complex revealed that the His548 side chain formed a more stable hydrogen bond structure than did Arg548, reducing the flexibility of the rifampin-resistant cluster II region of RpoB, suggesting that the RpoB Arg548His mutant does not effectively interact with rifampin and results in bacterial resistance to the drug. This is the first report on the relationship between the mutation in codon 548 of RpoB and rifampin resistance in tuberculosis. The novel mutational profile of the rpoB gene described here will contribute to the comprehensive understanding of rifampin resistance patterns and to the development of a useful tool for simple and rapid drug susceptibility tests.

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

The recurrence rate of Mycobacterium tuberculosis in Taiwan is 3%. Here, we present the draft genome sequences of M. tuberculosis strains A2 and A4 from a relapse patient. The draft genome sequences comprise 4,443,031 bp and 4,487,096 bp, revealing 4,220 and 4,143 coding sequences for A2 and A4, respectively, as well as 49 tRNA genes for the both isolates.

Tuberculosis remains a major infectious disease in Taiwan. Here we present the draft genome sequence of the Mycobacterium tuberculosis C2 strain, belonging to the Latin American–Mediterranean lineage. The draft genome sequence comprises 4,453,307 bp with a G+C content of 65.6%, revealing 4,390 coding genes and 45 tRNA genes.

The T cell receptor (TCR) is a heterodimeric molecule expressed on the surface of T cells and recognizes foreign peptides presented by the major histocompatibility complex on the surface of antigen-presenting cells or virus-infected cells. Analysis of TCR usage by T cells which recognize hepatitis B virus (HBV) provides further insight into the participation of T cell populations during the course of disease. In this study, we examined the T-cell-proliferative response and the TCR Vbeta gene usage of peripheral blood mononuclear cells in 3 patients with clinical evidence typical of chronic hepatitis B. All 3 patients had significant T-cell proliferative responses against HBV core antigen (HBcAg) during the remission stage, while no responses were detected during the acute exacerbation stage. In addition, the TCR Vbeta7 gene was utilized more frequently in T cells recognizing HBcAg during remission, while TCR Vbeta1 and Vbeta2 were utilized at a higher percentage during acute exacerbation. On the contrary, the T cell proliferative response against HBV surface antigen was undetectable and no specific Vbeta gene was utilized more frequently by all 3 patients, regardless of disease state. Our longitudinal studies, although based on a small sample of patients, demonstrate that the population of HBcAg-activated T cells alters during the course of disease in chronic hepatitis B patients.