Prediction and analyses of HLA-II restricted Mycobacterium tuberculosis CD4+ T cell epitopes in the Chinese population.

The Bacillus Calmette-Guérin (BCG) vaccine has been used to prevent tuberculosis (TB), but it cannot prevent adults against TB. The Mycobacterium tuberculosis Beijing strain is the most popular strain in China, but no vaccine is designed for the Beijing strain. It is vital to design a multiepitopes-based vaccine against the Beijing strain for the Chinese population. The bioinformatics tools were used to predict CD4+ T-cell epitopes in five protective antigens based on the Chinese population-specific alleles. The antigenicity, allergenicity, toxicity, IFN-γ level, population coverage, and three-dimensional structure were predicted using Vaxijen, AllerTOP, ToxinPred, IFN-γ epitope server, IEDB, and I-TASSER, respectively. One-hundred one promiscuous epitopes were obtained from Rv1813c, Rv2608, Rv3131, and Rv3628 proteins. After screening with antigenicity, allergenicity, toxicity, and IFN-γ level, seven epitopes from Rv2608 and Rv3131 proteins were selected to be vaccine candidates. Further study determined their three-dimensional structure and the coverage in the Chinese population as high as 99%. Our study predicted seven CD4+ T-cell dominant epitopes from the proteins Rv2608 and Rv3131 of M. tuberculosis Beijing strain for the first time, which may provide a basis for improving the design of multiepitopes-based vaccines for TB.

Japanese encephalitis virus in India: An update on virus genotypes.

Japanese encephalitis (JE) is a leading cause of viral encephalitis in Southeast Asia. It is a serious public health issue in India, and cases have been emerging in newer areas of the country. Although vaccination efforts have already been initiated in the country since 2006 and later through the Universal Immunization Programme in 2011, still a significant reduction in the number of cases has to be achieved since an escalating trend of JE incidence has been reported in certain States such as Assam, Uttar Pradesh and West Bengal. Moreover, fresh cases of JE have been reported from certain pockets in Odisha as well. Despite the mass JE vaccination programme implemented in prioritized endemic zones in the country in 2011, a shift in the age group of JE virus (JEV) infection was noticed affecting the adult population in West Bengal. The recent detection of the circulation of genotype I (GI) in Gorakhpur, Uttar Pradesh and the co-circulation of GI and genotype III (GIII) in West Bengal are probably a warning signal for the public health personnel to strengthen the surveillance system in all endemic hotspots in the country. The abrupt emergence of JEV genotype V (GV) in China and Korea in 2009, after its first detection in Malaya in 1952, endemic countries have been cautioned to strengthen their surveillance, because GV has been suspected of getting dispersed efficiently in other parts of Asia. Moreover, the reduced protection efficiency of the JEV GIII-based vaccine against the JEV genotype V further warrants careful evaluation of the ongoing vaccination strategies in the endemic countries, anticipating the possible incursion of GV and its impact on future control strategies. In view of the above facts, the present communication reviews the current knowledge on the molecular epidemiology of JEV in India vis-a-vis the global scenario and discusses the future priorities in JEV research in India for effectively designing control strategies.

Tuberculosis outbreak in a primary school, Milan, Italy.

Investigation of an outbreak of tuberculosis (TB) in a primary school in Milan, Italy, found 15 schoolchildren had active TB disease and 173 had latent TB infection. TB was also identified in 2 homeless men near the school. Diagnostic delay, particularly in the index case-patient, contributed to the transmission of infection.

Host cell transcriptomic response to the multidrug-resistant Mycobacterium tuberculosis clonal outbreak Beijing strain reveals its pathogenic features.

The upsurge of multidrug-resistant infections has rendered tuberculosis the principal cause of death among infectious diseases. A clonal outbreak multidrug-resistant triggering strain of Mycobacterium tuberculosis was identified in Kanchanaburi Province, labelled "MKR superspreader," which was found to subsequently spread to other regions, as revealed by prior epidemiological reports in Thailand. Herein, we showed that the MKR displayed a higher growth rate upon infection into host macrophages in comparison with the H37Rv reference strain. To further elucidate MKR's biology, we utilized RNA-Seq and differential gene expression analyses to identify host factors involved in the intracellular viability of the MKR. A set of host genes function in the cellular response to lipid pathway was found to be uniquely up-regulated in host macrophages infected with the MKR, but not those infected with H37Rv. Within this set of genes, the IL-36 cytokines which regulate host cell cholesterol metabolism and resistance against mycobacteria attracted our interest, as our previous study revealed that the MKR elevated genes associated with cholesterol breakdown during its growth inside host macrophages. Indeed, when comparing macrophages infected with the MKR to H37Rv-infected cells, our RNA-Seq data showed that the expression ratio of IL-36RN, the negative regulator of the IL-36 pathway, to that of IL-36G was greater in macrophages infected with the MKR. Furthermore, the MKR's intracellular survival and increased intracellular cholesterol level in the MKR-infected macrophages were diminished with decreased IL-36RN expression. Overall, our results indicated that IL-36RN could serve as a new target against this emerging multidrug-resistant M. tuberculosis strain.

The Utility of a real-time polymerase chain reaction kit for differentiating between Mycobacterium tuberculosis and the Beijing familythe.

Background: Tuberculosis (TB) is a severe public health challenge in Korea. Of all Mycobacterium tuberculosis (M. tb) strains, the Beijing genotype strain reportedly correlates with hypervirulence and drug resistance. Hence, an early identification of the Beijing genotype strain of M. tb plays a significant role in initial TB treatment. Kogenebiotech® (KoRT-polymerase chain reaction [PCR]) has developed a real-time PCR 17 18 kit to determine the Beijing genotype strain classified as M. tb. To determine the feasibility of the commercially produced KoRT-PCR kit in identifying the M. tb strain. Methods: We used 100 clinical isolates of M. tb and 100 non-M. tb samples for the assessment. We evaluated the overall concordance between the KoRT-PCR kit and the mycobacterial interspersed repetitive unite variable number tandem repeat typing kit (GenoScreen, Lille, France). Moreover, we measured the detection limits based on the chromosomal DNA copies for the KoRT-PCR kit. In addition, we determined the reproducibility among individual technicians using the KoRT-PCR. Results: The KoRT-PCR kit successfully discriminated all M. tb (confidence interval [CI]: 96.38%-100.00% for both sensitivity and specificity) and Beijing genotype strain (CI: 95.70%-100.00% for sensitivity and 96.87%-100.00% for specificity). We confirmed no significant deviation in the reproducibility between the technicians. Conclusions: The KoRT-PCR kit displayed sufficient capability of discriminating the Beijing genotype strain, which enabled the rapid identification of the Beijing genotype strain from the M. tb clinical isolates.

Comparison on Major Gene Mutations Related to Rifampicin and Isoniazid Resistance between Beijing and Non-Beijing Strains of Mycobacterium tuberculosis: A Systematic Review and Bayesian Meta-Analysis.

Objective: The Beijing strain of Mycobacterium tuberculosis (MTB) is controversially presented as the predominant genotype and is more drug resistant to rifampicin and isoniazid compared to the non-Beijing strain. We aimed to compare the major gene mutations related to rifampicin and isoniazid drug resistance between Beijing and non-Beijing genotypes, and to extract the best evidence using the evidence-based methods for improving the service of TB control programs based on genetics of MTB. Method: Literature was searched in Google Scholar, PubMed and CNKI Database. Data analysis was conducted in R software. The conventional and Bayesian random-effects models were employed for meta-analysis, combining the examinations of publication bias and sensitivity. Results: Of the 8785 strains in the pooled studies, 5225 were identified as Beijing strains and 3560 as non-Beijing strains. The maximum and minimum strain sizes were 876 and 55, respectively. The mutations prevalence of rpoB, katG, inhA and oxyR-ahpC in Beijing strains was 52.40% (2738/5225), 57.88% (2781/4805), 12.75% (454/3562) and 6.26% (108/1724), respectively, and that in non-Beijing strains was 26.12% (930/3560), 28.65% (834/2911), 10.67% (157/1472) and 7.21% (33/458), separately. The pooled posterior value of OR for the mutations of rpoB was 2.72 ((95% confidence interval (CI): 1.90, 3.94) times higher in Beijing than in non-Beijing strains. That value for katG was 3.22 (95% CI: 2.12, 4.90) times. The estimate for inhA was 1.41 (95% CI: 0.97, 2.08) times higher in the non-Beijing than in Beijing strains. That for oxyR-ahpC was 1.46 (95% CI: 0.87, 2.48) times. The principal patterns of the variants for the mutations of the four genes were rpoB S531L, katG S315T, inhA-15C > T and oxyR-ahpC intergenic region. Conclusion: The mutations in rpoB and katG genes in Beijing are significantly more common than that in non-Beijing strains of MTB. We do not have sufficient evidence to support that the prevalence of mutations of inhA and oxyR-ahpC is higher in non-Beijing than in Beijing strains, which provides a reference basis for clinical medication selection.

Bacterial Strain-Dependent Dissociation of Cell Recruitment and Cell-to-Cell Spread in Early M. tuberculosis Infection.

In the initial stage of respiratory infection, Mycobacterium tuberculosis traverses from alveolar macrophages to phenotypically diverse monocyte-derived phagocytes and neutrophils in the lung parenchyma. Here, we compare the in vivo kinetics of early bacterial growth and cell-to-cell spread of two strains of M. tuberculosis: a lineage 2 strain, 4334, and the widely studied lineage 4 strain H37Rv. Using flow cytometry, live cell sorting of phenotypic subsets, and quantitation of bacteria in cells of the distinct subsets, we found that 4334 induces less leukocyte influx into the lungs but demonstrates earlier population expansion and cell-to-cell spread. The earlier spread of 4334 to recruited cells, including monocyte-derived dendritic cells, is accompanied by earlier and greater magnitude of CD4+ T cell activation. The results provide evidence that strain-specific differences in interactions with lung leukocytes can shape adaptive immune responses in vivo. IMPORTANCE Tuberculosis is a leading infectious disease killer worldwide and is caused by Mycobacterium tuberculosis. After exposure to M. tuberculosis, outcomes range from apparent elimination to active disease. Early innate immune responses may contribute to differences in outcomes, yet it is not known how bacterial strains alter the early dynamics of innate immune and T cell responses. We infected mice with distinct strains of M. tuberculosis and discovered striking differences in innate cellular recruitment, cell-to-cell spread of bacteria in the lungs, and kinetics of initiation of antigen-specific CD4 T cell responses. We also found that M. tuberculosis can spread beyond alveolar macrophages even before a large influx of inflammatory cells. These results provide evidence that distinct strains of M. tuberculosis can exhibit differential kinetics in cell-to-cell spread which is not directly linked to early recruitment of phagocytes but is subsequently linked to adaptive immune responses.

Revised nomenclature and SNP barcode for Mycobacterium tuberculosis lineage 2.

Mycobacterium tuberculosis (Mtb) lineage 2 (L2) strains are present globally, contributing to a widespread tuberculosis (TB) burden, particularly in Asia where both prevalence of TB and numbers of drug resistant TB are highest. The increasing availability of whole-genome sequencing (WGS) data worldwide provides an opportunity to improve our understanding of the global genetic diversity of Mtb L2 and its association with the disease epidemiology and pathogenesis. However, existing L2 sublineage classification schemes leave >20 % of the Modern Beijing isolates unclassified. Here, we present a revised SNP-based classification scheme of L2 in a genomic framework based on phylogenetic analysis of >4000 L2 isolates from 34 countries in Asia, Eastern Europe, Oceania and Africa. Our scheme consists of over 30 genotypes, many of which have not been described before. In particular, we propose six main genotypes of Modern Beijing strains, denoted L2.2.M1-L2.2.M6. We also provide SNP markers for genotyping L2 strains from WGS data. This fine-scale genotyping scheme, which can classify >98 % of the studied isolates, serves as a basis for more effective monitoring and reporting of transmission and outbreaks, as well as improving genotype-phenotype associations such as disease severity and drug resistance. This article contains data hosted by Microreact.

Severe Clinical Outcomes of Tuberculosis in Kharkiv Region, Ukraine, Are Associated with Beijing Strains of Mycobacterium tuberculosis.

Genotypic variation in Beijing lineages of Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB), has been associated with hyper virulence and the spread of extensively and multiple drug (X/MDR) resistant MTB strains in Eastern Europe, Central Asia, and East Asia. The clinical outcomes of 215 new cases of TB among the population of the Kharkiv region of Eastern Ukraine were analyzed to uncover factors associated with severe infection. Infecting MTB strains were profiled by 5 locus exact tandem repeats (ETRs) and 15 locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) genotyping. Among diverse MTB genotypes discovered in Ukraine, the Beijing genotype (MIRU-VNTR 42425) was significantly associated with risk factors for severe outcomes of disease in the study population, including TB/HIV co-infection and treatment failure. Strain replacement (superinfection) was observed in 10 patients, suggesting repeated exposure to novel MTB strains in hospital or community settings. Inclusion of MTB genotyping data may identify at-risk patients and improve treatment adherence to prevent X/MDR development for effective public health response against tuberculosis in Ukraine.

CLONING AND EXPRESSION OF MCE1A GENE FROM MYCOBACTERIUM TUBERCULOSIS BEIJING AND H37RV STRAIN FOR VACCINE CANDIDATE DEVELOPMENT.

BACKGROUND: Tuberculosis remains the leading cause of death in the world, especially wherever poverty, malnutrition and poor housing prevail. Mycobacterium tuberculosis Beijing strain is the most common strain that causes tuberculosis in Indonesia. The wide spread of tuberculosis has been further aggravated by HIV-AIDS and drug resistance. Unfortunately, Bacille Calmette-Guerin (BCG) as the current vaccine has different protection function and efficacy. According to function analysis, mce1A gene was predicted to have a role in host invasion and survival of Mycobacterium tuberculosis in human macrophages. MATERIALS AND METHODS: We performed cloning and protein expression of Mce1A gene of Mycobacterium tuberculosis Beijing strain as local isolate and standard strain H37Rv as a comparison on the expression system Escherichia coli BL21(DE3). Mce1A gene from the strains were amplified by PCR and inserted into the vector pET28a. Each resulting recombinant plasmid was subsequently transformed into E. coli BL21(DE3) and Mce1A protein was expressed with IPTG induction. RESULTS: E. coli BL21(DE3) was succesfully transformed with a recombinant plasmid that contains the Mce1A gene insert with correct orientation and reading frame. There was no mutation found in the amino acids sequence for B and T cell epitope. Mce1A expression in E. coli BL21(DE3) showed a protein band that was higher than expected. The protein was confirmed with Western blotting using anti-His detector. CONCLUSION: We assumed that Mce1A recombinant protein that has been expressed in E. coli BL21(DE3) is in their dimeric form or alternatively formed aggregates of different sizes.

Protection by novel vaccine candidates, Mycobacterium tuberculosis ΔmosR and ΔechA7, against challenge with a Mycobacterium tuberculosis Beijing strain.

Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), infects over two billion people, claiming around 1.5 million lives annually. The only vaccine approved for clinical use against this disease is the Bacillus Calmette-Guérin (BCG) vaccine. Unfortunately, BCG has limited efficacy against the adult, pulmonary form of tuberculosis. This vaccine was developed from M. bovis with antigen expression and host specificity that differ from M. tuberculosis. To address these problems, we have designed two novel, live attenuated vaccine (LAV) candidates on an M. tuberculosis background: ΔmosR and ΔechA7. These targeted genes are important to M. tuberculosis pathogenicity during infection. To examine the efficacy of these strains, C57BL/6 mice were vaccinated subcutaneously with either LAV, BCG, or PBS. Both LAV strains persisted up to 16 weeks in the spleens or lungs of vaccinated mice, while eliciting minimal pathology prior to challenge. Following challenge with a selected, high virulence M. tuberculosis Beijing strain, protection was notably greater for both groups of LAV vaccinated animals as compared to BCG at both 30 and 60 days post-challenge. Additionally, vaccination with either ΔmosR or ΔechA7 elicited an immune response similar to BCG. Although these strains require further development to meet safety standards, this first evidence of protection by these two new, live attenuated vaccine candidates shows promise.

Complete genome sequence of Mycobacterium tuberculosis K from a Korean high school outbreak, belonging to the Beijing family.

Mycobacterium tuberculosis K, a member of the Beijing family, was first identified in 1999 as the most prevalent genotype in South Korea among clinical isolates of M. tuberculosis from high school outbreaks. M. tuberculosis K is an aerobic, non-motile, Gram-positive, and non-spore-forming rod-shaped bacillus. A transmission electron microscopy analysis displayed an abundance of lipid bodies in the cytosol. The genome of the M. tuberculosis K strain was sequenced using two independent sequencing methods (Sanger and Illumina). Here, we present the genomic features of the 4,385,518-bp-long complete genome sequence of M. tuberculosis K (one chromosome, no plasmid, and 65.59 % G + C content) and its annotation, which consists of 4194 genes (3447 genes with predicted functions), 48 RNA genes (3 rRNA and 45 tRNA) and 261 genes with peptide signals.

Application of Deletion- Targeted Multiplex PCR technique for detection of Mycobacterium tuberculosis Beijing strains in samples from tuberculosis patients.

BACKGROUND AND OBJECTIVE: Molecular epidemiological studies have shown that certain genotypes of Mycobacterium tuberculosis (MTB) are over-represented in limited geographical regions, suggesting of evolution of certain genotypes with increasing virulence and pathogenicity. Beijing strain of MTB was initially described by its potential to cause outbreaks worldwide and its association with drug resistance. Due to tuberculosis (TB)-related mortality which is associated with Beijing genotype, this study was designed with the aim to detect the MTB Beijing genotype in the region of study. MATERIALS AND METHODS: A total of 170 clinical isolates of MTB were collected from the TB reference laboratory of Khuzestan province, Iran, over one year period from February 2010 to February 2011. Phenotypic tests were used for preliminary detection of MTB. Culture positive MTB isolates were confirmed by multiplex PCR based on IS6110 gene with subsequent screening for resistance to isoniazid (INH), and rifampin (RIF) by PCR using relevant primers. Three set of primers were used to differentiate Beijing from non-Beijing strains by using Deletion- Targeted Multiplex (DTM) PCR. RESULTS: From 160 PCR-confirmed MTB isolates, 18 (11.25%) showed mutation in katG gene related to INH resistance and 20 (12.5%), associated with mutation in rpoB gene related to RIF resistance, and 8 (5%) were detected as Beijing strain using multiplex PCR. The majority of detected Beijing strains (6/8[75%]) comprised mutation in katG gene with the prevalent mutation specifically in codon 315. In 4 Beijing strains (2.5%), mutation in rpoB gene were also detected. CONCLUSION: Using DTM- PCR, the rate of Beijing strains in the region of study was determined as 5%. Although for detection of MTB antimicrobial resistance, it is advised to use a combination of conventional antimicrobial susceptibility testing and molecular techniques, however for time saving, it seems that DTM-PCR, is a simple technique for use in areas of the world where Beijing strains are highly prevalent.

Testing for the presence of Mycobacterium tuberculosis Beijing genotype strains in Syrian samples.

The Beijing family of Mycobacterium tuberculosis (MTB) has been reported to have an exceptional capacity to spread tuberculosis (TB) and induce multi-drug resistance. A method has been developed to distinguish this family from the rest of the MTB families through real-time DNA amplification and subsequent analysis of the melting point of an amplicon. Two pools of multi-drug resistant (MDR) MTB samples collected at two different time periods from various regions in Syria have been selected. This preliminary screening indicated a complete absence of the Beijing family in all samples. This research presents an effective differentiation of bacterial Beijing strains, with minimal effort and cost through analysis of differential amplicon melting points.

Sublineages of Beijing Strain of Mycobacterium tuberculosis in Sri Lanka.

Strains of the Beijing/W genotype of Mycobacterium tuberculosis have been responsible for large outbreaks of tuberculosis around the world, sometimes involving multi-drug resistance. It has been shown that more recently evolved Beijing sublineages are prone to cause outbreaks. Furthermore Beijing is the single predominant cluster in Sri Lanka. The present study identifies that recently evolved sublineages of Beijing strains are present in the study population. The majority of Beijing isolates (92.85%) were pan-susceptible. However, these findings may have important implications for the control and prevention of tuberculosis in Sri Lanka.