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Sample records for mycobacterium tuberculosis genotype

  1. Drug susceptibility of Mycobacterium tuberculosis Beijing genotype and association with MDR TB

    NARCIS (Netherlands)

    Steenwinkel, J.E. de; Kate, M.T. Ten; Knegt, G.J. de; Kremer, K.; Aarnoutse, R.E.; Boeree, M.J.; Verbrugh, H.A.; Soolingen, D. van; Bakker-Woudenberg, I.A.

    2012-01-01

    To determine differences in the ability of Mycobacterium tuberculosis strains to withstand antituberculosis drug treatment, we compared the activity of antituberculosis drugs against susceptible Beijing and East-African/Indian genotype M. tuberculosis strains. Beijing genotype strains showed high ra

  2. Drug susceptibility of mycobacterium tuberculosis Beijing genotype and association with MDR TB

    NARCIS (Netherlands)

    J.E.M. de Steenwinkel (Jurriaan); M.T. ten Kate (Marian); G.J. de Knegt (Gerjo); K. Kremer (Kristin); E.J. Aarnoutse (E. J.); M. Boeree (Martin); H.A. Verbrugh (Henri); D. van Soolingen (Dick); I.A.J.M. Bakker-Woudenberg (Irma)

    2012-01-01

    textabstractTo determine differences in the ability of Mycobacterium tuberculosis strains to withstand antituberculosis drug treatment, we compared the activity of antituberculosis drugs against susceptible Beijing and East-African/Indian genotype M. tuberculosis strains. Beijing genotype strains sh

  3. Drug Susceptibility of Mycobacterium tuberculosis Beijing Genotype and Association with MDR TB

    OpenAIRE

    2012-01-01

    To determine differences in the ability of Mycobacterium tuberculosis strains to withstand antituberculosis drug treatment, we compared the activity of antituberculosis drugs against susceptible Beijing and East-African/Indian genotype M. tuberculosis strains. Beijing genotype strains showed high rates of mutation within a wide range of drug concentrations, possibly explaining this genotype's association with multidrug-resistant tuberculosis.

  4. Drug susceptibility of Mycobacterium tuberculosis Beijing genotype and association with MDR TB.

    Science.gov (United States)

    de Steenwinkel, Jurriaan E M; ten Kate, Marian T; de Knegt, Gerjo J; Kremer, Kristin; Aarnoutse, Rob E; Boeree, Martin J; Verbrugh, Henri A; van Soolingen, Dick; Bakker-Woudenberg, Irma A J M

    2012-04-01

    To determine differences in the ability of Mycobacterium tuberculosis strains to withstand antituberculosis drug treatment, we compared the activity of antituberculosis drugs against susceptible Beijing and East-African/Indian genotype M. tuberculosis strains. Beijing genotype strains showed high rates of mutation within a wide range of drug concentrations, possibly explaining this genotype's association with multidrug-resistant tuberculosis.

  5. Tuberculosis relapse in Vietnam is significantly associated with Mycobacterium tuberculosis Beijing genotype infections.

    NARCIS (Netherlands)

    Huyen, M.N.; Buu, T.N.; Tiemersma, E.; Lan, N.T.; Dung, N.H.; Kremer, K.; Soolingen, D.V.; Cobelens, F.G.

    2013-01-01

    BACKGROUND: In Vietnam, the Mycobacterium tuberculosis Beijing genotype is associated with multi-drug resistance and is emerging. A possible explanation for this genotype's success is an increased rate of relapse. METHODS: In a prospective cohort study, isolates from patients with smear-positive tub

  6. Population structure of mixed Mycobacterium tuberculosis infection is strain genotype and culture medium dependent.

    NARCIS (Netherlands)

    Hanekom, M.; Streicher, E.M.; Berg, D. Van den; Cox, H.; McDermid, C.; Bosman, M.; Pittius, N.C. Gey van; Victor, T.C.; Kidd, M.; Soolingen, D. van; Helden, P.D. van; Warren, R.M.

    2013-01-01

    BACKGROUND: Molecular genotyping methods have shown infection with more than one Mycobacterium tuberculosis strain genotype in a single sputum culture, indicating mixed infection. AIM: This study aimed to develop a PCR-based genotyping tool to determine the population structure of M. tuberculosis st

  7. Differential activation of dendritic cells by Mycobacterium tuberculosis Beijing genotype.

    Science.gov (United States)

    Reyes-Martínez, Juana Elizabeth; Nieto-Patlán, Erik; Nieto-Patlán, Alejandro; Gonzaga-Bernachi, Job; Santos-Mendoza, Teresa; Serafín-López, Jeanet; Chávez-Blanco, Alma; Sandoval-Montes, Claudia; Flores-Romo, Leopoldo; Estrada-Parra, Sergio; Estrada-García, Iris; Chacón-Salinas, Rommel

    2014-01-01

    Mycobacterium tuberculosis (Mtb) inhibits dendritric cells (DC) function in order to delay T cell response. Furthermore, there is increasing evidence that genetic diversity of Mtb strains can affect their interaction with the immune system. Beijing genotype has attracted attention because of its high prevalence and multi-drug resistance. Although it is known that this genotype is hypervirulent and differentially activates macrophages when compared to other genotypes, little is known about its interaction with DC. In order to address this issue, murine bone marrow derived DC (BMDC) were stimulated with soluble extracts (SE) from BCG, H37Rv, Canetti and Beijing genotypes. We observed that unlike other mycobacteria strains, SE-Beijing was unable to induce maturation of DC as assessed by cell surface MHC-II expression. DC stimulated with SE-Beijing failed to produce IL-12 and TNF-α, but did secrete IL-10. Interestingly, SE-Beijing induced CCR7 and PDL-1 on BMDC, but did not induce the expression of CD86. When BMDC stimulated with SE-Beijing were used to activate CD4+ cells they were unable to induce a Th1 response when compared with less virulent genotypes. These results indicate that Beijing is able to modulate DC activation and function, which may be related to the pathogenesis induced by this genotype.

  8. Molecular epidemiology and genotyping of Mycobacterium tuberculosis isolated in Baghdad.

    Science.gov (United States)

    Mustafa Ali, Ruqaya; Trovato, Alberto; Couvin, David; Al-Thwani, Amina N; Borroni, Emanuele; Dhaer, Fahim H; Rastogi, Nalin; Cirillo, Daniela M

    2014-01-01

    Tuberculosis (TB) remains a major health problem in Iraq but the strains responsible for the epidemic have been poorly characterized. Our aim was to characterize the TB strains circulating in Bagdad (Iraq). A total of 270 Mycobacterium tuberculosis complex (MTBC) strains isolated between 2010 and 2011 from TB patients attending the Center of Chest and Respiratory diseases in Baghdad were analyzed by Spoligotyping. The analysis indicated that 94.1% of the isolates belong to known genotype clades: CAS 39.6%, ill-defined T clade 29.6%, Manu 7.4%, Haarlem 7%, Ural 4.1%, LAM 3.3%, X 0.7%, LAM7-TUR 0.7%, EAI 0.7%, S 0.7%, and unknown 5.9%. Comparison with the international multimarker database SITVIT2 showed that SIT 309 (CAS1-Delhi) and SIT1144 (T1) were the most common types. In addition, 44 strains were included in SITVIT2 database under 16 new Spoligotype International Types (SITs); of these, 6 SITs (SIT3346, SIT3497, SIT3708, SIT3790, SIT3791, and SIT3800) (n = 32 strains) were created within the present study and 10 were created after a match with an orphan in the database. By using 24-loci MIRU-VNTR-typing on a subset of 110 samples we found a high recent transmission index (RTI) of 33.6%. In conclusion, we present the first unifying framework for both epidemiology and evolutionary analysis of M. tuberculosis in Iraq.

  9. Molecular Epidemiology and Genotyping of Mycobacterium tuberculosis Isolated in Baghdad

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    Ruqaya Mustafa Ali

    2014-01-01

    Full Text Available Tuberculosis (TB remains a major health problem in Iraq but the strains responsible for the epidemic have been poorly characterized. Our aim was to characterize the TB strains circulating in Bagdad (Iraq. A total of 270 Mycobacterium tuberculosis complex (MTBC strains isolated between 2010 and 2011 from TB patients attending the Center of Chest and Respiratory diseases in Baghdad were analyzed by Spoligotyping. The analysis indicated that 94.1% of the isolates belong to known genotype clades: CAS 39.6%, ill-defined T clade 29.6%, Manu 7.4%, Haarlem 7%, Ural 4.1%, LAM 3.3%, X 0.7%, LAM7-TUR 0.7%, EAI 0.7%, S 0.7%, and unknown 5.9%. Comparison with the international multimarker database SITVIT2 showed that SIT 309 (CAS1-Delhi and SIT1144 (T1 were the most common types. In addition, 44 strains were included in SITVIT2 database under 16 new Spoligotype International Types (SITs; of these, 6 SITs (SIT3346, SIT3497, SIT3708, SIT3790, SIT3791, and SIT3800 (n = 32 strains were created within the present study and 10 were created after a match with an orphan in the database. By using 24-loci MIRU-VNTR-typing on a subset of 110 samples we found a high recent transmission index (RTI of 33.6%. In conclusion, we present the first unifying framework for both epidemiology and evolutionary analysis of M. tuberculosis in Iraq.

  10. Mycobacterium tuberculosis strains of the Beijing genotype are rarely observed in tuberculosis patients in South America

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    Viviana Ritacco

    2008-08-01

    Full Text Available The frequency of the Beijing genotype of Mycobacterium tuberculosis as a cause of tuberculosis (TB in South America was determined by analyzing genotypes of strains isolated from patients that had been diagnosed with the disease between 1997 and 2003 in seven countries of the subcontinent. In total, 19 of the 1,202 (1.6% TB cases carried Beijing isolates, including 11 of the 185 patients from Peru (5.9%, five of the 512 patients from Argentina (1.0%, two of the 252 Brazilian cases (0.8%, one of the 166 patients from Paraguay (0.6% and none of the samples obtained from Chile (35, Colombia (36 and Ecuador (16. Except for two patients that were East Asian immigrants, all cases with Beijing strains were native South Americans. No association was found between carrying a strain with the Beijing genotype and having drug or multi-drug resistant disease. Our data show that presently transmission of M. tuberculosis strains of the Beijing genotype is not frequent in Latin America. In addition, the lack of association of drug resistant TB and infection with M. tuberculosis of the Beijing genotype observed presently demands efforts to define better the contribution of the virulence and lack of response to treatment to the growing spread of Beijing strains observed in other parts of the world.

  11. Mycobacterium genotypes in pulmonary tuberculosis infections and their detection by trained African giant pouched rats.

    Science.gov (United States)

    Mgode, Georgies F; Cohen-Bacrie, Stéphan; Bedotto, Marielle; Weetjens, Bart J; Cox, Christophe; Jubitana, Maureen; Kuipers, Dian; Machang'u, Robert S; Kazwala, Rudovick; Mfinanga, Sayoki G; Kaufmann, Stefan H E; Drancourt, Michel

    2015-02-01

    Tuberculosis (TB) diagnosis in low-income countries is mainly done by microscopy. Hence, little is known about the diversity of Mycobacterium spp. in TB infections. Different genotypes or lineages of Mycobacterium tuberculosis vary in virulence and induce different inflammatory and immune responses. Trained Cricetomys rats show a potential for rapid diagnosis of TB. They detect over 28 % of smear-negative, culture-positive TB. However, it is unknown whether these rats can equally detect sputa from patients infected with different genotypes of M. tuberculosis. A 4-month prospective study on diversity of Mycobacterium spp. was conducted in Dar es Salaam, Tanzania. 252 sputa from 161 subjects were cultured on Lowenstein-Jensen medium and thereafter tested by rats. Mycobacterial isolates were subjected to molecular identification and multispacer sequence typing (MST) to determine species and genotypes. A total of 34 Mycobacterium spp. isolates consisting of 32 M. tuberculosis, 1 M. avium subsp. hominissuis and 1 M. intracellulare were obtained. MST analyses of 26 M. tuberculosis isolates yielded 10 distinct MST genotypes, including 3 new genotypes with two clusters of related patterns not grouped by geographic areas. Genotype MST-67, shared by one-third of M. tuberculosis isolates, was associated with the Mwananyamala clinic. This study shows that diverse M. tuberculosis genotypes (n = 10) occur in Dar es Salaam and trained rats detect 80 % of the genotypes. Sputa with two M. tuberculosis genotypes (20 %), M. avium hominissuis and M. intracellulare were not detected. Therefore, rats detect sputa with different M. tuberculosis genotypes and can be used to detect TB in resource-poor countries.

  12. The influence of host and bacterial genotype on the development of disseminated disease with Mycobacterium tuberculosis.

    OpenAIRE

    2008-01-01

    The factors that govern the development of tuberculosis disease are incompletely understood. We hypothesized that some strains of Mycobacterium tuberculosis (M. tuberculosis) are more capable of causing disseminated disease than others and may be associated with polymorphisms in host genes responsible for the innate immune response to infection. We compared the host and bacterial genotype in 187 Vietnamese adults with tuberculous meningitis (TBM) and 237 Vietnamese adults with uncomplicated p...

  13. Population structure of mixed Mycobacterium tuberculosis infection is strain genotype and culture medium dependent.

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    Madeleine Hanekom

    Full Text Available BACKGROUND: Molecular genotyping methods have shown infection with more than one Mycobacterium tuberculosis strain genotype in a single sputum culture, indicating mixed infection. AIM: This study aimed to develop a PCR-based genotyping tool to determine the population structure of M. tuberculosis strain genotypes in primary Mycobacterial Growth Indicator Tubes (MGIT and Löwenstein-Jensen (LJ cultures to identify mixed infections and to establish whether the growth media influenced the recovery of certain strain genotypes. METHOD: A convenience sample of 206 paired MGIT and LJ M. tuberculosis cultures from pulmonary tuberculosis patients resident in Khayelitsha, South Africa were genotyped using an in-house PCR-based method to detect defined M. tuberculosis strain genotypes. RESULTS: The sensitivity and specificity of the PCR-based method for detecting Beijing, Haarlem, S-family, and LAM genotypes was 100%, and 75% and 50% for detecting the Low Copy Clade, respectively. Thirty-one (15% of the 206 cases showed the presence of more than one M. tuberculosis strain genotype. Strains of the Beijing and Haarlem genotypes were significantly more associated with a mixed infection (on both media when compared to infections with a single strain (Beijing MGIT p = 0.02; LJ, p<0.01 and (Haarlem: MGIT p<0.01; LJ, p = 0.01. Strains with the Beijing genotype were less likely to be with "other genotype" strains (p<0.01 while LAM, Haarlem, S-family and LCC occurred independently with the Beijing genotype. CONCLUSION: The PCR-based method was able to identify mixed infection in at least 15% of the cases. LJ media was more sensitive in detecting mixed infections than MGIT media, implying that the growth characteristics of M. tuberculosis on different media may influence our ability to detect mixed infections. The Beijing and Haarlem genotypes were more likely to occur in a mixed infection than any of the other genotypes tested suggesting pathogen

  14. Phenotypic and genotypic analysis of anti-tuberculosis drug resistance in Mycobacterium tuberculosis isolates in Myanmar.

    Science.gov (United States)

    Aung, Wah Wah; Ei, Phyu Win; Nyunt, Wint Wint; Swe, Thyn Lei; Lwin, Thandar; Htwe, Mi Mi; Kim, Kyung Jun; Lee, Jong Seok; Kim, Chang Ki; Cho, Sang Nae; Song, Sun Dae; Chang, Chulhun L

    2015-09-01

    Tuberculosis (TB) is one of the most serious health problems in Myanmar. Because TB drug resistance is associated with genetic mutation(s) relevant to responses to each drug, genotypic methods for detecting these mutations have been proposed to overcome the limitations of classic phenotypic drug susceptibility testing (DST). We explored the current estimates of drug-resistant TB and evaluated the usefulness of genotypic DST in Myanmar. We determined the drug susceptibility of Mycobacterium tuberculosis isolated from sputum smear-positive patients with newly diagnosed pulmonary TB at two main TB centers in Myanmar during 2013 by using conventional phenotypic DST and the GenoType MTBDRplus assay (Hain Lifescience, Germany). Discrepant results were confirmed by sequencing the genes relevant to each type of resistance (rpoB for rifampicin; katG and inhA for isoniazid). Of 191 isolates, phenotypic DST showed that 27.7% (n=53) were resistant to at least one first-line drug and 20.9% (n=40) were resistant to two or more, including 18.3% (n=35) multidrug-resistant TB (MDR-TB) strains. Monoresistant strains accounted for 6.8% (n=13) of the samples. Genotypic assay of 189 isolates showed 17.5% (n=33) MDR-TB and 5.3% (n=10) isoniazid-monoresistant strains. Genotypic susceptibility results were 99.5% (n=188) concordant and agreed almost perfectly with phenotypic DST (kappa=0.99; 95% confidence interval 0.96-1.01). The results highlight the burden of TB drug resistance and prove the usefulness of the genotypic DST in Myanmar.

  15. The influence of host and bacterial genotype on the development of disseminated disease with Mycobacterium tuberculosis.

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    Maxine Caws

    2008-03-01

    Full Text Available The factors that govern the development of tuberculosis disease are incompletely understood. We hypothesized that some strains of Mycobacterium tuberculosis (M. tuberculosis are more capable of causing disseminated disease than others and may be associated with polymorphisms in host genes responsible for the innate immune response to infection. We compared the host and bacterial genotype in 187 Vietnamese adults with tuberculous meningitis (TBM and 237 Vietnamese adults with uncomplicated pulmonary tuberculosis. The host genotype of tuberculosis cases was also compared with the genotype of 392 cord blood controls from the same population. Isolates of M. tuberculosis were genotyped by large sequence polymorphisms. The hosts were defined by polymorphisms in genes encoding Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP and Toll-like receptor-2 (TLR-2. We found a significant protective association between the Euro-American lineage of M. tuberculosis and pulmonary rather than meningeal tuberculosis (Odds ratio (OR for causing TBM 0.395, 95% confidence intervals (C.I. 0.193-0.806, P = 0.009, suggesting these strains are less capable of extra-pulmonary dissemination than others in the study population. We also found that individuals with the C allele of TLR-2 T597C allele were more likely to have tuberculosis caused by the East-Asian/Beijing genotype (OR = 1.57 [95% C.I. 1.15-2.15] than other individuals. The study provides evidence that M. tuberculosis genotype influences clinical disease phenotype and demonstrates, for the first time, a significant interaction between host and bacterial genotypes and the development of tuberculosis.

  16. The Influence of Host and Bacterial Genotype on the Development of Disseminated Disease with Mycobacterium tuberculosis

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    Caws, Maxine; Thwaites, Guy; Dunstan, Sarah; Hawn, Thomas R.; Thi Ngoc Lan, Nguyen; Thuong, Nguyen Thuy Thuong; Stepniewska, Kasia; Huyen, Mai Nguyet Thu; Bang, Nguyen Duc; Huu Loc, Tran; Gagneux, Sebastien; van Soolingen, Dick; Kremer, Kristin; van der Sande, Marianne; Small, Peter; Thi Hoang Anh, Phan; Chinh, Nguyen Tran; Thi Quy, Hoang; Thi Hong Duyen, Nguyen; Quang Tho, Dau; Hieu, Nguyen T.; Torok, Estee; Hien, Tran Tinh; Dung, Nguyen Huy; Thi Quynh Nhu, Nguyen; Duy, Phan Minh; van Vinh Chau, Nguyen; Farrar, Jeremy

    2008-01-01

    The factors that govern the development of tuberculosis disease are incompletely understood. We hypothesized that some strains of Mycobacterium tuberculosis (M. tuberculosis) are more capable of causing disseminated disease than others and may be associated with polymorphisms in host genes responsible for the innate immune response to infection. We compared the host and bacterial genotype in 187 Vietnamese adults with tuberculous meningitis (TBM) and 237 Vietnamese adults with uncomplicated pulmonary tuberculosis. The host genotype of tuberculosis cases was also compared with the genotype of 392 cord blood controls from the same population. Isolates of M. tuberculosis were genotyped by large sequence polymorphisms. The hosts were defined by polymorphisms in genes encoding Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) and Toll-like receptor-2 (TLR-2). We found a significant protective association between the Euro-American lineage of M. tuberculosis and pulmonary rather than meningeal tuberculosis (Odds ratio (OR) for causing TBM 0.395, 95% confidence intervals (C.I.) 0.193–0.806, P = 0.009), suggesting these strains are less capable of extra-pulmonary dissemination than others in the study population. We also found that individuals with the C allele of TLR-2 T597C allele were more likely to have tuberculosis caused by the East-Asian/Beijing genotype (OR = 1.57 [95% C.I. 1.15–2.15]) than other individuals. The study provides evidence that M. tuberculosis genotype influences clinical disease phenotype and demonstrates, for the first time, a significant interaction between host and bacterial genotypes and the development of tuberculosis. PMID:18369480

  17. Tuberculosis associated factors caused by Mycobacterium tuberculosis of the RDRio genotype

    Science.gov (United States)

    Moraes, Eloise Brasil; Slompo, Letícia; Finardi, Amanda Juliane; da Silveira, Heloisa Paro Pedro; Ruiz, Luciana; Gomes, Harrison Magdinier; Richini, Virginia Bodelão; Suffys, Philip; Fortaleza, Carlos Magno Castelo Branco; Cavalcanti, Ricardo; Baptista, Ida Maria Foschiani Dias

    2017-01-01

    BACKGROUND Tuberculosis (TB) continues to be a disease that affects many countries around the world, including Brazil. Recently, a subtype of Latin American-Mediterranean family strain was identified and characterised by RDRio. The strain has been associated with different characteristics of the disease. OBJECTIVES In the present study we investigated the association of epidemiological, clinical, radiological and bacteriological variables with pulmonary tuberculosis caused by RDRio Mycobacterium tuberculosis strain in large regions of São Paulo. METHODS We conducted a cross-sectional study in 530 patients with pulmonary tuberculosis, diagnosed using sputum culture, from two regions of the São Paulo state in Brazil. The samples were brought to São Paulo reference laboratories for epidemiological, clinical, radiological and bacteriological analyses, and the data were obtained from a TB notification system. RDRio genotyping and Spoligotyping of the samples were performed. For the analysis of the categorical variables we used the chi-square test or the Fisher’s exact test, and for the continuous variables, the Mann-Whitney test. In addition, a logistic regression was used for multivariate analysis. Differences with p < 0.05 were considered significant. FINDINGS The RDRio deletion was identified in 152 (28.7%) samples. In the univariate analysis, both the age groups above 25 years and alcohol consumption were associated with the RDRio deletion. The multivariate analysis confirmed the association of the RDRio deletion with the age groups: 25-35 years old [OR: 2.28 (1.02-5.07; p = 0.04)] and 36-60 years old (OR: 2.36 (1.11-5.05); p = 0.03], and also with alcohol consumption [OR: 1.63 (1.05-2.54); p = 0,03]. MAIN CONCLUSIONS In this study, we identified new factors associated with the M. tuberculosis of the RDRio deletion strains infection. PMID:28225901

  18. A Mycobacterium tuberculosis cluster demonstrating the use of genotyping in urban tuberculosis control

    NARCIS (Netherlands)

    G. de Vries (Gerard); R.M. van Hest (Reinier); C.C.A. Burdo (Conny); D. van Soolingen (Dick); J.H. Richardus (Jan Hendrik)

    2009-01-01

    textabstractBackground: DNA fingerprinting of Mycobacterium tuberculosis isolates offers better opportunities to study links between tuberculosis (TB) cases and can highlight relevant issues in urban TB control in low-endemic countries. Methods: A medium-sized molecular cluster of TB cases with iden

  19. Mycobacterium tuberculosis Beijing genotype induces differential cytokine production by peripheral blood mononuclear cells of healthy BCG vaccinated individuals.

    NARCIS (Netherlands)

    Rivera-Ordaz, A.; Gonzaga-Bernachi, J.; Serafin-Lopez, J.; Hernandez-Pando, R.; Soolingen, D. van; Estrada-Parra, S.; Estrada-Garcia, I.; Chacon-Salinas, R.

    2012-01-01

    Members of the Mycobacterium tuberculosis (Mtb) Beijing genotype are a major concern due to their high prevalence in tuberculosis patients and their high rate of multi-drug resistance. Although it has been shown that Beijing modifies macrophage behavior, little is known about how this genotype could

  20. Drug-Susceptible Mycobacterium tuberculosis Beijing Genotype Does Not Develop Mutation-Conferred Resistance to Rifampin at an Elevated Rate

    OpenAIRE

    2003-01-01

    The Mycobacterium tuberculosis Beijing genotype has drawn attention because it is often strongly associated with multidrug-resistant tuberculosis (MDR-TB). A possible reason is that the Beijing strains may have an enhanced capacity to develop drug resistance. In this study, we used the Luria-Delbrück fluctuation test to investigate whether strains of Beijing and non-Beijing genotypes exhibit differences in the acquisition of drug resistance. The M. tuberculosis reference strain H37Rv and 12 f...

  1. Consequences of Noncompliance for Therapy Efficacy and Emergence of Resistance in Murine Tuberculosis Caused by the Beijing Genotype of Mycobacterium tuberculosis

    OpenAIRE

    2012-01-01

    Despite great effort by health organizations worldwide in fighting tuberculosis (TB), morbidity and mortality are not declining as expected. One of the reasons is related to the evolutionary development of Mycobacterium tuberculosis, in particular the Beijing genotype strains. In a previous study, we showed the association between the Beijing genotype and an increased mutation frequency for rifampin resistance. In this study, we use a Beijing genotype strain and an East-African/Indian genotyp...

  2. First insight into the genotypic diversity of clinical Mycobacterium tuberculosis isolates from Gansu Province, China.

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    Jie Liu

    Full Text Available BACKGROUND: Investigations of Mycobacterium tuberculosis genetic diversity in China have indicated a significant regional distribution. The aim of this study was to characterize the genotypes of clinical M. tuberculosis isolates obtained from Gansu, which has a special geographic location in China. METHODOLOGY/PRINCIPAL FINDINGS: A total of 467 clinical M. tuberculosis strains isolated in Gansu Province were genotyped by 15-locus mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTR and spoligotyping. The results showed that 445 isolates belonged to six known spoligotype lineages, whereas 22 isolates were unknown. The Beijing genotype was the most prevalent (87.58%, n = 409, while the shared type 1 was the dominant genotype (80.94%, n = 378. The second most common lineage was the T lineage, with 25 isolates (5.35%, followed by the H lineage with 5 isolates (1.07%, the MANU family (0.64%, 3 isolates, the U family (0.43%, 2 isolates and the CAS lineage with 1 isolate (0.21%. By using the VNTR15China method, we observed 15 groups and 228 genotypes among the 467 isolates. We found no association between the five larger groups (including the Beijing genotype and sex, age, or treatment status, and there was no noticeable difference in the group analysis in different areas. In the present study, seven of the 15 MIRU-VNTR loci were highly or moderately discriminative according to their Hunter-Gaston discriminatory index. CONCLUSIONS/SIGNIFICANCE: The Beijing genotype is the predominant genotype in Gansu province. We confirm that VNTR15China is suitable for typing Beijing strains in China and that it has a better discriminatory power than spoligotyping. Therefore, the use of both methods is the most suitable for genotyping analysis of M. tuberculosis.

  3. Genotypic characterization and historical perspective of Mycobacterium tuberculosis among older and younger Finns, 2008-2011.

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    Smit, P W; Haanperä, M; Rantala, P; Couvin, D; Lyytikäinen, O; Rastogi, N; Ruutu, P; Soini, H

    2014-11-01

    The Mycobacterium tuberculosis genotypes obtained from elderly Finns were assessed and compared with those obtained from younger Finns to comprehend the epidemiology of tuberculosis (TB) in Finland. From 2008 to 2011, a total of 1021 M. tuberculosis isolates were characterized by spoligotyping and 15-locus mycobacterial interspersed repetitive units-variable number tandem repeat typing. In total, 733 Finnish-born cases were included in the study, of which 466 (64%) were born before 1945 (older Finns). Of these, 63 (14%) shared an M. tuberculosis genotype with foreign-born or younger Finnish cases (born after 1945), and 59 (13%) shared a genotype with older Finnish cases. Eighty-five per cent had a unique genotypic profile while 70% belonged to T or Haarlem families, suggesting that ongoing transmission is infrequent among young and elderly Finns. Simultaneous reactivation of TB among older Finns was the most likely cause for clustering. As most isolates belonged to Haarlem or T, Finland was most likely affected by a similar TB epidemic at the beginning of the twentieth century as that seen in Sweden and Norway. Younger Finns were significantly more likely to be clustered (56% versus 27%, ptuberculosis isolates from elderly Finns were associated with dominant lineages of the early twentieth century and differed from the heterogeneous lineages found among younger TB patients. Additionally, younger TB patients were more likely to transmit TB than elderly Finns.

  4. Evolutionary History and Ongoing Transmission of Phylogenetic Sublineages of Mycobacterium tuberculosis Beijing Genotype in China

    Science.gov (United States)

    Yin, Qing-qin; Liu, Hai-can; Jiao, Wei-wei; Li, Qin-jing; Han, Rui; Tian, Jian-ling; Liu, Zhi-guang; Zhao, Xiu-qin; Li, Ying-jia; Wan, Kang-lin; Shen, A-dong; Mokrousov, Igor

    2016-01-01

    Mycobacterium tuberculosis Beijing genotype originated in China and has undergone a dramatic population growth and global spread in the last century. Here, a collection of M. tuberculosis Beijing family isolates from different provinces across all China was genotyped by high-resolution (24-MIRU-VNTR) and low-resolution, high-rank (modern and ancient sublineages) markers. The molecular profiles and global and local phylogenies were compared to the strain phenotype and patient data. The phylogeographic patterns observed in the studied collection demonstrate that large-scale (but not middle/small-scale) distance remains one of the decisive factors of the genetic divergence of M. tuberculosis populations. Analysis of diversity and network topology of the local collections appears to corroborate a recent intriguing hypothesis about Beijing genotype originating in South China. Placing our results within the Eurasian context suggested that important Russian B0/W148 and Asian/Russian A0/94-32 epidemic clones of the Beijing genotype could trace their origins to the northeastern and northwestern regions of China, respectively. The higher clustering of the modern isolates in children and lack of increased MDR rate in any sublineage suggest that not association with drug resistance but other (e.g., speculatively, virulence-related) properties underlie an enhanced dissemination of the evolutionarily recent, modern sublineage of the Beijing genotype in China. PMID:27681182

  5. Genotype MTBDR plus assay for molecular detection of rifampicin and isoniazid resistance in Mycobacterium tuberculosis

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    Soniya Sharma

    2014-01-01

    Full Text Available Aim: This study was performed for the rapid identification of Mycobacterium tuberculosis complex and its resistance to rifampicin and isoniazid, directly from the sputum samples of pulmonary tuberculosis patients. Materials and Methods: A commercially available genotype MTBDR plus assay was used for the identification and detection of mutations in Mycobacterial isolates. A total of 100 sputum samples of pulmonary tuberculosis patients were analyzed by using the genotype MTBDR plus assay. The MTBDR plus assay is designed to detect the mutations in the hotspot region of rpoB gene, katG and regulatory region of inhA gene. Results: The genotype MTBDR plus assay detected 22% multidrug resistant (MDR, 2% rifampicin (RMP monoresistant and 1% isoniazid (INH monoresistant isolates. In 22 MDR isolates, the codons most frequently involved in RMP-associated mutations were codon 531 (54.55%, 516 (31.82% and 526 (13.63%, and 90.90% of MDR isolates showed KatG S315T mutations and 9.1% showed inhA C-15T mutations associated with INH resistance. Conclusion: The new genotype MTBDR plus assay represents a rapid, reliable tool for the detection of MDR-TB, wherein results are obtained in 5 h allowing early and appropriate treatment, which is essential to cut the transmission path and reduce the spread of MDR-TB. The genotype MTBDR plus assay can readily be included in a routine laboratory work for the early diagnosis and control of MDR-TB.

  6. A study of Mycobacterium tuberculosis genotypic diversity & drug resistance mutations in Varanasi, north India

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    Anamika Gupta

    2014-01-01

    Full Text Available Background & objectives: One-fifth of the world′s new tuberculosis (TB cases and two-thirds of cases in the South East Asian region occur in India. Molecular typing of Mycobacterium tuberculosis isolates has greatly facilitated to understand the transmission of TB. This study was aimed to investigate the molecular epidemiology of M. tuberculosis genotypes in Varanasi, north India, and their association with clinical presentation among patients with pulmonary TB. Methods: M. tuberculosis isolates from 104 TB patients attending a tertiary referral hospital of north India were screened for susceptibility to isoniazid (INH, rifampicin (RIF, ethambutol (EMB and streptomycin (STR by proportion method and multiplex-allele-specific-polymerase chain reaction (MAS-PCR. These were genotyped by spoligotyping. The spoligotype patterns were compared with those in the international SITVIT2 spoligotyping database. Results: Eighty three of 104 isolates were distributed in 38 SITs, of which SIT3366 was newly created within the present study. The mass of ongoing transmission with MDR-TB isolates in Varanasi, northern India, was linked to Beijing genotype followed by the CAS1_Delhi lineage. HIV-seropositive patients had a significantly higher proportion of clustered isolates than HIV-seronegative patients and compared with the wild type(wt isolates, the isolates with katG315Thr mutation were considerably more likely to be clustered. Interpretation & conclusions: This study gives an insight into the M. tuberculosis genetic biodiversity in Varanasi, north India, the predominant spoligotypes and their impact on disease transmission. In this region of north India, TB is caused by a wide diversity of spoligotypes with predominance of four genotype lineages: Beijing, CAS, EAI and T. The Beijing genotype was the most frequent single spoligotype and strongly associated with multi drug resistant (MDR-TB isolates. These findings may have important implications for control

  7. Consequences of noncompliance for therapy efficacy and emergence of resistance in murine tuberculosis caused by the Beijing genotype of Mycobacterium tuberculosis

    NARCIS (Netherlands)

    J.E.M. de Steenwinkel (Jurriaan); M.T. ten Kate (Marian); G.J. de Knegt (Gerjo); H.A. Verbrugh (Henri); E.J. Aarnoutse (E. J.); M. Boeree (Martin); M.A. den Bakker (Michael); D. van Soolingen (Dick); I.A.J.M. Bakker-Woudenberg (Irma)

    2012-01-01

    textabstractDespite great effort by health organizations worldwide in fighting tuberculosis (TB), morbidity and mortality are not declining as expected. One of the reasons is related to the evolutionary development of Mycobacterium tuberculosis, in particular the Beijing genotype strains. In a previ

  8. Consequences of Noncompliance for Therapy Efficacy and Emergence of Resistance in Murine Tuberculosis Caused by the Beijing Genotype of Mycobacterium tuberculosis.

    NARCIS (Netherlands)

    Steenwinkel, J.E. de; Kate, M.T. Ten; Knegt, G.J. de; Verbrugh, H.A.; Aarnoutse, R.E.; Boeree, M.J.; Bakker, M.A. den; Soolingen, D. van; Bakker-Woudenberg, I.A.

    2012-01-01

    Despite great effort by health organizations worldwide in fighting tuberculosis (TB), morbidity and mortality are not declining as expected. One of the reasons is related to the evolutionary development of Mycobacterium tuberculosis, in particular the Beijing genotype strains. In a previous study, w

  9. Clonality and micro-diversity of a nationwide spreading genotype of Mycobacterium tuberculosis in Japan.

    Directory of Open Access Journals (Sweden)

    Takayuki Wada

    Full Text Available Mycobacterium tuberculosis transmission routes can be estimated from genotypic analysis of clinical isolates from patients. In Japan, still a middle-incidence country of TB, a unique genotype strain designated as 'M-strain' has been isolated nationwide recently. To ascertain the history of the wide spread of the strain, 10 clinical isolates from different areas were subjected to genome-wide analysis based on deep sequencers. Results show that all isolates possessed common mutations to those of referential strains. The greatest number of accumulated single nucleotide variants (SNVs from the oldest coalescence was 13 nucleotides, indicating high clonality of these isolates. When an SNV common to the isolates was used as a surrogate marker of the clone, authentic clonal isolates with variation in a reliable subset of variable number of tandem repeat (VNTR genotyping method can be selected successfully from clinical isolates populations of M. tuberculosis. When the authentic clones can also be assigned to sub-clonal groups by SNVs derived from the genomic comparison, they are classifiable into three sub-clonal groups with a bias of geographical origins. Feedback from genomic analysis of clinical isolates of M. tuberculosis to genotypic markers will be an efficient strategy for the big data in various settings for public health actions against TB.

  10. Clonality and micro-diversity of a nationwide spreading genotype of Mycobacterium tuberculosis in Japan.

    Science.gov (United States)

    Wada, Takayuki; Iwamoto, Tomotada; Tamaru, Aki; Seto, Junji; Ahiko, Tadayuki; Yamamoto, Kaori; Hase, Atushi; Maeda, Shinji; Yamamoto, Taro

    2015-01-01

    Mycobacterium tuberculosis transmission routes can be estimated from genotypic analysis of clinical isolates from patients. In Japan, still a middle-incidence country of TB, a unique genotype strain designated as 'M-strain' has been isolated nationwide recently. To ascertain the history of the wide spread of the strain, 10 clinical isolates from different areas were subjected to genome-wide analysis based on deep sequencers. Results show that all isolates possessed common mutations to those of referential strains. The greatest number of accumulated single nucleotide variants (SNVs) from the oldest coalescence was 13 nucleotides, indicating high clonality of these isolates. When an SNV common to the isolates was used as a surrogate marker of the clone, authentic clonal isolates with variation in a reliable subset of variable number of tandem repeat (VNTR) genotyping method can be selected successfully from clinical isolates populations of M. tuberculosis. When the authentic clones can also be assigned to sub-clonal groups by SNVs derived from the genomic comparison, they are classifiable into three sub-clonal groups with a bias of geographical origins. Feedback from genomic analysis of clinical isolates of M. tuberculosis to genotypic markers will be an efficient strategy for the big data in various settings for public health actions against TB.

  11. Beijing genotype of Mycobacterium tuberculosis is significantly associated with high-level fluoroquinolone resistance in Vietnam.

    Science.gov (United States)

    Duong, Duy An; Nguyen, Thi Hong Duyen; Nguyen, Thi Ngoc Lan; Dai, Viet Hoa; Dang, Thi Minh Ha; Vo, Sy Kiet; Do, Dang Anh Thu; Nguyen, Van Vinh Chau; Nguyen, Huy Dung; Dinh, Ngoc Sy; Farrar, Jeremy; Caws, Maxine

    2009-11-01

    Consecutive fluoroquinolone (FQ)-resistant isolates (n = 109) identified at the Pham Ngoc Thach Hospital for Tuberculosis, Ho Chi Minh City, Vietnam, were sequenced in the quinolone resistance-determining regions of the gyrA and gyrB genes and typed by large sequence polymorphism typing and spoligotyping to identify the Beijing genotype of Mycobacterium tuberculosis. Beijing genotype prevalence was compared with 109 consecutive isolates from newly presenting patients with pulmonary tuberculosis from the hospital outpatient department. Overall, 82.6% (n = 90/109) of isolates had mutations in gyrAB. Nine novel mutations were identified in gyrB (S486F, N538T, T539P, D500A, D500H, D500N, G509A, E540V, and E540D). The influence of these novel gyrB mutations on FQ resistance is not proven. The Beijing genotype was significantly associated with FQ resistance (odds ratio [OR], 2.39 [95% confidence interval {CI}, 1.34 to 4.25]; P = 0.003). Furthermore, Beijing genotype FQ-resistant isolates were significantly more likely than FQ-resistant isolates of other genotypes to have gyrA mutations (OR, 7.75 [95% CI, 2.84 to 21.15]; P = 0.0001) and high-level (>8 microg/ml) FQ resistance (OR, 11.0 [95% CI, 2.6 to 47.0]; P = 0.001). The underlying mechanism of the association of the Beijing genotype with high-level FQ resistance in this setting remains to be determined. The association of the Beijing genotype with relatively high-level FQ resistance conferred by specific gyrA mutations reported here is of grave concern given the epidemic spread of the Beijing genotype and the current hopes for shorter first-line treatment regimens based on FQs.

  12. Genotypes of Mycobacterium tuberculosis in patients at risk of drug resistance in Bolivia.

    Science.gov (United States)

    Monteserin, Johana; Camacho, Mirtha; Barrera, Lucía; Palomino, Juan Carlos; Ritacco, Viviana; Martin, Anandi

    2013-07-01

    Bolivia ranks among the 10 Latin American countries with the highest rates of tuberculosis (TB) and multidrug resistant (MDR) TB. In view of this, and of the lacking information on the population structure of Mycobacterium tuberculosis in the country, we explored genotype associations with drug resistance and clustering by analyzing isolates collected in 2010 from 100 consecutive TB patients at risk of drug resistance in seven of the nine departments in which Bolivia is divided. Fourteen isolates were MDR, 29 had other drug resistance profiles, and 57 were pansusceptible. Spoligotype family distribution was: Haarlem 39.4%, LAM 26.3%, T 22.2%, S 2.0%, X 1.0%, orphan 9.1%, with very low intra-family diversity and absence of Beijing genotypes. We found 66 different MIRU-VNTR patterns; the most frequent corresponded to Multiple Locus Variable Analysis (MLVA) MtbC15 patterns 860, 372 and 873. Twelve clusters, each with identical MIRU-VNTR and spoligotypes, gathered 35 patients. We found no association of genotype with drug resistant or MDR-TB. Clustering associated with SIT 50 and the H3 subfamily to which it belongs (pBolivia. However, results should be taken cautiously because the sample is small and includes a particular subset of M. tuberculosis population. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. DRUG RESISTANCE, VIABILITY AND VIRULENCE IN VITRO ОF MYCOBACTERIUM TUBERCULOSIS STRAINS OF DIFFERENT GENOTYPES

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    O. A. Manicheva

    2011-01-01

    Full Text Available Abstract. The drug resistance, mutation spectrum caused resistance to rifampicin and isoniazid, viability, cytotoxicity were studied as well as 111 clinical isolates of Mycobacterium tuberculosis (MBT were genotyped. Spoligotyping revealed 28 spoligotypes; the greatest number of strains belonged to genetic families Beijing and LAM. The typing of 59 strains of spoligotype SIT1 (Beijing allowed to differentiate 19 variants of IS6110-RFLP-profiles: 13 of them were individual and 6 were presented by clusters. The clusters А0 and В0 included the greatest number of M. tuberculosis strains — 21 (35,6% and 17 (28,8% accordingly. The high frequency of MLU and SLU of Beijing strains was associated with mutations rpoB Ser531→Leu and katG Ser315→Thr. The strains of M. tuberculosis belonged to another genetic families (T, H, Ural, U have shown drug resistance more often. The level of resistance to isoniazid in vitro in MLU/SLU strains of MBT of different genotypes, especially, was high in case of mixed mutations such as katG Ser315→Thr и inhA_T15. The rates of viability and cytotoxicity of MBT strains of studied genotypes with different spectrum of mutations and phenotypic drug resistance were not substantially distinguished.

  14. Transmission of extensively drug-resistant and multidrug resistant Mycobacterium tuberculosis in families identified by genotyping

    Institute of Scientific and Technical Information of China (English)

    YAN Li-ping; QIN Lian-hua; ZHANG Qing; SUN Hua; HAN Min; XIAO He-ping

    2013-01-01

    Background Diagnosis and appropriate treatment of multidrug-resistant tuberculosis (MDR-TB) remain major challenges.We sought to elucidate that persons who share a household with drug resistance tuberculosis patients are at high risk for primary drug resistance tuberculosis and how to prevent these outbreaks.Methods We used 12-locus mycobacterial interspersed repetitive unit and 7-locus variable-number tandem repeat to identify household transmission of extensively drug resistant and multiple drug resistant Mycobacterium tuberculosis in three families admitted in Shanghai Pulmonary Hospital affiliated with Tongji University.Drug susceptibility tests were done by the modified proportion method in the MGIT 960 system in the same time.Clinical data were also obtained from the subjects' medical records.Results All of the six strains were defined as Beijing genotype by the deletion-targeted multiplex PCR (DTM-PCR) identification on the genomic deletion RD105.Strains from family-1 had the same minisatellite interspersed repetitive unit (MIRU) pattem (232225172531) and the same MIRU pattern (3677235).Strains from family-2 had the same MIRU pattern (2212261553323) and the same MIRU pattern (3685134).Strains from family-3 did not have the same MIRU pattern and they differed at only one locus (223326173533,223325173533),and did not have the same VNTR pattern with two locus differed (3667233,3677234).Conclusions Household transmission exists in the three families.A clear chain of tuberculosis transmission within family exists.Tuberculosis susceptibility should be considered when there is more than one tuberculosis patients in a family.Household tuberculosis transmission could be prevented with adequate treatment of source patients.

  15. Genotypic analysis of Mycobacterium tuberculosis isolates from a Lisbon hospital in Portugal.

    Science.gov (United States)

    Perdigão, João; Milho, Catarina; Carrilho, Lurdes; Brum, Laura; Portugal, Isabel

    2009-01-01

    Portugal has one of the highest tuberculosis notification rates of the European Union with Lisbon Health Region having an incidence rate well above the national average. The present study analyses the transmission, drug susceptibility and characteristics of a study population from a Central Lisbon's Hospital. One hundred and thirty -two Mycobacterium tuberculosis clinical isolates were previously tested for drug susceptibility to first -line drugs. The multidrug (MDR) resistance rate was found to be 3.0%, while 13.6% of the isolates were resistant to one or more first -line drugs. HIV serology was available for 98 patients, 26 (26.5%) were positive. Genotyping was performed by MIRU -VNTR and 53 (40.2%) out of the 132 isolates were found to be distributed through 17 MIRU -VNTR clusters of two or more isolates. Lisboa strains accounted for 25.8% of all strains. We conclude that transmission of resistant and susceptible Mycobacterium tuberculosis strains is occurring, with special concern for Lisboa strains.

  16. Increased level of acute phase reactants in patients infected with modern Mycobacterium tuberculosis genotypes in Mwanza, Tanzania

    DEFF Research Database (Denmark)

    Stavrum, Ruth; PrayGod, George; Range, Nyagosya;

    2014-01-01

    BACKGROUND: There is increasing evidence to suggest that different Mycobacterium tuberculosis lineages cause variations in the clinical presentation of tuberculosis (TB). Certain M. tuberculosis genotypes/lineages have been shown to be more likely to cause active TB in human populations from...... a distinct genetic ancestry. This study describes the genetic biodiversity of M. tuberculosis genotypes in Mwanza city, Tanzania and the clinical presentation of the disease caused by isolates of different lineages. METHODS: Two-hundred-fifty-two isolates from pulmonary TB patients in Mwanza, Tanzania were...... characterized by spoligotyping, and 45 isolates were further characterized by mycobacterium interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR). The patients' level of the acute phase reactants AGP, CRP and neutrophil counts, in addition to BMI, were measured and compared to the M...

  17. Diverse Molecular Genotypes of Mycobacterium tuberculosis Complex Isolates Circulating in the Free State, South Africa

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    Anneke Van der Spoel van Dijk

    2016-01-01

    Full Text Available Tuberculosis is a serious public health concern especially in Africa and Asia. Studies describing strain diversity are lacking in the Free State region of South Africa. The aim of the study was to describe the diversity of Mycobacterium tuberculosis (M. tuberculosis strain families in the Free State province of South Africa. A total of 86 M. tuberculosis isolates were genotyped using spoligotyping. A 12-locus mycobacterial interspersed repetitive units-variable-number tandem repeats (MIRU-VNTRs typing was used to further characterize the resulting spoligotyping clusters. SITVITWEB identified 49 different patterns with allocation to six lineages including Latin-American-Mediterranean (LAM (18 isolates, T (14 isolates, Beijing (five isolates, S (six isolates, Haarlem (one isolate, and X (five isolates, while 37 (43.0% orphans were identified. Eight clusters included 37 isolates with identical spoligotypes (2 to 13/cluster. MIRU-VNTR typing further differentiated three spoligotyping clusters: SIT1/Beijing/MIT17, SIT33/LAM3/MIT213, and confirmed one SIT34/S/MIT311. In addition, SpolDB3/RIM assignment of the orphan strains resulted in a further 10 LAM and 13 T families. In total, LAM (28 isolates and T (27 isolates cause 63% of the individual cases of MTB in our study. The Free State has a highly diverse TB population with LAM being predominant. Further studies with inclusion of multidrug-resistant strains with larger sample size are warranted.

  18. [Discriminatory power of variable number on tandem repeats loci for genotyping Mycobacterium tuberculosis strains in China].

    Science.gov (United States)

    Chen, H X; Cai, C; Liu, J Y; Zhang, Z G; Yuan, M; Jia, J N; Sun, Z G; Huang, H R; Gao, J M; Li, W M

    2017-06-10

    Objective: Using the standard genotype method, variable number of tandem repeats (VNTR), we constructed a VNTR database to cover all provinces and proposed a set of optimized VNTR loci combinations for each province, in order to improve the preventive and control programs on tuberculosis, in China. Methods: A total of 15 loci VNTR was used to analyze 4 116 Mycobacterium tuberculosis strains, isolated from national survey of Drug Resistant Tuberculosis, in 2007. Hunter-Gaston Index (HGI) was also used to analyze the discriminatory power of each VNTR site. A set combination of 12-VNTR, 10-VNTR, 8-VNTR and 5-VNTR was respectively constructed for each province, based on 1) epidemic characteristics of M. tuberculosis lineages in China, with high discriminatory power and genetic stability. Results: Through the completed 15 loci VNTR patterns of 3 966 strains under 96.36% (3 966/4 116) coverage, we found seven high HGI loci (including QUB11b and MIRU26) as well as low stable loci (including QUB26, MIRU16, Mtub21 and QUB11b) in several areas. In all the 31 provinces, we found an optimization VNTR combination as 10-VNTR loci in Inner Mongolia, Chongqing and Heilongjiang, but with 8-VNTR combination shared in other provinces. Conclusions: It is necessary to not only use the VNTR database for tracing the source of infection and cluster of M. tuberculosis in the nation but also using the set of optimized VNTR combinations in monitoring those local epidemics and M. tuberculosis (genetics in local) population.

  19. Online tools for polyphasic analysis of Mycobacterium tuberculosis complex genotyping data: now and next.

    Science.gov (United States)

    Weniger, Thomas; Krawczyk, Justina; Supply, Philip; Harmsen, Dag; Niemann, Stefan

    2012-06-01

    Molecular diagnostics and genotyping of pathogens have become indispensable tools in clinical microbiology and disease surveillance. For isolates of the Mycobacterium tuberculosis complex (MTBC, causative agents of tuberculosis), multilocus variable number tandem repeat analysis (MLVA) targeting mycobacterial interspersed repetitive units (MIRU) has been internationally adopted as the new standard, portable, reproducible, and discriminatory typing method. Here, we review new sets of specialized web based bioinformatics tools that have become available for analyzing MLVA data especially in combination with other, complementary genotyping markers (polyphasic analysis). Currently, there are only two databases available that are not restricted to store one kind of genotyping data only, namely SITVIT/SpolDB4 and MIRU-VNTRplus. SITVIT/SpolDB4 (http://www.pasteur-guadeloupe.fr:8081/SITVITDemo) contains spoligotyping data from a large number of strains of diverse origin. However, besides options to query the data, the actual version of SITVIT/SpolDB4 offers no functionality for more complex analysis e.g. tree-based analysis. In comparison, the MIRU-VNTRplus web application (http://www.miru-vntrplus.org), represents a freely accessible service that enables users to analyze genotyping data of their strains alone or in comparison with a currently limited but well characterized reference database of strains representing the major MTBC lineages. Data (MLVA-, spoligotype-, large sequence polymorphism, and single nucleotide polymorphism) can be visualized and analyzed using just one genotyping method or a weighted combination of several markers. A variety of analysis tools are available such as creation of phylogenetic and minimum spanning trees, semi-automated phylogenetic lineage identification based on comparison with the reference database and mapping of geographic information. To facilitate scientific communication, a universal, expanding genotype nomenclature (MLVA MtbC15

  20. Genotyping and drug resistance patterns of Mycobacterium tuberculosis strains observed in a tuberculosis high-burden municipality in Northeast, Brazil

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    Roberta dos Santos Silva Luiz

    Full Text Available OBJECTIVES: This study has used a combination of clinical information, spoligotyping, and georeferencing system to elucidate the genetic diversity of the Mycobacterium tuberculosis isolates circulating in a TB-prevalent municipality of Northeast Brazil. METHODS: A total of 115 M. tuberculosis strains were isolated from pulmonary tuberculosis patients from January 2007 to March 2008 in Fortaleza. Drug susceptibility and spoligotyping assays were performed and place of residence of the patients were georeferenced. RESULTS: Of the M. tuberculosis strains studied, 51 (44.3% isolates were resistant to at least one drug (R-TB and 64 (55.7% were sensitive to all the drugs tested (S-TB. A high frequency of resistance was found in previously treated cases (84% and among new cases (16%; p < 0.001. a total of 74 (64% isolates were grouped into 22 spoligotyped lineages, while 41 (36% isolates were identified as new. among the predominant genotypes, 33% were latim american mediterranean (lam, 12% haarlem (h, and 5% u. there was no association of geographic distribution of rt-tb patients as compared to the controls and also the geographic location to the spoligotype patterns. the geospatial analysis revealed that 24 (23% patients (hot spot zones either shared the same residence or lived in a close neighborhood of a case. among these concentration zones, the patients lived in the same residence and shared a common genotype pattern and resistance pattern. DISCUSSION: it was observed that the spoligopatterns family distribution was similar to that reported for south america, prevailing the lam and h lineages. a high rate-case among the resistant TB group occurs as a result of transmitted and acquired resistance. A more effective surveillance program is needed in order to succeed in reducing tuberculosis in Northeast Brazil.

  1. A Mycobacterium tuberculosis cluster demonstrating the use of genotyping in urban tuberculosis control

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    Burdo Conny CA

    2009-09-01

    Full Text Available Abstract Background DNA fingerprinting of Mycobacterium tuberculosis isolates offers better opportunities to study links between tuberculosis (TB cases and can highlight relevant issues in urban TB control in low-endemic countries. Methods A medium-sized molecular cluster of TB cases with identical DNA fingerprints was used for the development of a visual presentation of epidemiologic links between cases. Results Of 32 cases, 17 (53% were linked to the index case, and 11 (34% to a secondary case. The remaining four (13% could not be linked and were classified as possibly caused by the index patient. Of the 21 cases related to the index case, TB developed within one year of the index diagnosis in 11 patients (52%, within one to two years in four patients (19%, and within two to five years in six patients (29%. Conclusion Cluster analysis underscored several issues for TB control in an urban setting, such as the recognition of the outbreak, the importance of reinfections, the impact of delayed diagnosis, the contribution of pub-related transmissions and its value for decision-making to extend contact investigations. Visualising cases in a cluster diagram was particularly useful in finding transmission locations and the similarities and links between patients.

  2. Genotypic detection of rifampicin and isoniazid resistant Mycobacterium tuberculosis strains by DNA sequencing: a randomized trial

    Directory of Open Access Journals (Sweden)

    El mashad Noha

    2009-01-01

    Full Text Available Abstract Background Tuberculosis is a growing international health concern. It is the biggest killer among the infectious diseases in the world today. Early detection of drug resistance allows starting of an appropriate treatment. Resistance to drugs is due to particular genomic mutations in specific genes of Mycobacterium tuberculosis(MTB. The aim of this study was to identify the presence of Isoniazid (INH and Rifampicin(RIF drug resistance in new and previously treated tuberculosis (TB cases using DNA sequencing. Methods This study was carried out on 153 tuberculous patients with positive Bactec 460 culture for acid fast bacilli. Results Of the 153 patients, 105 (68.6% were new cases and 48 (31.4% were previously treated cases. Drug susceptibility testing on Bactec revealed 50 resistant cases for one or more of the first line antituberculous. Genotypic analysis was done only for rifampicin resistant specimens (23 cases and INH resistant specimens (26 cases to detect mutations responsible for drug resistance by PCR amplification of rpoB gene for rifampicin resistant cases and KatG gene for isoniazid resistant cases. Finally, DNA sequencing was done for detection of mutation within rpoB and KatG genes. Genotypic analysis of RIF resistant cases revealed that 20/23 cases (86.9% of RIF resistance were having rpoB gene mutation versus 3 cases (13.1% having no mutation with a high statistical significant difference between them (P Conclusion We can conclude that rifampicin resistance could be used as a useful surrogate marker for estimation of multidrug resistance. In addition, Genotypic method was superior to that of the traditional phenotypic method which is time-consuming taking several weeks or longer.

  3. Drug resistant Mycobacterium tuberculosis of the Beijing genotype does not spread in Sweden.

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    Solomon Ghebremichael

    Full Text Available BACKGROUND: Drug resistant (DR and multi-drug resistant (MDR tuberculosis (TB is increasing worldwide. In some parts of the world 10% or more of new TB cases are MDR. The Beijing genotype is a distinct genetic lineage of Mycobacterium tuberculosis, which is distributed worldwide, and has caused large outbreaks of MDR-TB. It has been proposed that certain lineages of M. tuberculosis, such as the Beijing lineage, may have specific adaptive advantages. We have investigated the presence and transmission of DR Beijing strains in the Swedish population. METHODOLOGY/PRINCIPAL FINDINGS: All DR M. tuberculosis complex isolates between 1994 and 2008 were studied. Isolates that were of Beijing genotype were investigated for specific resistance mutations and phylogenetic markers. Seventy (13% of 536 DR strains were of Beijing genotype. The majority of the patients with Beijing strains were foreign born, and their country of origin reflects the countries where the Beijing genotype is most prevalent. Multidrug-resistance was significantly more common in Beijing strains than in non-Beijing strains. There was a correlation between the Beijing genotype and specific resistance mutations in the katG gene, the mabA-inhA-promotor and the rpoB gene. By a combined use of RD deletions, spoligotyping, IS1547, mutT gene polymorphism and Rv3135 gene analysis the Beijing strains could be divided into 11 genomic sublineages. Of the patients with Beijing strains 28 (41% were found in altogether 10 clusters (2-5 per cluster, as defined by RFLP IS6110, while 52% of the patients with non-Beijing strains were in clusters. By 24 loci MIRU-VNTR 31 (45% of the patients with Beijing strains were found in altogether 7 clusters (2-11 per cluster. Contact tracing established possible epidemiological linkage between only two patients with Beijing strains. CONCLUSIONS/SIGNIFICANCE: Although extensive outbreaks with non-Beijing TB strains have occurred in Sweden, Beijing strains have not

  4. Evaluation of the Genotype MTBDR Assay for Rapid Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Isolates

    OpenAIRE

    2006-01-01

    A novel PCR-based reverse hybridization method Genotype MTBDR assay (Hain Lifescience GmbH, Nehren, Germany) was evaluated for rapid detection of rifampin (RIF) and isoniazid (INH) resistance in Turkish Mycobacterium tuberculosis isolates. The Genotype MTBDR assay is designed to detect mutations within the 81-bp hotspot region of rpoB and mutations at katG codon 315. A total of 41 RIF-resistant M. tuberculosis isolates with rpoB mutations that were previously tested by the INNO-LiPA Rif.TB ki...

  5. Standard Genotyping Overestimates Transmission of Mycobacterium tuberculosis among Immigrants in a Low-Incidence Country.

    Science.gov (United States)

    Stucki, David; Ballif, Marie; Egger, Matthias; Furrer, Hansjakob; Altpeter, Ekkehardt; Battegay, Manuel; Droz, Sara; Bruderer, Thomas; Coscolla, Mireia; Borrell, Sonia; Zürcher, Kathrin; Janssens, Jean-Paul; Calmy, Alexandra; Mazza Stalder, Jesica; Jaton, Katia; Rieder, Hans L; Pfyffer, Gaby E; Siegrist, Hans H; Hoffmann, Matthias; Fehr, Jan; Dolina, Marisa; Frei, Reno; Schrenzel, Jacques; Böttger, Erik C; Gagneux, Sebastien; Fenner, Lukas

    2016-07-01

    Immigrants from regions with a high incidence of tuberculosis (TB) are a risk group for TB in low-incidence countries such as Switzerland. In a previous analysis of a nationwide collection of 520 Mycobacterium tuberculosis isolates from 2000 to 2008, we identified 35 clusters comprising 90 patients based on standard genotyping (24-locus mycobacterial interspersed repetitive-unit-variable-number tandem-repeat [MIRU-VNTR] typing and spoligotyping). Here, we used whole-genome sequencing (WGS) to revisit these transmission clusters. Genome-based transmission clusters were defined as isolate pairs separated by ≤12 single nucleotide polymorphisms (SNPs). WGS confirmed 17/35 (49%) MIRU-VNTR typing clusters; the other 18 clusters contained pairs separated by >12 SNPs. Most transmission clusters (3/4) of Swiss-born patients were confirmed by WGS, as opposed to 25% (4/16) of the clusters involving only foreign-born patients. The overall clustering proportion was 17% (90 patients; 95% confidence interval [CI], 14 to 21%) by standard genotyping but only 8% (43 patients; 95% CI, 6 to 11%) by WGS. The clustering proportion was 17% (67/401; 95% CI, 13 to 21%) by standard genotyping and 7% (26/401; 95% CI, 4 to 9%) by WGS among foreign-born patients and 19% (23/119; 95% CI, 13 to 28%) and 14% (17/119; 95% CI, 9 to 22%), respectively, among Swiss-born patients. Using weighted logistic regression, we found weak evidence of an association between birth origin and transmission (adjusted odds ratio of 2.2 and 95% CI of 0.9 to 5.5 comparing Swiss-born patients to others). In conclusion, standard genotyping overestimated recent TB transmission in Switzerland compared to WGS, particularly among immigrants from regions with a high TB incidence, where genetically closely related strains often predominate. We recommend the use of WGS to identify transmission clusters in settings with a low incidence of TB.

  6. MIRU-VNTRplus: a web tool for polyphasic genotyping of Mycobacterium tuberculosis complex bacteria.

    Science.gov (United States)

    Weniger, Thomas; Krawczyk, Justina; Supply, Philip; Niemann, Stefan; Harmsen, Dag

    2010-07-01

    Harmonized typing of bacteria and easy identification of locally or internationally circulating clones are essential for epidemiological surveillance and disease control. For Mycobacterium tuberculosis complex (MTBC) species, multi-locus variable number tandem repeat analysis (MLVA) targeting mycobacterial interspersed repetitive units (MIRU) has been internationally adopted as the new standard, portable, reproducible and discriminatory typing method. However, no specialized bioinformatics web tools are available for analysing MLVA data in combination with other, complementary typing data. Therefore, we have developed the web application MIRU-VNTRplus (http://www.miru-vntrplus.org). This freely accessible service allows users to analyse genotyping data of their strains alone or in comparison with a reference database of strains representing the major MTBC lineages. Analysis and comparisons of genotypes can be based on MLVA-, spoligotype-, large sequence polymorphism and single nucleotide polymorphism data, or on a weighted combination of these markers. Tools for data exploration include search for similar strains, creation of phylogenetic and minimum spanning trees and mapping of geographic information. To facilitate scientific communication, an expanding genotype nomenclature (MLVA MtbC15-9 type) that can be queried via a web- or a SOAP-interface has been implemented. An extensive documentation guides users through all application functions.

  7. Mycobacterium tuberculosis Beijing genotype is associated with HIV infection in Mozambique.

    Science.gov (United States)

    Viegas, Sofia O; Machado, Adelina; Groenheit, Ramona; Ghebremichael, Solomon; Pennhag, Alexandra; Gudo, Paula S; Cuna, Zaina; Langa, Egídio; Miotto, Paolo; Cirillo, Daniela M; Rastogi, Nalin; Warren, Rob M; van Helden, Paul D; Koivula, Tuija; Källenius, Gunilla

    2013-01-01

    The Beijing genotype is a lineage of Mycobacterium tuberculosis that is distributed worldwide and responsible for large epidemics, associated with multidrug-resistance. However, its distribution in Africa is less understood due to the lack of data. Our aim was to investigate the prevalence and possible transmission of Beijing strains in Mozambique by a multivariate analysis of genotypic, geographic and demographic data. A total of 543 M. tuberculosis isolates from Mozambique were spoligotyped. Of these, 33 were of the Beijing lineage. The genetic relationship between the Beijing isolates were studied by identification of genomic deletions within some Regions of Difference (RD), Restriction Fragment Length Polymorphism (RFLP) and Mycobacterial Interspersed Repetivie Unit - variable number tandem repeat (MIRU-VNTR). Beijing strains from South Africa, representing different sublineages were included as reference strains. The association between Beijing genotype, Human Immunodeficiency Virus (HIV) serology and baseline demographic data was investigated. HIV positive serostatus was significantly (p=0.023) more common in patients with Beijing strains than in patients with non-Beijing strains in a multivariable analysis adjusted for age, sex and province (14 (10.9%) of the 129 HIV positive patients had Beijing strains while 6/141 (4.3%) of HIV negative patients had Beijing strains). The majority of Beijing strains were found in the Southern region of Mozambique, particularly in Maputo City (17%). Only one Beijing strain was drug resistant (multi-drug resistant). By combined use of RD and spoligotyping, three genetic sublineages could be tentatively identified where 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 South African "sublineage 7" by RFLP and MIRU-VNTR, suggesting that this sublineage could have been recently introduced in Mozambique from South Africa

  8. Multiplex agarose gel electrophoresis system for variable number of tandem repeats genotyping: analysis example using Mycobacterium tuberculosis.

    Science.gov (United States)

    Wada, Takayuki; Maeda, Shinji

    2013-04-01

    As one genotyping method for Mycobacterium tuberculosis, variable number of tandem repeats (VNTR) is a promising tool to trace the undefined transmission of tuberculosis, but it often requires large equipment such as a genetic analyzer for DNA fragment analysis or CE system to conduct systematic analyses. For convenient genotyping at low cost in laboratories, we designed a multiplex PCR system that is applicable to agarose gel electrophoresis using fluorescent PCR primers. For tuberculosis genotyping by VNTR, the copy quantities of minisatellite DNA must be determined in more than 12 loci. The system can halve laborious electrophoresis processes by presenting an image of two VNTR amplicons on a single lane. No expensive equipment is necessary for this method. Therefore, it is useful even in developing countries. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Application of infrequent-restriction-site amplification for genotyping of Mycobacterium tuberculosis and non-tuberculous mycobacterium.

    OpenAIRE

    Choi, Tae Yeal; Kang, Jung Oak

    2002-01-01

    Infrequent restriction site amplification (IRS-PCR) is a method of amplifying DNA sequences, which flank an infrequent restriction site, and produces a strain-specific electrophoretic pattern. We studied the use of IRS-PCR to characterize Mycobacterium tuberculosis and non-tuberculous mycobactria (NTM). One-hundred and sixteen M. tuberculosis and nine NTM isolated at Hanyang University Hospital in Seoul, Korea were used in this study. IRS-PCR using AH1 and PX-G primers produced unique pattern...

  10. Genetic diversity of Mycobacterium tuberculosis from Guadalajara, Mexico and identification of a rare multidrug resistant Beijing genotype.

    Science.gov (United States)

    Flores-Treviño, Samantha; Morfín-Otero, Rayo; Rodríguez-Noriega, Eduardo; González-Díaz, Esteban; Pérez-Gómez, Héctor R; Bocanegra-García, Virgilio; Vera-Cabrera, Lucio; Garza-González, Elvira

    2015-01-01

    Determining the genetic diversity of M. tuberculosis strains allows identification of the distinct Mycobacterium tuberculosis genotypes responsible for tuberculosis in different regions. Several studies have reported the genetic diversity of M. tuberculosis strains in Mexico, but little information is available from the state of Jalisco. Therefore, the aim of this study was to determine the genetic diversity of Mycobacterium tuberculosis clinical isolates from Western Mexico. Sixty-eight M. tuberculosis isolates were tested for susceptibility to first-line drugs using manual Mycobacteria Growth Indicator Tube method and genotyped using spoligotyping and IS6110-restriction fragment length polymorphism (RFLP) pattern analyses. Forty-seven (69.1%) isolates were grouped into 10 clusters and 21 isolates displayed single patterns by spoligotyping. Three of the 21 single patterns corresponded to orphan patterns in the SITVITWEB database, and 1 new type that contained 2 isolates was created. The most prevalent lineages were T (38.2%), Haarlem (17.7%), LAM (17.7%), X (7.4%), S (5.9%), EAI (1.5%) and Beijing (1.5%). Six (12.8%) of the clustered isolates were MDR, and type 406 of the Beijing family was among the MDR isolates. Seventeen (26.2%) isolates were grouped into 8 clusters and 48 isolates displayed single patterns by IS6110-RFLP. Combination of IS6110-RFLP and spoligotyping reduced the clustering rate to 20.0%. The results show that T, Haarlem, and LAM are predominant lineages among clinical isolates of M. tuberculosis in Guadalajara, Mexico. Clustering rates indicated low transmission of MDR strains. We detected a rare Beijing genotype, SIT406, which was a highly resistant strain. This is the first report of this Beijing genotype in Latin America.

  11. Performance assessment of the GenoType MTBDRplus test and DNA sequencing in detection of multidrug-resistant Mycobacterium tuberculosis.

    Science.gov (United States)

    Huang, Wei-Lun; Chen, Huang-Yau; Kuo, Yuh-Min; Jou, Ruwen

    2009-08-01

    To facilitate the management of multidrug-resistant (MDR) tuberculosis, two nucleic acid sequence-based methods, the GenoType MTBDRplus test and DNA sequencing, were assessed for the rapid detection of drug-resistant Mycobacterium tuberculosis for the first time in the Asia-Pacific region. The performances of these two assays in detecting the presence of rifampin (rifampicin) (RIF) and isoniazid (INH) resistance-associated mutations in the rpoB, katG, inhA regulatory region, inhA, and oxyR-ahpC genes were compared to that of a conventional agar proportion drug susceptibility test. A total of 242 MDR and 30 pansusceptible M. tuberculosis isolates were evaluated in this study. The sensitivities obtained for RIF-resistant detection by the GenoType MTBDRplus test and by resistance gene sequencing were 95.5% and 97.9%, respectively. The sensitivities for INH resistance detection by the GenoType MTBDRplus test and by resistance gene sequencing were 81.8% and 93.4%, respectively. Together, the sensitivity for MDR tuberculosis detection was 78.5% with the GenoType MTBDRplus test and 91.3% by resistance gene sequencing. The specificity for RIF resistance, INH resistance, and MDR detection was 100% by both methods. The GenoType MTBDRplus test has the advantage of a short turnaround time for drug-resistant M. tuberculosis detection. Overall, the two assays performed equally well in detecting RIF resistance (P = 0.13). However, DNA sequencing demonstrated superior performance in detecting INH resistance (P tuberculosis (P GenoType MTBDRplus test, especially for different geographic areas with genetically diverse M. tuberculosis strains.

  12. Real-time PCR assay for rapid detection of epidemiologically and clinically significant Mycobacterium tuberculosis Beijing genotype isolates.

    Science.gov (United States)

    Mokrousov, Igor; Vyazovaya, Anna; Zhuravlev, Viacheslav; Otten, Tatiana; Millet, Julie; Jiao, Wei-Wei; Shen, A-Dong; Rastogi, Nalin; Vishnevsky, Boris; Narvskaya, Olga

    2014-05-01

    Mycobacterium tuberculosis Beijing genotype strains are rapidly disseminating, frequently hypervirulent, and multidrug resistant. Here, we describe a method for their rapid detection by real-time PCR that targets the specific IS6110 insertion in the dnaA-dnaN genome region. The method was evaluated with a geographically and genetically diverse collection representing areas in East Asia and the former Soviet Union in which the Beijing genotype is endemic and epidemic (i.e., major foci of its global propagation) and with clinical specimens.

  13. Clonal expansion of both modern and ancient genotypes of Mycobacterium tuberculosis in southern Taiwan.

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    Jia-Ru Chang

    Full Text Available 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.

  14. Genotyping of Mycobacterium tuberculosis clinical isolates in two cities of Turkey: Description of a new family of genotypes that is phylogeographically specific for Asia Minor

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    Durmaz Riza

    2005-07-01

    Full Text Available Abstract Background Population-based bacterial genetics using repeated DNA loci is an efficient approach to study the biodiversity and phylogeographical structure of human pathogens, such as Mycobacterium tuberculosis, the agent of tuberculosis. Indeed large genetic diversity databases are available for this pathogen and are regularly updated. No population-based polymorphism data were yet available for M. tuberculosis in Turkey, at the crossroads of Eurasia. Results A total of 245 DNAs from Mycobacterium tuberculosis clinical isolates from tuberculosis patients residing in Turkey (Malatya n = 147 or Ankara n = 98 were genotyped by spoligotyping, a high-throughput genotyping method based on the polymorphism of the Direct Repeat locus. Thirty-three spoligotyping-defined clusters including 206 patients and 39 unique patterns were found. The ST41 cluster, as designated according to the international SpolDB3 database project, represented one fourth and when gathered to three genotypes, ST53, ST50 and ST284, one half of all the isolates. Out of 34 clinical isolates harboring ST41 which were further genotyped by IS6110 and by MIRU-VNTR typing, a typical 2-copy IS6110-RFLP pattern and a "215125113322" MIRU-VNTR pattern were observed among 21 clinical isolates. Further search in various databases confirms the likely Turkish-phylogeographical specificity of this clonal complex. Conclusion We described a new phylogeographically-specific clone of M. tuberculosis, designated LAM7-TUR. Further investigations to assess its frequency within all regions of Turkey and its phylogeographical origin and phylogenetic position within the global M. tuberculosis phylogenetic tree will shed new light on its endemicity in Asia Minor.

  15. Difference in Antibody Responses to Mycobacterium tuberculosis Antigens in Japanese Tuberculosis Patients Infected with the Beijing/Non-Beijing Genotype

    Science.gov (United States)

    Zhao, Jingge; Okumura, Masao; Yanai, Hideki; Matsumoto, Makoto; Mizuno, Kazue; Ono, Kenji; Oda, Tetsuya; Ashino, Yugo; Matsuba, Takashi; Yoshiyama, Takashi; Suzuki, Yasuhiko

    2017-01-01

    The Beijing genotype Mycobacterium tuberculosis (MTB), notorious for its virulence and predisposition to relapse, could be identified by spoligotyping based on genetic heterogeneity. The plasma samples from 20 cases of Beijing and 16 cases of non-Beijing MTB infected individuals and 24 healthy controls (HCs) were collected, and antibodies against 11 antigens (Rv0679c142Asn, Rv0679c142Lys, Ag85B, Ag85A, ARC, TDM-M, TDM-K, HBHA, MDP-1, LAM, and TBGL) were measured by ELISA. Compared to the HCs, the MTB infected subjects showed higher titers of anti-Ag85B IgG (positivity 58.2%) and anti-ACR IgG (positivity 48.2%). Of note, anti-ACR IgG showed higher titer in Beijing MTB infected tuberculosis (TB) patients than in HC (Kruskal–Wallis test, p < 0.05), while the levels of anti-Ag85B, anti-TBGL, anti-TDM-K, and anti-TDM-M IgG were higher in non-Beijing TB patients than in HC. Moreover, anti-Ag85B IgG showed higher response in non-Beijing TB patients than in Beijing TB patients (p < 0.05; sensitivity, 76.9% versus 44.4%). The sensitivity and specificity analysis showed that 78.8% Beijing infected individuals were negative in anti-TBGL-IgG or/and anti-Ag85B-IgG, while 75.0% of those were positive in anti-TBGL-IgA or/and anti-ACR-IgG tests. These results indicate the possibility of developing antibody-based test to identify Beijing MTB. PMID:28182078

  16. A first insight into the genotypic diversity of Mycobacterium tuberculosis from Rwanda

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    Gafirita James

    2012-11-01

    Full Text Available Abstract Background Mycobacterium tuberculosis complex (MTC is the causative agent of tuberculosis (TB. Globally, increasing evidence shows that in M. tuberculosis, transmission varies from strain to strain and that different strains exhibit a range of geographical and host specificities, pathogenicity, and drug susceptibility. Therefore rapid and accurate differentiation of the members of MTC is critical in guiding treatment and public health decisions. We carried out a study at different health units and the National Reference Laboratory in Rwanda identify Mycobacterium tuberculosis complex species prevalent in TB patients in Rwanda. We further characterized the isolates using spoligotyping in order to gain an insight into the strain diversity of drug resistant and susceptible isolates of M. tuberculosis in this setting. Methods A total of 151 isolates from culture positive sputum samples were harvested, heat killed at 80°C for two hours, and then shipped to Makerere University College of Health Sciences, Uganda, for speciation and typing. Species identification was achieved by regions of difference (RD analysis, while Spoligotyping was done to identify strain types. Results Region of difference analysis identified all the 151 isolates as M. tuberculosis. Spoligotyping revealed predominance of the T2 family (58.3%, 88/151, with SIT 52 being the most prevalent strain (31.8%, 48/151. Among the 151 isolates, 64 (42.4% were multidrug resistant (MDR with 3 cases on mono-resistance. Of 94 retreatment cases, 48 (51.1% were MDR and of 46 newly presenting cases 14 (30.4% were MDR. There was a significant difference (p=0.01 in anti-TB drug resistance between new and retreatment cases in the sample. However, there was no significant relationship between HIV serostatus and the two major strain types SIT 52 (p =0.15and SIT 152 (p = 0.41. Conclusion Mycobacterium tuberculosis is the most prevalent species of Mycobacterium tuberculosis complex in Rwanda

  17. First baseline of circulating genotypic lineages of Mycobacterium tuberculosis in patients from the brazilian borders with Argentina and Paraguay.

    Science.gov (United States)

    Machado, Luzia Neri C; Marcondes, Nadir R; Leite, Clarice Q Fijimura; Santos, Adolfo C Barreto; Pavan, Fernando Rogério; Baldin, Vanessa Pietrowski; Castilho, Aline Lemes; Siqueira, Vera Lúcia D; Baeza, Lilian Cristiane; Berghs, Henri; Cardoso, Rosilene Fressatti

    2014-01-01

    At the triple border Brazil/Paraguay/Argentina there is easy mobility from one city to another for economic and tourism activities. This constant and fast population mobility is mainly to visit Iguazu Falls, in the Iguazu River, on the border of the Brazilian state of Paraná and the Argentina. As the incidence of tuberculosis is high in this setting, our study aimed to establish a first baseline of circulating genotypic lineages of Mycobacterium tuberculosis. This study included 120 patients from 10 cities in southwestern Paraná, Brazil with pulmonary symptoms, from July 2009 to July 2011. Information about sex, age, clinical features and address was collected by reviewing the national tuberculosis notification database. Of these, 96 (80%) isolates were identified as M. tuberculosis and 22 (22.9%) were drug resistant (20, 20.8% INH mono-resistant and 2, 2.1% multidrug-resistant). All isolates were subjected to genotyping by Spoligotyping and MIRU-VNTR typing. The distribution of the isolates analyzed by spoligotyping revealed 30 distinct patterns. The four mainly detected clades were Latin American and Mediterranean (LAM), ill-defined T, Haarlem (H) and S. The MIRU-VNTR showed 85 distinct patterns. Spoligotyping combined to MIRU-VNTR allowed 90 distinct patterns. Our study demonstrated that there is significant molecular diversity in circulating M. tuberculosis, with predominance of the LAM and T clades in cities of southwestern Paraná, Brazil, bordering Argentina and Paraguay.

  18. Phenotypic and Genotypic Analysis of Multidrug-Resistant Mycobacterium tuberculosis Isolates from Sudanese Patients

    Science.gov (United States)

    Salih, Mohamed Ahmed; Ali, Manasik; EL-Zaki, Salah-Eldin; Abuzeid, Nadir; Elgadi, Zeinab Abubaker Mohammed; Altayb, Hisham N.; Elegail, Asrar M. A.; Ibrahim, Nuha Y.; Elamin, Bahaeldin K.

    2017-01-01

    Background. Currently, mutations in rpoB, KatG, and rrs genes and inhA promoter were considered to be involved in conferring resistance to rifampicin, isoniazid, and streptomycin in Mycobacterium tuberculosis (MTB). Objective. The aims of this study were to detect the prevalence of first-line tuberculosis (TB) drug resistance among a group of previously treated and newly detected TB patients, to determine the association between prevalence of multidrug resistance (MDR) and demographic information (age and sex), to explain genes correlated with MDR Mycobacterium tuberculosis, and to characterize MTB via 16S ribosomal RNA (16S rRNA) analysis. Methods. A hundred MTB isolates from Sudanese pulmonary TB patients were included in the study. The proportional method of drug susceptibility test was carried out on Löwenstein-Jensen media. Multiplex PCR of rpoB and KatG genes and inhA promoter was conducted; then rrs genes were amplified by conventional PCR and were sequenced. The sequences of the PCR product were compared with known rrs gene sequences in the GenBank database by multiple sequence alignment tools. Result. The prevalence of MDR was 14.7% among old cases and 5.3% among newly diagnosed cases. Conclusion. Mutations in rrs could be considered as a diagnostic marker. PMID:28197340

  19. Phenotypic and Genotypic Analysis of Multidrug-Resistant Mycobacterium tuberculosis Isolates from Sudanese Patients

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    Solima M. A. Sabeel

    2017-01-01

    Full Text Available Background. Currently, mutations in rpoB, KatG, and rrs genes and inhA promoter were considered to be involved in conferring resistance to rifampicin, isoniazid, and streptomycin in Mycobacterium tuberculosis (MTB. Objective. The aims of this study were to detect the prevalence of first-line tuberculosis (TB drug resistance among a group of previously treated and newly detected TB patients, to determine the association between prevalence of multidrug resistance (MDR and demographic information (age and sex, to explain genes correlated with MDR Mycobacterium tuberculosis, and to characterize MTB via 16S ribosomal RNA (16S rRNA analysis. Methods. A hundred MTB isolates from Sudanese pulmonary TB patients were included in the study. The proportional method of drug susceptibility test was carried out on Löwenstein-Jensen media. Multiplex PCR of rpoB and KatG genes and inhA promoter was conducted; then rrs genes were amplified by conventional PCR and were sequenced. The sequences of the PCR product were compared with known rrs gene sequences in the GenBank database by multiple sequence alignment tools. Result. The prevalence of MDR was 14.7% among old cases and 5.3% among newly diagnosed cases. Conclusion. Mutations in rrs could be considered as a diagnostic marker.

  20. Consequences of noncompliance for therapy efficacy and emergence of resistance in murine tuberculosis caused by the Beijing genotype of Mycobacterium tuberculosis.

    Science.gov (United States)

    de Steenwinkel, Jurriaan E M; ten Kate, Marian T; de Knegt, Gerjo J; Verbrugh, Henri A; Aarnoutse, Rob E; Boeree, Martin J; den Bakker, Michael A; van Soolingen, Dick; Bakker-Woudenberg, Irma A J M

    2012-09-01

    Despite great effort by health organizations worldwide in fighting tuberculosis (TB), morbidity and mortality are not declining as expected. One of the reasons is related to the evolutionary development of Mycobacterium tuberculosis, in particular the Beijing genotype strains. In a previous study, we showed the association between the Beijing genotype and an increased mutation frequency for rifampin resistance. In this study, we use a Beijing genotype strain and an East-African/Indian genotype strain to investigate with our mouse TB model whether the higher mutation frequency observed in a Beijing genotype strain is associated with treatment failure particularly during noncompliance therapy. Both genotype strains showed high virulence in comparison to that of M. tuberculosis strain H37Rv, resulting in a highly progressive infection with a rapid lethal outcome in untreated mice. Compliance treatment was effective without relapse of TB irrespective of the infecting strain, showing similar decreases in the mycobacterial load in infected organs and similar histopathological changes. Noncompliance treatment, simulated by a reduced duration and dosing frequency, resulted in a relapse of infection. Relapse rates were correlated with the level of noncompliance and were identical for Beijing infection and East African/Indian infection. However, only in Beijing-infected mice, isoniazid-resistant mutants were selected at the highest level of noncompliance. This is in line with the substantial selection of isoniazid-resistant mutants in vitro in a wide isoniazid concentration window observed for the Beijing strain and not for the EAI strain. These results suggest that genotype diversity of M. tuberculosis may be involved in emergence of resistance and indicates that genotype-tailor-made treatment should be investigated.

  1. Increased transmission of Mycobacterium tuberculosis Beijing genotype strains associated with resistance to streptomycin: a population-based study.

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    Tran N Buu

    Full Text Available BACKGROUND: Studies have shown that the Mycobacterium tuberculosis Beijing genotype is an emerging pathogen that is frequently associated with drug resistance. This suggests that drug resistant Beijing strains have a relatively high transmission fitness compared to other drug-resistant strains. METHODS AND FINDINGS: We studied the relative transmission fitness of the Beijing genotype in relation to anti-tuberculosis drug resistance in a population-based study of smear-positive tuberculosis patients prospectively recruited and studied over a 4-year period in rural Vietnam. Transmission fitness was analyzed by clustering of cases on basis of three DNA typing methods. Of 2531 included patients, 2207 (87% were eligible for analysis of whom 936 (42% were in a DNA fingerprint cluster. The clustering rate varied by genotype with 292/786 (37% for the Beijing genotype, 527/802 (67% for the East-African Indian (EAI genotype, and 117/619 (19% for other genotypes. Clustering was associated with the EAI compared to the Beijing genotype (adjusted odds ratio (OR(adj 3.4: 95% CI 2.8-4.4. Patients infected with streptomycin-resistant strains were less frequently clustered than patients infected with streptomycin-susceptible strains when these were of the EAI genotype (OR(adj 0.6, 95% CI 0.4-0.9, while this pattern was reversed for strains of the Beijing genotype (OR(adj 1.3, 95% CI 1.0-1.8, p for difference 0.002. The strong association between Beijing and MDR-TB (OR(adj 7.2; 95% CI 4.2-12.3 existed only if streptomycin resistance was present. CONCLUSIONS: Beijing genotype strains showed less overall transmissibility than EAI strains, but when comparisons were made within genotypes, Beijing strains showed increased transmission fitness when streptomycin-resistant, while the reverse was observed for EAI strains. The association between MDR-TB and Beijing genotype in this population was strongly dependent on resistance to streptomycin. Streptomycin resistance may

  2. Genotypic characterization of multi-drug-resistant Mycobacterium tuberculosis isolates in Myanmar.

    Science.gov (United States)

    Aye, Khin Saw; Nakajima, Chie; Yamaguchi, Tomoyuki; Win, Min Min; Shwe, Mu Mu; Win, Aye Aye; Lwin, Thandar; Nyunt, Wint Wint; Ti, Ti; Suzuki, Yasuhiko

    2016-03-01

    The number of multi-drug-resistant tuberculosis (MDR-TB) cases is rising worldwide. As a countermeasure against this situation, the implementation of rapid molecular tests to identify MDR-TB would be effective. To develop such tests, information on the frequency and distribution of mutations associating with phenotypic drug resistance in Mycobacterium tuberculosis is required in each country. During 2010, the common mutations in the rpoB, katG and inhA of 178 phenotypically MDR M. tuberculosis isolates collected by the National Tuberculosis Control Program (NTP) in Myanmar were investigated by DNA sequencing. Mutations affecting the 81-bp rifampicin (RIF) resistance-determining region (RRDR) of the rpoB were identified in 127 of 178 isolates (71.3%). Two of the most frequently affected codons were 531 and 526, with percentages of 48.3% and 14.0% respectively. For isoniazid (INH) resistance, 114 of 178 MDR-TB isolates (64.0%) had mutations in the katG in which a mutation-conferring amino acid substitution at codon 315 from Ser to Thr was the most common. Mutations in the inhA regulatory region were also detected in 20 (11.2%) isolates, with the majority at position -15. Distinct mutation rate and pattern from surrounding countries might suggest that MDR-TB has developed and spread domestically in Myanmar.

  3. First baseline of circulating genotypic lineages of Mycobacterium tuberculosis in patients from the brazilian borders with Argentina and Paraguay.

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    Luzia Neri C Machado

    Full Text Available BACKGROUND: At the triple border Brazil/Paraguay/Argentina there is easy mobility from one city to another for economic and tourism activities. This constant and fast population mobility is mainly to visit Iguazu Falls, in the Iguazu River, on the border of the Brazilian state of Paraná and the Argentina. As the incidence of tuberculosis is high in this setting, our study aimed to establish a first baseline of circulating genotypic lineages of Mycobacterium tuberculosis. METHODOLOGY/PRINCIPAL FINDINGS: This study included 120 patients from 10 cities in southwestern Paraná, Brazil with pulmonary symptoms, from July 2009 to July 2011. Information about sex, age, clinical features and address was collected by reviewing the national tuberculosis notification database. Of these, 96 (80% isolates were identified as M. tuberculosis and 22 (22.9% were drug resistant (20, 20.8% INH mono-resistant and 2, 2.1% multidrug-resistant. All isolates were subjected to genotyping by Spoligotyping and MIRU-VNTR typing. The distribution of the isolates analyzed by spoligotyping revealed 30 distinct patterns. The four mainly detected clades were Latin American and Mediterranean (LAM, ill-defined T, Haarlem (H and S. The MIRU-VNTR showed 85 distinct patterns. Spoligotyping combined to MIRU-VNTR allowed 90 distinct patterns. CONCLUSIONS/SIGNIFICANCE: Our study demonstrated that there is significant molecular diversity in circulating M. tuberculosis, with predominance of the LAM and T clades in cities of southwestern Paraná, Brazil, bordering Argentina and Paraguay.

  4. Genotypic and phenotypic characteristics of aminoglycoside-resistant Mycobacterium tuberculosis isolates in Latvia.

    Science.gov (United States)

    Bauskenieks, Matiss; Pole, Ilva; Skenders, Girts; Jansone, Inta; Broka, Lonija; Nodieva, Anda; Ozere, Iveta; Kalvisa, Adrija; Ranka, Renate; Baumanis, Viesturs

    2015-03-01

    Mutations causing resistance to aminoglycosides, such as kanamycin (KAN), amikacin (AMK), and streptomycin, are not completely understood. In this study, polymorphisms of aminoglycoside resistance influencing genes such as rrs, eis, rpsL, and gidB in 41 drug-resistant and 17 pan-sensitive Mycobacterium tuberculosis clinical isolates in Latvia were analyzed. Mutation A1400G in rrs gene was detected in 92% isolates with high resistance level to KAN and diverse MIC level to AMK. Mutations in promoter region of eis were detected in 80% isolates with low-level MIC of KAN. The association of K43R mutation in rpsL gene, a mutation in the rrs gene at position 513, and various polymorphisms in gidB gene with distinct genetic lineages of M. tuberculosis was observed. The results of this study suggest that association of different controversial mutations of M. tuberculosis genes to the drug resistance phenotype should be done in respect to genetic lineages.

  5. Evolution of Mycobacterium tuberculosis.

    Science.gov (United States)

    Behr, Marcel A

    2013-01-01

    Genomic studies have provided a refined understanding of the genetic diversity within the Mycobacterium genus, and more specifically within Mycobacterium tuberculosis. These results have informed a new perspective on the macro- and micro-evolution of the tubercle bacillus. In the first step, a M. kansasii-like opportunistic pathogen acquired new genes, through horizontal gene transfer, that enabled it to better exploit an intracellular niche and ultimately evolve into a professional pathogen. In the second step, different subspecies and strains of the M. tuberculosis complex emerged through mutation and deletion of unnecessary DNA. Understanding the differences between M. tuberculosis and related less pathogenic mycobacteria is expected to reveal key bacterial virulence mechanisms and provide opportunities to understand host resistance to mycobacterial infection. Understanding differences within the M. tuberculosis complex and the evolutionary forces shaping these differences is important for investigating the basis of its success as both a symbiont and a pathogen.

  6. Comparative study on genotypic and phenotypic second-line drug resistance testing of Mycobacterium tuberculosis complex isolates.

    NARCIS (Netherlands)

    Ingen, J. van; Simons, S.O.; Zwaan, R. de; Laan, T. van der; Kamst-van Agterveld, M.; Boeree, M.J.; Soolingen, D. van

    2010-01-01

    The mycobacterium growth indicator tube (MGIT960) automated liquid medium testing method is becoming the international gold standard for second-line drug susceptibility testing of multidrug- and extensively drug-resistant Mycobacterium tuberculosis complex isolates. We performed a comparative study

  7. Beijing genotype of Mycobacterium tuberculosis is significantly associated with linezolid resistance in multidrug-resistant and extensively drug-resistant tuberculosis in China.

    Science.gov (United States)

    Zhang, Zhijian; Pang, Yu; Wang, Yufeng; Liu, Changting; Zhao, Yanlin

    2014-03-01

    Linezolid (LNZ) is a promising antimicrobial agent for the treatment of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB). To investigate the efficacy of LNZ among MDR-TB and XDR-TB in China, the LNZ susceptibility of 158 MDR-TB isolates from the national drug resistance survey was determined by the minimum inhibitory concentration method. The 158 MDR-TB isolates were also sequenced in the 23S rRNA, rplC and rplD genes conferring LNZ resistance and were typed using spoligotyping to identify the Beijing genotype of Mycobacterium tuberculosis. Overall, the prevalence of LNZ-resistant isolates was 10.8% (17/158) among MDR-TB isolates circulating in China. Beijing genotype was significantly associated with LNZ resistance in MDR-TB and XDR-TB (odds ratio=4.66, 95% confidence interval 1.03-21.16; P=0.033). In addition, a higher frequency of LNZ-resistant isolates was observed among XDR-TB strains (60%) compared with the MDR (5.6%; PMutations in 23S rRNA and rplC were responsible for only 29.4% of LNZ-resistant M. tuberculosis among MDR-TB isolates, and a novel non-synonymous substitution His155Asp in rplC was first identified to be contributing to low-level LNZ resistance (2μg/mL) in M. tuberculosis. The unsatisfactory correlation between mutant genotypes highlights the urgent need to investigate another mechanism for LNZ resistance that has not yet been described.

  8. Mutations in the regulatory network underlie the recent clonal expansion of a dominant subclone of the Mycobacterium tuberculosis Beijing genotype.

    Science.gov (United States)

    Schürch, Anita C; Kremer, Kristin; Warren, Robin M; Hung, Nguyen V; Zhao, Yanlin; Wan, Kanglin; Boeree, Martin J; Siezen, Roland J; Smith, Noel H; van Soolingen, Dick

    2011-04-01

    The Beijing genotype family is an epidemiologically important sub-group of Mycobacterium tuberculosis. It has been suggested that the high frequency of the Beijing isolates in some areas could be explained by selective advantages. Some evidence suggests that the emerging and most frequently isolated "Typical Beijing" lineage has the ability to circumvent BCG-induced immunity. To investigate the phylogeny of the Beijing genotype of M. tuberculosis, the genome of six Beijing strains from three different countries was sequenced with next-generation sequencing. The phylogeny of these strains was established using single nucleotide polymorphisms (SNPs). The three Typical Beijing strains clustered very tightly in the Beijing phylogeny suggesting that Typical Beijing strains represent a monophyletic lineage and resulted from recent diversification. Typing of 150 M. tuberculosis strains with a subset of the SNPs and comparison of the IS6110 restriction-fragment length polymorphism (RFLP) patterns of these strains to a database of 1522 Beijing RFLP patterns revealed that about 80% of all Beijing strains belong to the Typical Beijing subclone, which indicates clonal expansion. To identify the genomic changes that are characteristic for all Typical Beijing strains and to reconstruct their most recent common ancestor, the presence of SNPs was assayed in other Beijing strains. We identified 51 SNPs that define the minimal set of polymorphisms for all Typical Beijing strains. Nonsynonymous polymorphisms in genes coding for the regulatory network were over-represented in this set of mutations. We suggest that alterations in the response to environmental signals may have enabled Typical Beijing strains to develop the emerging phenotype.

  9. Underestimation of the resistance of Mycobacterium tuberculosis to second-line drugs by the new GenoType MTBDRsl test.

    Science.gov (United States)

    Jin, Jialin; Shen, Yaojie; Fan, Xiaoping; Diao, Ni; Wang, Feifei; Wang, Sen; Weng, Xinhua; Zhang, Wenhong

    2013-01-01

    The GenoType MTBDRsl is a new-generation PCR-based line-probe assay for the detection of extensively drug-resistant tuberculosis (XDR-TB). This study evaluated the performance of MTBDRsl in detecting genotypic resistance to ethambutol, kanamycin, and ofloxacin in Mycobacterium tuberculosis (MTB) strains. The drug resistance of 262 unique clinical MTB isolates from China was analyzed with MTBDRsl, traditional TB drug susceptibility testing (DST), and sequencing. Sensitivity of MTBDRsl was 62.4% (93/149; 95% CI = 54.1 to 70.2) for detection of ethambutol resistance, 57.9% (55/95; 95% CI = 47.3 to 68) for kanamycin resistance, and 81% (111/137; 95% CI = 73.4 to 87.2) for ofloxacin resistance; specificity was 76.8% (86/112; 95% CI = 67.9 to 84.2), 98.8% (164/166; 95% CI = 95.7 to 99.9), and 91.1% (113/124; 95% CI = 84.7 to 95.5), respectively. Sequencing suggested that 36.9% (55/149) of ethambutol-resistant strains had no embB306 mutation and that 26.8% (40/149) had embB497 mutation not covered by MTBDRsl. Furthermore, MTBDRsl indicated ethambutol resistance in 23.2% (26/112) of ethambutol-susceptible strains, of which 92.3% (24/26) were confirmed resistant by sequencing. This study demonstrated that genotypic resistance to ethambutol, kanamycin, and ofloxacin in MTB can be quickly determined with the MTBDRsl. As a rapid and convenient genetic method, this assay could function as a supplement to traditional DST. More relevant genetic markers are needed to improve sensitivity.

  10. Usefulness of ligation-mediated PCR genotyping in tracking outbreak-associated Mycobacterium tuberculosis strains.

    Science.gov (United States)

    Mardassi, H; Namouchi, A; Karbouli, A; Khabouchi, N; Haltiti, R; Zarrouk, M; Mhennii, B; Kaabi, S

    2005-01-01

    With the emergence of a multidrug resistant tuberculosis (MDR-TB) outbreak, the availability of a rapid typing method to carry out a nationwide prospective survey for the tracking of newly emerging MDR-TB foci became a priority. For this purpose, we have applied the IS6110 PCR-based genotyping assay, namely, LM-PCR (ligation-mediated PCR). The latter relies on ligation of a synthetic oligonucleotide priming site to a restriction site flanking IS6110. Sequences between the IS element and the restriction site are then amplified using an IS6110 specific outward primer and an oligonucleotide specific to the ligated priming site. Although it was found slightly less discriminative than the standard IS6110 restriction fragment length polymorphism analysis (IS6110 RFLP), LM-PCR allowed for the rapid and prospective identification of new outbreak-related cases within a large pool of circulating M. tuberculosis isolates. In comparison to IS6110 RFLP LM-PCR was found simple enough to justify its implementation in laboratories involved in MDR-TB surveillance at a nationwide scale.

  11. MIRU-VNTR Genotyping of Mycobacterium tuberculosis Strains Using QIAxcel Technology: A Multicentre Evaluation Study.

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    Vladyslav Nikolayevskyy

    Full Text Available Molecular genotyping of M.tuberculosis is an important laboratory tool in the context of emerging drug resistant TB. The standard 24-loci MIRU-VNTR typing includes PCR amplification followed by the detection and sizing of PCR fragments using capillary electrophoresis on automated sequencers or using agarose gels. The QIAxcel Advanced system might offer a cost-effective medium-throughput alternative.Performance characteristics of the QIAxcel Advanced platform for the standard 24 VNTR loci panel was evaluated at two centres on a total of 140 DNA specimens using automated capillary electrophoresis as a reference method. Additionally 4 hypervariable MIRU-VNTR loci were evaluated on 53 crude DNA extracts. The sizing accuracy, interlaboratory reproducibility and overall instrument's performance were assessed during the study.An overall concordance with the reference method was high reaching 98.5% and 97.6% for diluted genomic and crude DNA extracts respectively. 91.4% of all discrepancies were observed in fragments longer than 700bp. The concordance for hypervariable loci was lower except for locus 4120 (96.2%. The interlaboratory reproducibility agreement rates were 98.9% and 91.3% for standard and hypervariable loci, respectively. Overall performance of the QIAxcel platform for M.tuberculosis genotyping using a panel of standard loci is comparable to that of established methods for PCR fragments up to 700bp. Inaccuracies in sizing of longer fragments could be resolved through using in-house size markers or introduction of offset values. To conclude, the QiaXcel system could be considered an effective alternative to existing methods in smaller reference and regional laboratories offering good performance and shorter turnaround times.

  12. Mycobacterium tuberculosis Complex Genotype Diversity and Drug Resistance Profiles in a Pediatric Population in Mexico

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    Mercedes Macías Parra

    2011-01-01

    Full Text Available The aim of this study was to determine the frequency of drug resistance and the clonality of genotype patterns in M. tuberculosis clinical isolates from pediatric patients in Mexico (n=90 patients from 19 states; time period—January 2002 to December 2003. Pulmonary disease was the most frequent clinical manifestation (71%. Children with systemic tuberculosis (TB were significantly younger compared to patients with localized TB infections (mean 7.7±6.2 years versus 15±3.4 years P=0.001. Resistance to any anti-TB drug was detected in 24/90 (26.7% of the isolates; 21/90 (23.3% and 10/90 (11.1% were resistant to Isoniazid and Rifampicin, respectively, and 10/90 (11.1% strains were multidrug-resistant (MDR. Spoligotyping produced a total of 55 different patterns; 12/55 corresponded to clustered isolates (n=47, clustering rate of 52.2%, and 43/55 to unclustered isolates (19 patterns were designated as orphan by the SITVIT2 database. Database comparison led to designation of 36 shared types (SITs; 32 SITs (n=65 isolates matched a preexisting shared type in SITVIT2, whereas 4 SITs (n=6 isolates were newly created. Lineage classification based on principal genetic groups (PGG revealed that 10% of the strains belonged to PGG1 (Bovis and Manu lineages. Among PGG2/3 group, the most predominant clade was the Latin-American and Mediterranean (LAM in 27.8% of isolates, followed by Haarlem and T lineages. The number of single drug-resistant (DR and multidrug-resistant (MDR-TB isolates in this study was similar to previously reported in studies from adult population with risk factors. No association between the spoligotype, age, region, or resistance pattern was observed. However, contrary to a study on M. tuberculosis spoligotyping in Acapulco city that characterized a single cluster of SIT19 corresponding to the EAI2-Manila lineage in 70 (26% of patients, not a single SIT19 isolate was found in our pediatric patient population. Neither did we find any

  13. Mycobacterium tuberculosis Complex Genotype Diversity and Drug Resistance Profiles in a Pediatric Population in Mexico

    Science.gov (United States)

    Macías Parra, Mercedes; Kumate Rodríguez, Jesús; Arredondo García, José Luís; López-Vidal, Yolanda; Castañón-Arreola, Mauricio; Balandrano, Susana; Rastogi, Nalin; Gutiérrez Castrellón, Pedro

    2011-01-01

    The aim of this study was to determine the frequency of drug resistance and the clonality of genotype patterns in M. tuberculosis clinical isolates from pediatric patients in Mexico (n = 90 patients from 19 states; time period—January 2002 to December 2003). Pulmonary disease was the most frequent clinical manifestation (71%). Children with systemic tuberculosis (TB) were significantly younger compared to patients with localized TB infections (mean 7.7 ± 6.2 years versus 15 ± 3.4 years P = 0.001). Resistance to any anti-TB drug was detected in 24/90 (26.7%) of the isolates; 21/90 (23.3%) and 10/90 (11.1%) were resistant to Isoniazid and Rifampicin, respectively, and 10/90 (11.1%) strains were multidrug-resistant (MDR). Spoligotyping produced a total of 55 different patterns; 12/55 corresponded to clustered isolates (n = 47, clustering rate of 52.2%), and 43/55 to unclustered isolates (19 patterns were designated as orphan by the SITVIT2 database). Database comparison led to designation of 36 shared types (SITs); 32 SITs (n = 65 isolates) matched a preexisting shared type in SITVIT2, whereas 4 SITs (n = 6 isolates) were newly created. Lineage classification based on principal genetic groups (PGG) revealed that 10% of the strains belonged to PGG1 (Bovis and Manu lineages). Among PGG2/3 group, the most predominant clade was the Latin-American and Mediterranean (LAM) in 27.8% of isolates, followed by Haarlem and T lineages. The number of single drug-resistant (DR) and multidrug-resistant (MDR-TB) isolates in this study was similar to previously reported in studies from adult population with risk factors. No association between the spoligotype, age, region, or resistance pattern was observed. However, contrary to a study on M. tuberculosis spoligotyping in Acapulco city that characterized a single cluster of SIT19 corresponding to the EAI2-Manila lineage in 70 (26%) of patients, not a single SIT19 isolate was found in our pediatric patient population. Neither did we

  14. Phenotypic and genotypic variant of MDR-Mycobacterium tuberculosis multiple isolates in the same tuberculosis episode, Rio de Janeiro, Brazil

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    M.K.N. Andrade

    2009-05-01

    Full Text Available Assuming that the IS6110-restriction fragment length polymorphism (RFLP changes at a constant rate of 3.2 years, this methodology was applied to demonstrate, for the first time, variant patterns of Mycobacterium tuberculosis (MTB in multiple isolates obtained at short time intervals from sputum and blood of an HIV+ patient with multiple admissions to the Emergency Room and to the multidrug-resistant tuberculosis (MDR-TB Reference Center of a secondary-care hospital in Rio de Janeiro, Brazil. In sputum, the IS6110-RFLP appeared in isolates with two variant patterns with 10 and 13 IS6110 copies. However, blood presented only the pattern corresponding to 10 copies, suggesting compartmentalization. With regard to the exact match of 10 of 13 bands, this may be a subpopulation with the same clonal origin and this may be related to the IS6110 transposition. A susceptibility test demonstrated an MDR profile (INH R, RIF R, SM R, and EMB R, with the sputum isolate also exhibiting EMB S (R = resistant; S = sensitive. A gene mutation confirmed resistance only to streptomycin. There was agreement between the results of the phenotypic test and the clinical response to MDR-TB treatment, suggesting serious implications with regard to treatment administration based exclusively on molecular methods, and calling attention to the fact that more effective control strategies against the emergence of MDR strains must be implemented by the TB control program to prevent transmission of MDR-MTB strains at health facilities in areas highly endemic for TB.

  15. [Molecular epidemiology of Mycobacterium tuberculosis and its prospect based on variable number of tandem repeat (VNTR) genotyping--a strategy in Osaka City, Japan].

    Science.gov (United States)

    Wada, Takayuki; Hase, Atsushi

    2010-12-01

    The methodological establishment of variable number of tandem repeat(s) (VNTR) genotyping of Mycobacterium tuberculosis has opened a new era of molecular epidemiology against tuberculosis (TB). The method can provide simple, rapid and accurate identification of clinical isolates from TB patients that makes it possible to compare the isolates among different laboratories. Such advantages of VNTR not only help us certify the identification of isolates in putative outbreaks easily but also promote the reasonable estimation of unidentified transmissions in surveillance studies. Recently, the Japan Anti-Tuberculosis Association (JATA) (12)-VNTR has become a standard genotyping method of M. tuberculosis, and its spread has been expected in Japan. In Osaka City, located in the western part of the country, JATA (12)-VNTR has been applied to molecular epidemiological study of TB. Moreover, the additional 12 VNTR loci have been analyzed for various purposes, such as to enhance the discriminatory power (public health needs) or to further analyze the population genetic structure (research needs). As the nationwide findings of VNTR genotyping of M. tuberculosis are accumulated, this technology will be increasingly useful for detecting transmission of any specific strain in large geographic areas that could not be recognized by conventional epidemiological methods. The needs for the VNTR genotyping of M. tuberculosis and its practical uses are expected to expand drastically in the future.

  16. Pre-existing isoniazid resistance, but not the genotype of Mycobacterium tuberculosis drives rifampicin resistance codon preference in vitro.

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    Indra Bergval

    Full Text Available Both the probability of a mutation occurring and the ability of the mutant to persist will influence the distribution of mutants that arise in a population. We studied the interaction of these factors for the in vitro selection of rifampicin (RIF-resistant mutants of Mycobacterium tuberculosis. We characterised two series of spontaneous RIF-resistant in vitro mutants from isoniazid (INH-sensitive and -resistant laboratory strains and clinical isolates, representing various M. tuberculosis genotypes. The first series were selected from multiple parallel 1 ml cultures and the second from single 10 ml cultures. RIF-resistant mutants were screened by Multiplex Ligation-dependent Probe Amplification (MLPA or by sequencing the rpoB gene. For all strains the mutation rate for RIF resistance was determined with a fluctuation assay. The most striking observation was a shift towards rpoB-S531L (TCG→TTG mutations in a panel of laboratory-generated INH-resistant mutants selected from the 10-ml cultures (p<0.001. All tested strains showed similar mutation rates (1.33×10⁻⁸ to 2.49×10⁻⁷ except one of the laboratory-generated INH mutants with a mutation rate measured at 5.71×10⁻⁷, more than 10 times higher than that of the INH susceptible parental strain (5.46-7.44×10⁻⁸. No significant, systematic difference in the spectrum of rpoB-mutations between strains of different genotypes was observed. The dramatic shift towards rpoB-S531L in our INH-resistant laboratory mutants suggests that the relative fitness of resistant mutants can dramatically impact the distribution of (subsequent mutations that accumulate in a M. tuberculosis population, at least in vitro. We conclude that, against specific genetic backgrounds, certain resistance mutations are particularly likely to spread. Molecular screening for these (combinations of mutations in clinical isolates could rapidly identify these particular pathogenic strains. We therefore recommend that

  17. Pre-existing isoniazid resistance, but not the genotype of Mycobacterium tuberculosis drives rifampicin resistance codon preference in vitro.

    Science.gov (United States)

    Bergval, Indra; Kwok, Brian; Schuitema, Anja; Kremer, Kristin; van Soolingen, Dick; Klatser, Paul; Anthony, Richard

    2012-01-01

    Both the probability of a mutation occurring and the ability of the mutant to persist will influence the distribution of mutants that arise in a population. We studied the interaction of these factors for the in vitro selection of rifampicin (RIF)-resistant mutants of Mycobacterium tuberculosis. We characterised two series of spontaneous RIF-resistant in vitro mutants from isoniazid (INH)-sensitive and -resistant laboratory strains and clinical isolates, representing various M. tuberculosis genotypes. The first series were selected from multiple parallel 1 ml cultures and the second from single 10 ml cultures. RIF-resistant mutants were screened by Multiplex Ligation-dependent Probe Amplification (MLPA) or by sequencing the rpoB gene. For all strains the mutation rate for RIF resistance was determined with a fluctuation assay. The most striking observation was a shift towards rpoB-S531L (TCG→TTG) mutations in a panel of laboratory-generated INH-resistant mutants selected from the 10-ml cultures (pmutation rates (1.33×10⁻⁸ to 2.49×10⁻⁷) except one of the laboratory-generated INH mutants with a mutation rate measured at 5.71×10⁻⁷, more than 10 times higher than that of the INH susceptible parental strain (5.46-7.44×10⁻⁸). No significant, systematic difference in the spectrum of rpoB-mutations between strains of different genotypes was observed. The dramatic shift towards rpoB-S531L in our INH-resistant laboratory mutants suggests that the relative fitness of resistant mutants can dramatically impact the distribution of (subsequent) mutations that accumulate in a M. tuberculosis population, at least in vitro. We conclude that, against specific genetic backgrounds, certain resistance mutations are particularly likely to spread. Molecular screening for these (combinations of) mutations in clinical isolates could rapidly identify these particular pathogenic strains. We therefore recommend that isolates are screened for the distribution of resistance

  18. Validation of genotype cluster investigations for Mycobacterium tuberculosis: application results for 44 clusters from four heterogeneous United States jurisdictions.

    Science.gov (United States)

    Teeter, Larry D; Vempaty, Padmaja; Nguyen, Duc T M; Tapia, Jane; Sharnprapai, Sharon; Ghosh, Smita; Kammerer, J Steven; Miramontes, Roque; Cronin, Wendy A; Graviss, Edward A

    2016-10-21

    Tracking the dissemination of specific Mycobacterium tuberculosis (Mtb) strains using genotyped Mtb isolates from tuberculosis patients is a routine public health practice in the United States. The present study proposes a standardized cluster investigation method to identify epidemiologic-linked patients in Mtb genotype clusters. The study also attempts to determine the proportion of epidemiologic-linked patients the proposed method would identify beyond the outcome of the conventional contact investigation. The study population included Mtb culture positive patients from Georgia, Maryland, Massachusetts and Houston, Texas. Mtb isolates were genotyped by CDC's National TB Genotyping Service (NTGS) from January 2006 to October 2010. Mtb cluster investigations (CLIs) were conducted for patients whose isolates matched exactly by spoligotyping and 12-locus MIRU-VNTR. CLIs were carried out in four sequential steps: (1) Public Health Worker (PHW) Interview, (2) Contact Investigation (CI) Evaluation, (3) Public Health Records Review, and (4) CLI TB Patient Interviews. Comparison between patients whose links were identified through the study's CLI interviews (Step 4) and patients whose links were identified earlier in CLI (Steps 1-3) was conducted using logistic regression. Forty-four clusters were randomly selected from the four study sites (401 patients in total). Epidemiologic links were identified for 189/401 (47 %) study patients in a total of 201 linked patient-pairs. The numbers of linked patients identified in each CLI steps were: Step 1 - 105/401 (26.2 %), Step 2 - 15/388 (3.9 %), Step 3 - 41/281 (14.6 %), and Step 4 - 28/119 (30 %). Among the 189 linked patients, 28 (14.8 %) were not identified in previous CI. No epidemiologic links were identified in 13/44 (30 %) clusters. We validated a standardized and practical method to systematically identify epidemiologic links among patients in Mtb genotype clusters, which can be integrated into the TB control and

  19. Research progress on Mycobacterium tuberculosis genotyping technologies%结核分枝杆菌基因分型技术的研究进展

    Institute of Scientific and Technical Information of China (English)

    董毅; 吴利先

    2013-01-01

    Mycobacterium tuberculosis is the pathogen causing TB,and can infect body's tissues and organs.Due to the complexity of Mycobacterium tuberculosis genotyping and different virulence levels,the gene classification is important for the TB epidemic surveillance,source of infection tracking,transmission route verification,recurrence strains identification and cross contamination detection in the lab,which provide theoretical basis for clinical problem solution.The Mycobacterium tuberculosis classification technologies mainly divide into the non-nucleic acid methods and nucleic acid methods.The main nucleic acid methods for genotyping are summarized in this review.%结核分枝杆菌是引起结核病的病原体,可侵犯全身各组织器官.由于结核分枝杆菌基因型复杂,不同菌株毒力不同,其基因分型可用于结核病的疫情监测、传染源追踪、传播途径查证、复发菌株鉴定、实验室交叉污染检测等方面,为解决临床实际问题提供依据.结核杆菌分型技术主要分为非核酸法和核酸法.此文就目前主要的基因分型技术作一综述.

  20. Pre-existing isoniazid resistance, but not the genotype of Mycobacterium tuberculosis drives rifampicin resistance codon preference in vitro.

    NARCIS (Netherlands)

    Bergval, I.; Kwok, B.; Schuitema, A.; Kremer, K.; Soolingen, D. van; Klatser, P.; Anthony, R.

    2012-01-01

    Both the probability of a mutation occurring and the ability of the mutant to persist will influence the distribution of mutants that arise in a population. We studied the interaction of these factors for the in vitro selection of rifampicin (RIF)-resistant mutants of Mycobacterium tuberculosis. We

  1. Tuberculosis transmission of predominant genotypes of Mycobacterium tuberculosis in Northern suburbs of Buenos Aires city region Transmisión de la tuberculosis por genotipos predominantes de Mycobacterium tuberculosis en la región Gran Buenos Aires Norte

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    N. Morcillo

    2007-09-01

    Full Text Available In 2003, the incidence of tuberculosis in Argentina showed an increase compared to 2002. The severe national crisis at the end of the 90s has probably strongly contributed to this situation. The goal of this work was to estimate the extent of the spread of the most predominant Mycobacterium tuberculosis strains and to assess the spread of predominant M. tuberculosis clusters as determined by spoligotyping and IS6110 RFLP. The study involved 590 pulmonary, smear-positive TB cases receiving medical attention at health centers and hospitals in Northern Buenos Aires (NBA suburbs, from October 2001 to December 2002. From a total of 208 clinical isolates belonging to 6 major clusters, 63 (30.2% isolates had identical spoligotyping and IS6110 RFLP pattern. Only 22.2% were shown to have epidemiological connections with another member of their respective cluster. In these major clusters, 30.2% of the 208 TB cases studied by both molecular techniques and contact tracing could be convincingly attributable to a recently acquired infection. This knowledge may be useful to assess the clonal distribution of predominant M. tuberculosis clusters in Argentina, which may make an impact on TB control strategies.La incidencia de la tuberculosis en Argentina mostró en 2003 un incremento en comparación con 2002. La grave crisis nacional a fines de los 90 ha probablemente contribuido en gran medida a esta situación. El objetivo del presente trabajo fue determinar la diversidad genética de aislamientos de Mycobacterium tuberculosis y el grado de dispersión de algunas cepas mayoritarias genéticamente relacionadas. El estudio involucró 590 aislamientos clínicos provenientes de muestras respiratorias con examen directo positivo, de pacientes atendidos en los hospitales y centros de salud que conforman la región Gran Buenos Aires Norte (NBA, de octubre de 2001 a diciembre de 2002. De 208 aislamientos que se encontraron en los 6 mayores clusters, 63 (30,2% ten

  2. Whole genome sequencing versus traditional genotyping for investigation of a Mycobacterium tuberculosis outbreak: a longitudinal molecular epidemiological study.

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    Andreas Roetzer

    Full Text Available BACKGROUND: Understanding Mycobacterium tuberculosis (Mtb transmission is essential to guide efficient tuberculosis control strategies. Traditional strain typing lacks sufficient discriminatory power to resolve large outbreaks. Here, we tested the potential of using next generation genome sequencing for identification of outbreak-related transmission chains. METHODS AND FINDINGS: During long-term (1997 to 2010 prospective population-based molecular epidemiological surveillance comprising a total of 2,301 patients, we identified a large outbreak caused by an Mtb strain of the Haarlem lineage. The main performance outcome measure of whole genome sequencing (WGS analyses was the degree of correlation of the WGS analyses with contact tracing data and the spatio-temporal distribution of the outbreak cases. WGS analyses of the 86 isolates revealed 85 single nucleotide polymorphisms (SNPs, subdividing the outbreak into seven genome clusters (two to 24 isolates each, plus 36 unique SNP profiles. WGS results showed that the first outbreak isolates detected in 1997 were falsely clustered by classical genotyping. In 1998, one clone (termed "Hamburg clone" started expanding, apparently independently from differences in the social environment of early cases. Genome-based clustering patterns were in better accordance with contact tracing data and the geographical distribution of the cases than clustering patterns based on classical genotyping. A maximum of three SNPs were identified in eight confirmed human-to-human transmission chains, involving 31 patients. We estimated the Mtb genome evolutionary rate at 0.4 mutations per genome per year. This rate suggests that Mtb grows in its natural host with a doubling time of approximately 22 h (400 generations per year. Based on the genome variation discovered, emergence of the Hamburg clone was dated back to a period between 1993 and 1997, hence shortly before the discovery of the outbreak through epidemiological

  3. The genotypic study of Mycobacterium tuberculosis complex resistant to isoniazid: Galicia, Spain (2008-2013).

    Science.gov (United States)

    Pérez Del Molino, M L; Barbeito-Castiñeiras, G; Mejuto, B; Alonso, P; Fernández, A; González-Mediero, G

    2016-11-01

    Incorporation of rapid detection systems to identify mutations in M. tuberculosis complex that confer resistance to isoniazid and rifampicin has potentiated the knowledge of their distribution, given the geographical variability. We performed antibiograms of the 2,993 strains isolated in Galicia, Spain (2008-2013). In the strains resistant to isoniazid, a concentration of 0.4 mg/mL and MTBDRplus Genotype test (Hain Lifescience, Germany) were used. We found that 3.64 % of strains were resistant to isoniazid, while 0.43 % were resistant to isoniazid and rifampicin (multidrug resistant, MDR). The MTBDRplus test showed an overall sensitivity of 72.48 %, with 62.5 % sensitivity for non MDR isoniazid-resistant strains and 100 % sensitivity for MDR strains. The katG gene mutation was detected at codon 315 in 38.53 % of strains. The S315T mutation appeared in 61.54 % of MDR strains and 34.38 % of non-MDR strains. The 28.44 % had mutations in inhA, (93.55 % in C15T), and 38.46 % of MDR strains were mutated. In non-MDR strains, 37.50 % were wild-type, 35.42 % and 27.08 % had mutations in katG and inhA, respectively. The most frequent mutation in rpoβ was S531L (46.15 %). The 38.71 % and 41.9 % of strains with resistance to isoniazid and streptomycin had mutations in katG and inhA, respectively (2 strains with mutations in T8C and T8A). The distribution pattern of resistance among strains with high and low concentrations of isoniazid showed statistically significant differences in relation to the mutation in katG and wild-type. The sensitivity of the Genotype MTBDRplus test for non-MDR strains in our area was at the lower threshold described.

  4. Genetic signatures of Mycobacterium tuberculosis Nonthaburi genotype revealed by whole genome analysis of isolates from tuberculous meningitis patients in Thailand.

    Science.gov (United States)

    Coker, Olabisi Oluwabukola; Chaiprasert, Angkana; Ngamphiw, Chumpol; Tongsima, Sissades; Regmi, Sanjib Mani; Clark, Taane G; Ong, Rick Twee Hee; Teo, Yik-Ying; Prammananan, Therdsak; Palittapongarnpim, Prasit

    2016-01-01

    Genome sequencing plays a key role in understanding the genetic diversity of Mycobacterium tuberculosis (M.tb). The genotype-specific character of M. tb contributes to tuberculosis severity and emergence of drug resistance. Strains of M. tb complex can be classified into seven lineages. The Nonthaburi (NB) genotype, belonging to the Indo-Oceanic lineage (lineage 1), has a unique spoligotype and IS6110-RFLP pattern but has not previously undergone a detailed whole genome analysis. In addition, there is not much information available on the whole genome analysis of M. tb isolates from tuberculous meningitis (TBM) patients in public databases. Isolates CSF3053, 46-5069 and 43-13838 of NB genotype were obtained from the cerebrospinal fluids of TBM Thai patients in Siriraj Hospital, Bangkok. The whole genomes were subjected to high throughput sequencing. The sequence data of each isolate were assembled into draft genome. The sequences were also aligned to reference genome, to determine genomic variations. Single nucleotide polymorphisms (SNPs) were obtained and grouped according to the functions of the genes containing them. They were compared with SNPs from 1,601 genomes, representing the seven lineages of M. tb complex, to determine the uniqueness of NB genotype. Susceptibility to first-line, second-line and other antituberculosis drugs were determined and related to the SNPs previously reported in drug-resistant related genes. The assembled genomes have an average size of 4,364,461 bp, 4,154 genes, 48 RNAs and 64 pseudogenes. A 500 base pairs deletion, which includes ppe50, was found in all isolates. RD239, specific for members of Indo Oceanic lineage, and RD147c were identified. A total of 2,202 SNPs were common to the isolates and used to classify the NB strains as members of sublineage 1.2.1. Compared with 1,601 genomes from the seven lineages of M. tb complex, mutation G2342203C was found novel to the isolates in this study. Three mutations (T28910C, C1180580T

  5. Genetic signatures of Mycobacterium tuberculosis Nonthaburi genotype revealed by whole genome analysis of isolates from tuberculous meningitis patients in Thailand

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    Olabisi Oluwabukola Coker

    2016-04-01

    Full Text Available Genome sequencing plays a key role in understanding the genetic diversity of Mycobacterium tuberculosis (M.tb. The genotype-specific character of M. tb contributes to tuberculosis severity and emergence of drug resistance. Strains of M. tb complex can be classified into seven lineages. The Nonthaburi (NB genotype, belonging to the Indo-Oceanic lineage (lineage 1, has a unique spoligotype and IS6110-RFLP pattern but has not previously undergone a detailed whole genome analysis. In addition, there is not much information available on the whole genome analysis of M. tb isolates from tuberculous meningitis (TBM patients in public databases. Isolates CSF3053, 46-5069 and 43-13838 of NB genotype were obtained from the cerebrospinal fluids of TBM Thai patients in Siriraj Hospital, Bangkok. The whole genomes were subjected to high throughput sequencing. The sequence data of each isolate were assembled into draft genome. The sequences were also aligned to reference genome, to determine genomic variations. Single nucleotide polymorphisms (SNPs were obtained and grouped according to the functions of the genes containing them. They were compared with SNPs from 1,601 genomes, representing the seven lineages of M. tb complex, to determine the uniqueness of NB genotype. Susceptibility to first-line, second-line and other antituberculosis drugs were determined and related to the SNPs previously reported in drug-resistant related genes. The assembled genomes have an average size of 4,364,461 bp, 4,154 genes, 48 RNAs and 64 pseudogenes. A 500 base pairs deletion, which includes ppe50, was found in all isolates. RD239, specific for members of Indo Oceanic lineage, and RD147c were identified. A total of 2,202 SNPs were common to the isolates and used to classify the NB strains as members of sublineage 1.2.1. Compared with 1,601 genomes from the seven lineages of M. tb complex, mutation G2342203C was found novel to the isolates in this study. Three mutations (T28910

  6. Assessment of the quality of dna extracted by two techniques from Mycobacterium tuberculosis for fast molecular identification and genotyping

    Directory of Open Access Journals (Sweden)

    Marcelo Miyata

    2011-06-01

    Full Text Available We report a comparative study of two DNA extraction techniques, thermolysis and chemical lysis (CTAB, for molecular identification and genotyping of M. tuberculosis. Forty DNA samples were subjected to PCR and the results demonstrated that with thermolysis it is possible to obtain useful data that enables fast identification and genotyping.

  7. Association between genotype and drug resistance profiles of Mycobacterium tuberculosis strains circulating in China in a national drug resistance survey

    Science.gov (United States)

    Zhou, Yang; van den Hof, Susan; Wang, Shengfen; Pang, Yu; Zhao, Bing; Xia, Hui; Anthony, Richard; Ou, Xichao; Li, Qiang; Zheng, Yang; Song, Yuanyuan; Zhao, Yanlin; van Soolingen, Dick

    2017-01-01

    We describe the population structure of a representative collection of 3,133 Mycobacterium tuberculosis isolates, collected within the framework of a national resistance survey from 2007 in China. Genotyping data indicate that the epidemic strains in China can be divided into seven major complexes, of which 92% belonged to the East Asian (mainly Beijing strains) or the Euro-American lineage. The epidemic Beijing strains in China are closely related to the Beijing B0/W148 strain earlier described in Russia and a large cluster of these strains has spread national wide. The density of Beijing strains is high in the whole of China (average 70%), but the highest prevalence was found North of the Yellow river. The Euro-American lineage consists of three sublineages (sublineage_1, 2, and 3) and is more prevalent in the South. Beijing lineage showed the highest cluster rate of 48% and a significantly higher level of resistance to rifampicin (14%, p<0.001), ethambutol (9%, p = 0.001), and ofloxacin (5%, p = 0.011). Within the Euro-American Lineage, sublineage_3 revealed the highest cluster rate (28%) and presented a significantly elevated level of resistance to streptomycin (44%, p<0.001). Our findings suggest that standardised treatment in this region may have contributed to the successful spread of certain strains: sublineage_3 in the Euro-American lineage may have thrived when streptomycin was used without rifampicin for treatment, while later under DOTS based treatment, in which rifampicin plays a key role, Beijing lineage appears to be spreading. PMID:28333978

  8. Mycobacterium tuberculosis of the RDRio genotype is the predominant cause of tuberculosis and associated with multidrug resistance in Porto Alegre City, South Brazil.

    Science.gov (United States)

    Dalla Costa, Elis Regina; Lazzarini, Luiz Claudio Oliveira; Perizzolo, Paulo Fernado; Díaz, Chyntia Acosta; Spies, Fernanda S; Costa, Lucas Laux; Ribeiro, Andrezza W; Barroco, Caroline; Schuh, Sandra Jungblut; da Silva Pereira, Marcia Aparecida; Dias, Claudia F; Gomes, Harrison M; Unis, Gisela; Zaha, Arnaldo; Almeida da Silva, Pedro E; Suffys, Philip N; Rossetti, Maria L R

    2013-04-01

    Spoligotyping has shown Mycobacterium tuberculosis strains to be composed of different lineages, and some of them are not just geographically restricted but also affect specific ethnic populations and are associated with outbreaks and drug resistance. We recently described a particular subtype within the Latin American-Mediterranean (LAM) family, called RD(Rio), widespread in Brazil. Moreover, recent data also indicate that RD(Rio) is present in many countries on all continents and is associated with cavitary disease and multidrug resistance (MDR). To further explore the relationship between RD(Rio) and MDR, we conducted a study in a tuberculosis (TB) reference center responsible for the care of MDR patients in Rio Grande do Sul, the southernmost Brazilian state. From a collection of 237 clinical isolates, RD(Rio) alone was responsible for one-half of all MDR cases, including one large group composed of strains with identical IS6110-restriction fragment length polymorphism (RFLP) and having the LAM5 signature. We additionally had complete data records for 96 patients and could make comparisons between the presence and absence of RD(Rio). No difference in clinical, radiological or laboratory features was observed, but a significantly greater number of cases with MDR were described in patients infected with an RD(Rio) strain (P = 0.0015). Altogether, RD(Rio) was responsible for 38% of all TB cases. These data support and confirmed previous findings that RD(Rio) is the main agent responsible for TB in Brazil and is associated with drug resistance. Considering that RD(Rio) is a globally distributed genotype, such findings raise concern about the increase in MDR in certain human populations.

  9. Highest prevalence of the Mycobacterium tuberculosis Beijing genotype isolates in patients newly diagnosed with tuberculosis in the Novosibirsk oblast, Russian Federation.

    Science.gov (United States)

    Dymova, M A; Kinsht, V N; Cherednichenko, A G; Khrapov, E A; Svistelnik, A V; Filipenko, M L

    2011-07-01

    In order to assess the genetic diversity of Mycobacterium tuberculosis in the Novosibirsk Region and determine profiles of resistance, 106 M. tuberculosis isolates were analysed. Fifty (47 %) isolates were identified using variable number tandem repeat typing as being in the Beijing family, of which eight (16 %) were type M2 isolates with the genetic profile 233325153533424 and eight (16 %) were type M11 isolates with the genetic profile 233325173533424, both of which are widespread in Russia. Mutations associated with resistance to isoniazid and rifampicin were identified. Of 48 isolates with resistance to isoniazid, 42 (87.5 %) contained a Ser(315)→Thr substitution in the katG gene and one contained a T→A substitution at position -34 of the promoter region of the ahpC gene. Of 31 isolates with resistance to rifampicin, 19 (61 %) each contained a mutation (TCG→TTG) at codon 531 of the rpoB gene. Two isolates each contained a mutation (GAC→GTC) at codon 516 of the rpoB gene and two others each contained a substitution at codon 526 of the rpoB gene, leading to a His→Asn substitution in one case and a His→Asp substitution in another case. One isolate contained a mutation (CTG→CCG) at codon 533 of the rpoB gene. An association between the Beijing genotype and multidrug resistance was demonstrated (R = 0.2, P = 0.032). However, it was interesting to note that a significant proportion (46 %) of isolates were sensitive to all drugs tested.

  10. Within Host Evolution Selects for a Dominant Genotype of Mycobacterium tuberculosis while T Cells Increase Pathogen Genetic Diversity

    Science.gov (United States)

    Copin, Richard; Louie, Eddie; Escuyer, Vincent; Gagneux, Sebastien; Palmer, Guy H.

    2016-01-01

    Molecular epidemiological assessments, drug treatment optimization, and development of immunological interventions all depend on understanding pathogen adaptation and genetic variation, which differ for specific pathogens. Mycobacterium tuberculosis is an exceptionally successful human pathogen, yet beyond knowledge that this bacterium has low overall genomic variation but acquires drug resistance mutations, little is known of the factors that drive its population genomic characteristics. Here, we compared the genetic diversity of the bacteria that established infection to the bacterial populations obtained from infected tissues during murine M. tuberculosis pulmonary infection and human disseminated M. bovis BCG infection. We found that new mutations accumulate during in vitro culture, but that in vivo, purifying selection against new mutations dominates, indicating that M. tuberculosis follows a dominant lineage model of evolution. Comparing bacterial populations passaged in T cell-deficient and immunocompetent mice, we found that the presence of T cells is associated with an increase in the diversity of the M. tuberculosis genome. Together, our findings put M. tuberculosis genetic evolution in a new perspective and clarify the impact of T cells on sequence diversity of M. tuberculosis. PMID:27973588

  11. 21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Mycobacterium tuberculosis immunofluorescent... § 866.3370 Mycobacterium tuberculosis immunofluorescent reagents. (a) Identification. Mycobacterium... used to identify Mycobacterium tuberculosis directly from clinical specimens. The identification...

  12. Correlations between major risk factors and closely related Mycobacterium tuberculosis isolates grouped by three current genotyping procedures: a population-based study in northeast Mexico.

    Science.gov (United States)

    Peñuelas-Urquides, Katia; Martínez-Rodríguez, Herminia Guadalupe; Enciso-Moreno, José Antonio; Molina-Salinas, Gloria María; Silva-Ramírez, Beatriz; Padilla-Rivas, Gerardo Raymundo; Vera-Cabrera, Lucio; Torres-de-la-Cruz, Víctor Manuel; Martínez-Martínez, Yazmin Berenice; Ortega-García, Jorge Luis; Garza-Treviño, Elsa Nancy; Enciso-Moreno, Leonor; Saucedo-Cárdenas, Odila; Becerril-Montes, Pola; Said-Fernández, Salvador

    2014-09-01

    The characteristics of tuberculosis (TB) patients related to a chain of recent TB transmissions were investigated. Mycobacterium tuberculosis (MTB) isolates (120) were genotyped using the restriction fragment length polymorphism-IS6110 (R), spacer oligotyping (S) and mycobacterial interspersed repetitive units-variable number of tandem repeats (M) methods. The MTB isolates were clustered and the clusters were grouped according to the similarities of their genotypes. Spearman's rank correlation coefficients between the groups of MTB isolates with similar genotypes and those patient characteristics indicating a risk for a pulmonary TB (PTB) chain transmission were ana- lysed. The isolates showing similar genotypes were distributed as follows: SMR (5%), SM (12.5%), SR (1.67%), MR (0%), S (46.67%), M (5%) and R (0%). The remaining 35 cases were orphans. SMR exhibited a significant correlation (p < 0.05) with visits to clinics, municipalities and comorbidities (primarily diabetes mellitus). S correlated with drug consumption and M with comorbidities. SMR is needed to identify a social network in metropolitan areas for PTB transmission and S and M are able to detect risk factors as secondary components of a transmission chain of TB.

  13. The Genotype MTBDRplus ver. 2.0 test as a quick indicator of resistance to rifampicin and isoniazid in Mycobacterium tuberculosis strains

    Directory of Open Access Journals (Sweden)

    Salvatore Nisticò

    2013-08-01

    Full Text Available Tuberculosis is still a global emergency and a major public health problem, in some cases related to the appearance of strains of multi drug resistance (MDR and extensive drug resistance (XDR Mycobacterium tuberculosis complex.The correct determination of antibiotic sensitivity profiles is therefore crucial to carry out appropriate treatment aimed to decrease the infectivity of each patient and to reduce mortality. The poor adherence to treatment by the patient or the use of therapies based on a single drug, as a result of incorrect requirements, promote the development of drug-resistance. Have some time on the market of molecular diagnostic tests that allow, quickly and directly from biological sample to search for resistance genes some key drugs of anti-TB therapy (Rifampicin and Isoniazid. One of the tests in question is the Genotype MTBDRplus ver 2.0 which can reveal the presence of genes for resistance to Isoniazid (INH and Rifampin (RMP.The loci analyzed are those corresponding to the rpoB gene for rifampicin, katG and inhA for isoniazid. Our study is based on the analysis of 83 strains of tubercular Mycobacteria identified and isolated from patients with tuberculosis disease and subjected to the tests sensitivity, searching for mutations and phenotypic susceptibility testing for Rifampicin and Isoniazid.The comparison of the results has shown that the results obtained using the Genotype MTBDRplus ver 2.0 test, were similar to the results obtained by the traditional susceptibility testing.

  14. Tracing Mycobacterium tuberculosis transmission by whole genome sequencing in a high incidence setting

    DEFF Research Database (Denmark)

    Bjorn-Mortensen, K; Soborg, B; Koch, A;

    2016-01-01

    In East Greenland, a dramatic increase of tuberculosis (TB) incidence has been observed in recent years. Classical genotyping suggests a genetically similar Mycobacterium tuberculosis (Mtb) strain population as cause, however, precise transmission patterns are unclear. We performed whole genome...

  15. Feasibility of the GenoType MTBDRsl assay for fluoroquinolone, amikacin-capreomycin, and ethambutol resistance testing of Mycobacterium tuberculosis strains and clinical specimens.

    Science.gov (United States)

    Hillemann, Doris; Rüsch-Gerdes, Sabine; Richter, Elvira

    2009-06-01

    The new GenoType Mycobacterium tuberculosis drug resistance second line (MTBDRsl) assay (Hain Lifescience, Nehren, Germany) was tested on 106 clinical isolates and directly on 64 sputum specimens for the ability to detect resistance to fluoroquinolones, injectable drugs (amikacin or capreomycin), and ethambutol in Mycobacterium tuberculosis strains. A total of 63 strains harboring fluoroquinolone, amikacin/capreomycin, or ethambutol resistance and 43 fully susceptible strains were comparatively analyzed with the new MTBDRsl assay, by DNA sequencing, and by conventional drug susceptibility testing in liquid and solid media. No discrepancies were obtained in comparison with the DNA sequencing results. Fluoroquinolone resistance was detected in 29 (90.6%) of 32, amikacin/capreomycin resistance was detected in 39/39 (84.8%/86.7%) of 46/45, and ethambutol resistance was detected in 36 (69.2%) of 52 resistant strains. A total of 64 sputum specimens (42 smear positive, 12 scanty, and 10 smear negative) were tested with the new MTBDRsl assay, and the results were compared with those of conventional drug susceptibility testing. Fluoroquinolone resistance was detected in 8 (88.9%) of 9, amikacin/capreomycin resistance was detected in 6/7 (75.0%/87.5%) of 8, and ethambutol resistance was detected in 10 (38.5%) of 26 resistant strains. No mutation was detected in susceptible strains. The new GenoType MTBDRsl assay represents a reliable tool for the detection of fluoroquinolone and amikacin/capreomycin resistance and to a lesser extent also ethambutol resistance. In combination with a molecular test for detection of rifampin and isoniazid resistance, the potential for the detection of extensively resistant tuberculosis within 1 to 2 days can be postulated.

  16. Study of Mycobacterium tuberculosis complex genotypic diversity in Malaysia reveals a predominance of ancestral East-African-Indian lineage with a Malaysia-specific signature.

    Science.gov (United States)

    Ismail, Fazli; Couvin, David; Farakhin, Izzah; Abdul Rahman, Zaidah; Rastogi, Nalin; Suraiya, Siti

    2014-01-01

    Tuberculosis (TB) still constitutes a major public health problem in Malaysia. The identification and genotyping based characterization of Mycobacterium tuberculosis complex (MTBC) isolates causing the disease is important to determine the effectiveness of the control and surveillance programs. This study intended a first assessment of spoligotyping-based MTBC genotypic diversity in Malaysia followed by a comparison of strains with those prevailing in neighboring countries by comparison with an international MTBC genotyping database. Spoligotyping was performed on a total of 220 M. tuberculosis clinical isolates collected in Kelantan and Kuala Lumpur. The results were compared with the SITVIT2 international database of the Pasteur Institute of Guadeloupe. Spoligotyping revealed 77 different patterns: 22 corresponded to orphan patterns while 55 patterns containing 198 isolates were assigned a Spoligo International Type (SIT) designation in the database (the latter included 6 newly created SITs). The eight most common SITs grouped 141 isolates (5 to 56 strains per cluster) as follows: SIT1/Beijing, n = 56, 25.5%; SIT745/EAI1-SOM, n = 33, 15.0%; SIT591/EAI6-BGD1, n = 13, 5.9%; SIT256/EAI5, n = 12, 5.5%; SIT236/EAI5, n = 10, 4.6%; SIT19/EAI2-Manila, n = 9, 4.1%; SIT89/EAI2-Nonthaburi, n = 5, 2.3%; and SIT50/H3, n = 3, 1.4%. The association between city of isolation and lineages was statistically significant; Haarlem and T lineages being higher in Kuala Lumpur (pMalaysia, and its probable ongoing evolution with locally evolved strains sharing a specific signature characterized by absence of spacers 37, 38, and 40. Pending complementary genotyping confirmation, we propose that SIT745/EAI-SOM is tentatively reclassified as SIT745/EAI-MYS.

  17. Genotypic Diversity of Mycobacterium tuberculosis Clinical Isolates in the Multiethnic Area of the Xinjiang Uygur Autonomous Region in China

    Science.gov (United States)

    Liu, Jie; Li, Junlian; Liu, Jiao; Lian, Lulu; Lu, Bing; Yu, Qin; Zhang, Jingrui; Qi, Yingcheng

    2017-01-01

    Objectives. We studied the genetic diversity of clinical isolates from patients with tuberculosis in the multiethnic area of Xinjiang autonomous region in China. A total of 311 clinical M. tuberculosis isolates were collected in 2006 and 2011 and genotyped by two genotyping methods. All isolates were grouped into 68 distinct spoligotypes using the spoligotyping method. The Beijing family was dominant, followed by T1 and CAS. MIRU-VNTR results showed that a total of 195 different VNTR types were identified. Ten of the 15 loci were highly or moderately discriminant according to their HGDI scores, and 13 loci had good discriminatory power in non-Beijing family strains, whereas only two loci had good discriminatory power in Beijing family strains. Chi-square tests demonstrated that there were no correlations between four characteristics (sex, age, type of case, and treatment history) and the Beijing family. In summary, Beijing family strains were predominant in Xinjiang, and the VNTR-15China locus-set was suitable for genotyping all Xinjiang strains, but not for the Beijing family strains. Thus, these data suggested that different genotype distributions may exist in different regions; MLVA locus-sets should be adjusted accordingly, with newly added loci to increase resolution if necessary.

  18. Mycobacterium Tuberculosis in Spinal Tuberculosis

    Science.gov (United States)

    Kim, Sung-Sim; Moon, Han-Lim; Kim, Dong-Hyeon

    2017-01-01

    Even in an era of remarkable medical advances, there is an issue of why tuberculosis remains in the list of disastrous diseases, afflicting humans and causing suffering. There has not been a plausible answer to this, and it has been suggested that clinicians and medical scientists could presently not win the war against the tubercle bacilli. With regards to this issue, based on the authors' own clinical and research experiences, in this review, the available literature was revisited in order to address the raised questions and to provide recent information on characteristics of tubercle bacilli and possible ways to more effectively treat tuberculosis. PMID:28243382

  19. Mycobacterium tuberculosis: factores de virulencia

    OpenAIRE

    Reinier Borrero; Nadine Álvarez; Fátima Reyes; María Elena Sarmiento; Armando Acosta

    2011-01-01

    Mycobacterium tuberculosis es el agente causal de la tuberculosis, una de las enfermedades infecciosas más letales en el mundo. La única vacuna disponible para su control es el BCG, sin embargo, falla en la protección contra la tuberculosis pulmonar, siendo esta la forma más frecuente y responsable de la diseminación. La identificación de factores de virulencia del microorganismo causal pudiera ayudar en el desarrollo de un nuevo candidato vacunal que sea capaz de neutralizar la acción de eso...

  20. A Systematic Follow-Up of Mycobacterium tuberculosis Drug-Resistance and Associated Genotypic Lineages in the French Departments of the Americas over a Seventeen-Year Period

    Directory of Open Access Journals (Sweden)

    Julie Millet

    2014-01-01

    Full Text Available The population of the French Departments of the Americas (FDA is highly influenced by the intense migratory flows with mainland France and surrounding countries of the Caribbean and Latin America, some of which have high incidence rates of tuberculosis (Haiti: 230/100,000; Guyana: 111/100,000; and Suriname: 145/100,000 and drug resistance. Since the development of drug resistance to conventional antituberculous drugs has a major impact on the treatment success of tuberculosis, we therefore decided to review carefully Mycobacterium tuberculosis drug resistance and associated genotypic lineages in the FDA over a seventeen-year period (January 1995–December 2011. A total of 1239 cases were studied, including 153 drug-resistant and 26 multidrug-resistant- (MDR- TB cases, representing 12.3% and 2.1% of the TB cases in our study setting. A significantly higher proportion of M. tuberculosis isolates among relapse cases showed drug resistance to isoniazid (22.5%, P=0.002, rifampicin (20.0%, P<0.001, or both (MDR-TB, 17.5%; P<0.001. Determination of spoligotyping based phylogenetic clades showed that among the five major lineages observed—T family (30.1%; Latin-American and Mediterranean (LAM, 23.7%; Haarlem (H, 22.2%; East-African Indian (EAI, 7.2%; and X family (6.5%—two lineages, X and LAM, were overrepresented in drug-resistant and MDR-TB cases, respectively. Finally, 19 predominant spoligotypes were identified for the 1239 isolates of M. tuberculosis in our study among which 4 were significantly associated with drug resistance corresponding to SIT20/LAM1, SIT64/LAM6, SIT45/H1, and SIT46/undefined lineage.

  1. Population structure and circulating genotypes of drug-sensitive and drug-resistant Mycobacterium tuberculosis clinical isolates in São Paulo state, Brazil

    Science.gov (United States)

    Martins, Maria Conceição; Saraiva Giampaglia, Carmen M.; Oliveira, Rosângela S.; Simonsen, Vera; Latrilha, Fábio Oliveira; Moniz, Letícia Lisboa; Couvin, David; Rastogi, Nalin; Ferrazoli, Lucilaine

    2013-01-01

    São Paulo is the most populous Brazilian state and reports the largest number of tuberculosis cases in the country annually (over 18,500). This study included 193 isolates obtained during the 2nd Nationwide Survey on Mycobacterium tuberculosis Drug Resistance that was conducted in São Paulo state and 547 isolates from a laboratory based study of drug resistance that were analyzed by the Mycobacteria Reference Laboratory at the Institute Adolfo Lutz. Both studies were conducted from 2006 to 2008 and sought to determine the genetic diversity and pattern of drug resistance of M. tuberculosis isolates (MTC) circulating in São Paulo. The patterns obtained from the spoligotyping analysis demonstrated that 51/740 (6.9%) of the isolates corresponded to orphan patterns and that 689 (93.1%) of the isolates distributed into 144 shared types, including 119 that matched a preexisting shared type in the SITVIT2 database and 25 that were new isolates. A total of 77/144 patterns corresponded to unique isolates, while the remaining 67 corresponded to clustered patterns (n = 612 isolates clustered into groups of 2–84 isolates each). The evolutionarily ancient PGG1 lineages (Beijing, CAS1-DEL, EAI3-IND, and PINI2) were rarely detected in São Paulo and comprised only 13/740, or 1.76%, of the total isolates; all of the remaining 727/740, or 98.24%, of the MTC isolates from São Paulo state were from the recent PGG2/3 evolutionary isolates belonging to the LAM, T, S, X, and Haarlem lineages, i.e., the Euro-American group. This study provides the first overview of circulating genotypes of M. tuberculosis in São Paulo state and demonstrates that the clustered shared types containing seven or more M. tuberculosis isolates that are spread in São Paulo state included both resistant and susceptible isolates. PMID:23201043

  2. Population structure and circulating genotypes of drug-sensitive and drug-resistant Mycobacterium tuberculosis clinical isolates in São Paulo state, Brazil.

    Science.gov (United States)

    Martins, Maria Conceição; Giampaglia, Carmen M Saraiva; Oliveira, Rosângela S; Simonsen, Vera; Latrilha, Fábio Oliveira; Moniz, Letícia Lisboa; Couvin, David; Rastogi, Nalin; Ferrazoli, Lucilaine

    2013-03-01

    São Paulo is the most populous Brazilian state and reports the largest number of tuberculosis cases in the country annually (over 18,500). This study included 193 isolates obtained during the 2nd Nationwide Survey on Mycobacterium tuberculosis Drug Resistance that was conducted in São Paulo state and 547 isolates from a laboratory based study of drug resistance that were analyzed by the Mycobacteria Reference Laboratory at the Institute Adolfo Lutz. Both studies were conducted from 2006 to 2008 and sought to determine the genetic diversity and pattern of drug resistance of M. tuberculosis isolates (MTC) circulating in São Paulo. The patterns obtained from the spoligotyping analysis demonstrated that 51/740 (6.9%) of the isolates corresponded to orphan patterns and that 689 (93.1%) of the isolates distributed into 144 shared types, including 119 that matched a preexisting shared type in the SITVIT2 database and 25 that were new isolates. A total of 77/144 patterns corresponded to unique isolates, while the remaining 67 corresponded to clustered patterns (n=612 isolates clustered into groups of 2-84 isolates each). The evolutionarily ancient PGG1 lineages (Beijing, CAS1-DEL, EAI3-IND, and PINI2) were rarely detected in São Paulo and comprised only 13/740, or 1.76%, of the total isolates; all of the remaining 727/740, or 98.24%, of the MTC isolates from São Paulo state were from the recent PGG2/3 evolutionary isolates belonging to the LAM, T, S, X, and Haarlem lineages, i.e., the Euro-American group. This study provides the first overview of circulating genotypes of M. tuberculosis in São Paulo state and demonstrates that the clustered shared types containing seven or more M. tuberculosis isolates that are spread in São Paulo state included both resistant and susceptible isolates.

  3. GenoType MTBDRsl for molecular detection of second-line-drug and ethambutol resistance in Mycobacterium tuberculosis strains and clinical samples.

    Science.gov (United States)

    Lacoma, A; García-Sierra, N; Prat, C; Maldonado, J; Ruiz-Manzano, J; Haba, L; Gavin, P; Samper, S; Ausina, V; Domínguez, J

    2012-01-01

    The purpose of this study was to evaluate the GenoType MTBDRsl assay (Hain Lifescience GmbH, Nehren, Germany) for its ability to detect resistance to fluoroquinolones (FLQ), injectable second-line antibiotics [kanamycin (KM) and capreomycin (CM)], and ethambutol (EMB) in Mycobacterium tuberculosis clinical strains and directly in clinical samples. A total of 34 clinical strains were characterized with the Bactec 460 TB system. Fifty-four clinical samples from 16 patients (5 were smear negative and 49 were smear positive) were also tested directly. The corresponding isolates of the clinical specimens were also analyzed with the Bactec 460TB. When there was a discrepancy between assays, pyrosequencing was performed. The overall rates of concordance of the MTBDRsl and the Bactec 460TB for the detection of FLQ, KM/CM, and EMB susceptibility in clinical strains were 72.4% (21/29), 88.8% (24/27), and 67.6% (23/34), whereas for clinical samples, rates were 86.5% (45/52), 92.3% (48/52), and 56% (28/50), respectively. In conclusion, the GenoType MTBDRsl assay may be a useful tool for making early decisions regarding KM/CM susceptibility and to a lesser extent regarding FLQ and EMB susceptibility. The test is able to detect mutations in both clinical strains and samples with a short turnaround time. However, for correct management of patients with extensively drug-resistant tuberculosis, results must be confirmed by a phenotypical method.

  4. Two cases of pulmonary tuberculosis caused by Mycobacterium tuberculosis subsp canetti.

    Science.gov (United States)

    Miltgen, Jean; Morillon, Marc; Koeck, Jean-Louis; Varnerot, Anne; Briant, Jean-François; Nguyen, Gilbert; Verrot, Denis; Bonnet, Daniel; Vincent, Véronique

    2002-11-01

    We identified an unusual strain of mycobacteria from two patients with pulmonary tuberculosis by its smooth, glossy morphotype and, primarily, its genotypic characteristics. Spoligotyping and restriction fragment length polymorphism typing were carried out with the insertion sequence IS6110 patterns. All known cases of tuberculosis caused by Mycobacterium canetti have been contracted in the Horn of Africa.

  5. Clustering of Beijing genotype Mycobacterium tuberculosis isolates from the Mekong delta in Vietnam on the basis of variable number of tandem repeat versus restriction fragment length polymorphism typing

    Directory of Open Access Journals (Sweden)

    Huyen Mai NT

    2013-02-01

    Full Text Available Abstract Background In comparison to restriction fragment length polymorphism (RFLP typing, variable number of tandem repeat (VNTR typing is easier to perform, faster and yields results in a simple, numerical format. Therefore, this technique has gained recognition as the new international gold standard in typing of Mycobacterium tuberculosis. However, some reports indicated that VNTR typing may be less suitable for Beijing genotype isolates. We therefore compared the performance of internationally standardized RFLP and 24 loci VNTR typing to discriminate among 100 Beijing genotype isolates from the Southern Vietnam. Methods Hundred Beijing genotype strains defined by spoligotyping were randomly selected and typed by RFLP and VNTR typing. The discriminatory power of VNTR and RFLP typing was compared using the Bionumerics software. Results Among 95 Beijing strains available for analysis, 14 clusters were identified comprising 34 strains and 61 unique profiles in 24 loci VNTR typing ((Hunter Gaston Discrimination Index (HGDI = 0.994. 13 clusters containing 31 strains and 64 unique patterns in RFLP typing (HGDI = 0.994 were found. Nine RFLP clusters were subdivided by VNTR typing and 12 VNTR clusters were split by RFLP. Five isolates (5% revealing double alleles or no signal in two or more loci in VNTR typing could not be analyzed. Conclusions Overall, 24 loci VNTR typing and RFLP typing had similar high-level of discrimination among 95 Beijing strains from Southern Vietnam. However, loci VNTR 154, VNTR 2461 and VNTR 3171 had hardly added any value to the level of discrimination.

  6. Immune Responses in Cattle Inoculated with Mycobacterium bovis, Mycobacterium tuberculosis, or Mycobacterium kansasii

    Science.gov (United States)

    Cattle were inoculated with Mycobacterium bovis, Mycobacterium tuberculosis, or Mycobacterium kansasii to compare antigen-specific immune responses to varied patterns of mycobacterial disease. Disease expression ranged from colonization with associated pathology (M. bovis), colonization without path...

  7. Genotypic Detection of rpoB and katG Gene Mutations Associated with Rifampicin and Isoniazid Resistance in Mycobacterium Tuberculosis Isolates: A Local Scenario (Kelantan).

    Science.gov (United States)

    Ismail, Nurul-Ain; Ismail, Mohd Fazli; Noor, Siti Suraiya Md; Camalxaman, Siti Nazrina

    2016-01-01

    Drug resistant tuberculosis (DR-TB) remains a public health issue that is of major concern on a global scale. The characterisation of clinical isolates may provide key information regarding the underlying mechanisms of drug resistance, and helps to augment therapeutic options. This study aims to evaluate the frequency of gene mutations associated with Rifampicin (RIF) and Isoniazid (INH) resistance among nine clinical isolates. A total of nine drug resistant Mycobacterium tuberculosis clinical isolates were screened for genetic mutations in rpoB and katusing polymerase chain reaction (PCR) amplification and DNA sequencing. Genotypic analysis was performed to detect the mutations in the sequence of the target genes. Our findings reveal that 80% of the isolates possess mutations at codon 119 (His119Tyr) and 135 (Arg135Trp and Ser135Leu) within the rpoB gene; and 70% possess mutations in the katG gene at codon 238 with amino acid change (Leu238Arg). Findings from this study provide an overview of the current situation of RIF and INH resistance in a hospital Universiti Sains Malaysia (HUSM) located in Kelantan, Malaysia, which could facilitate molecular-based detection methods of drug-resistant strains. Further information regarding the molecular mechanisms involved in resistance in RR-/MDR-TB should be addressed in the near future.

  8. Rapid and simultaneous detection of Mycobacterium tuberculosis complex and Beijing/W genotype in sputum by an optimized DNA extraction protocol and a novel multiplex real-time PCR.

    Science.gov (United States)

    Leung, Eric T Y; Zheng, L; Wong, Rity Y K; Chan, Edward W C; Au, T K; Chan, Raphael C Y; Lui, Grace; Lee, Nelson; Ip, Margaret

    2011-07-01

    Rapid diagnosis and genotyping of Mycobacterium tuberculosis by molecular methods are often limited by the amount and purity of DNA extracted from body fluids. In this study, we evaluated 12 DNA extraction methods and developed a highly sensitive protocol for mycobacterial DNA extraction directly from sputa using surface-coated magnetic particles. We have also developed a novel multiplex real-time PCR for simultaneous identification of M. tuberculosis complex and the Beijing/W genotype (a hypervirulent sublineage of M. tuberculosis) by using multiple fluorogenic probes targeting both the M. tuberculosis IS6110 and the Rv0927c-pstS3 intergenic region. With reference strains and clinical isolates, our real-time PCR accurately identified 20 non-Beijing/W and 20 Beijing/W M. tuberculosis strains from 17 different species of nontuberculosis Mycobacterium (NTM). Further assessment of our DNA extraction protocol and real-time PCR with 335 nonduplicate sputum specimens correctly identified all 74 M. tuberculosis culture-positive specimens. In addition, 15 culture-negative specimens from patients with confirmed tuberculosis were also identified. No cross-reactivity was detected with NTM specimens (n = 31). The detection limit of the assay is 10 M. tuberculosis bacilli, as determined by endpoint dilution analysis. In conclusion, an optimized DNA extraction protocol coupled with a novel multiprobe multiplex real-time PCR for the direct detection of M. tuberculosis, including Beijing/W M. tuberculosis, was found to confer high sensitivity and specificity. The combined procedure has the potential to compensate for the drawbacks of conventional mycobacterial culture in routine clinical laboratory setting, such as the lengthy incubation period and the limitation to viable organisms.

  9. Study of Mycobacterium tuberculosis complex genotypic diversity in Malaysia reveals a predominance of ancestral East-African-Indian lineage with a Malaysia-specific signature.

    Directory of Open Access Journals (Sweden)

    Fazli Ismail

    Full Text Available Tuberculosis (TB still constitutes a major public health problem in Malaysia. The identification and genotyping based characterization of Mycobacterium tuberculosis complex (MTBC isolates causing the disease is important to determine the effectiveness of the control and surveillance programs.This study intended a first assessment of spoligotyping-based MTBC genotypic diversity in Malaysia followed by a comparison of strains with those prevailing in neighboring countries by comparison with an international MTBC genotyping database.Spoligotyping was performed on a total of 220 M. tuberculosis clinical isolates collected in Kelantan and Kuala Lumpur. The results were compared with the SITVIT2 international database of the Pasteur Institute of Guadeloupe.Spoligotyping revealed 77 different patterns: 22 corresponded to orphan patterns while 55 patterns containing 198 isolates were assigned a Spoligo International Type (SIT designation in the database (the latter included 6 newly created SITs. The eight most common SITs grouped 141 isolates (5 to 56 strains per cluster as follows: SIT1/Beijing, n = 56, 25.5%; SIT745/EAI1-SOM, n = 33, 15.0%; SIT591/EAI6-BGD1, n = 13, 5.9%; SIT256/EAI5, n = 12, 5.5%; SIT236/EAI5, n = 10, 4.6%; SIT19/EAI2-Manila, n = 9, 4.1%; SIT89/EAI2-Nonthaburi, n = 5, 2.3%; and SIT50/H3, n = 3, 1.4%. The association between city of isolation and lineages was statistically significant; Haarlem and T lineages being higher in Kuala Lumpur (p<0.01. However, no statistically significant differences were noted when comparing drug resistance vs. major lineages, nor between gender and clades.The ancestral East-African-Indian (EAI lineage was most predominant followed by the Beijing lineage. A comparison of strains with those prevailing in neighboring countries in South Asia, East Asia and South East Asia underlined the phylogeographical specificity of SIT745 for Malaysia, and its probable ongoing evolution

  10. Mycobacterium tuberculosis: factores de virulencia

    Directory of Open Access Journals (Sweden)

    Reinier Borrero

    2011-04-01

    Full Text Available Mycobacterium tuberculosis es el agente causal de la tuberculosis, una de las enfermedades infecciosas más letales en el mundo. La única vacuna disponible para su control es el BCG, sin embargo, falla en la protección contra la tuberculosis pulmonar, siendo esta la forma más frecuente y responsable de la diseminación. La identificación de factores de virulencia del microorganismo causal pudiera ayudar en el desarrollo de un nuevo candidato vacunal que sea capaz de neutralizar la acción de esos determinantes patogénicos. El empleo de diferentes modelos animales ha permitido reproducir las etapas de la enfermedad, así como medir o cuantificar la virulencia de las distintas cepas circulantes de Mycobacterium tuberculosis. Las mutaciones génicas y otras técnicas de biología molecular han posibilitado dilucidar los genes específicos involucrados en la virulencia de este microorganismo que codifican para múltiples y complejos factores de diferente naturaleza.

  11. High-Throughput Mycobacterial Interspersed Repetitive-Unit–Variable-Number Tandem-Repeat Genotyping for Mycobacterium tuberculosis Epidemiological Studies

    Science.gov (United States)

    Bidault, Floriane; Mosnier, Amandine; Bablishvili, Nino; Tukvadze, Nestani; Somphavong, Silaphet; Paboriboune, Phimpha; Ocheretina, Oksana; Pape, Jean William; Paranhos-Baccala, Glaucia; Berland, Jean-Luc

    2014-01-01

    The emergence of drug-resistant forms of tuberculosis (TB) represents a major public health concern. Understanding the transmission routes of the disease is a key factor for its control and for the implementation of efficient interventions. Mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) marker typing is a well-described method for lineage identification and transmission tracking. However, the conventional manual genotyping technique is cumbersome and time-consuming and entails many risks for errors, thus hindering its implementation and dissemination. We describe here a new approach using the QIAxcel system, an automated high-throughput capillary electrophoresis system that also carries out allele calling. This automated method was assessed on 1,824 amplicons from 82 TB isolates and tested with sets of markers of 15 or 24 loci. Overall allele-calling concordance between the methods from 140 to 1,317 bp was 98.9%. DNA concentrations and repeatability and reproducibility performances showed no biases in allele calling. Furthermore, turnaround time using this automated system was reduced by 81% compared to the conventional manual agarose gel method. In sum, this new automated method facilitates MIRU-VNTR genotyping and provides reliable results. Therefore, it is well suited for field genotyping. The implementation of this method will help to achieve accurate and cost-effective epidemiological studies, especially in countries with a high prevalence of TB, where the high number of strains complicates the surveillance of circulating lineages and requires efficient interventions to be carried out in an urgent manner. PMID:25428144

  12. [Evaluation of the genotype MTBDR assay for rapid detection of rifampin and isoniazid resistance in clinical Mycobacterium tuberculosis complex clinical isolates].

    Science.gov (United States)

    Aslan, Gönül; Tezcan, Seda; Emekdaş, Gürol

    2009-04-01

    Rapid identification of resistant Mycobacterium tuberculosis complex isolates is quite important for the establishment of early and appropriate therapy. The Genotype MTBDR (Hain Lifescience, Nehren, Germany) is a commercially available DNA strip assay designed for the rapid detection of rpoB and katG gene mutations in clinical isolates. This study was conducted to determine the mutation types of phenotypically drug resistant 26 M. tuberculosis complex clinical isolates [15 isoniazid (INH), 1 rifampin (RMP) and 10 INH and RMP resistant] by Genotype MTBDR (G-MTBDR) DNA strip assay and to compare the diagnostic performance of this test. Sixteen of 25 (64%) INH-resistant and 9 of 11 (81.8%) RMP-resistant clinical isolates were correctly identified with the presence of hybridization in mutation probe or lack of hybridization at least by one of the wild type probes, by G-MTBDR assay. Hybridization with mutation probes was detected in only 5 of the RMP resistant isolates. We observed rpoB MUT3 (S531L, Ser-->4Leu) mutation in 4 and rpoB MUT1 (D516V) in one of these isolates. In 56% (14/25) of the INH-resistant isolates, katG T1 (S315T1) hybridization pattern was observed at katG mutation probe. G-MTBDR assay couldn't identify two of the 11 (18.2%) RMP-resistant isolates and one of these iSolates was shown to have a mutation at codon 531 (TCG-GCG) and the other at codon 545 (CTG-->ATG), possibly not associated with resistance, by sequence analysis. In four of the eight (8/25; 32%) INH-resistant isolates not identified by G-MTBDR assay, DNA cycle sequencing revealed different nucleotide changes outside the most common mutation zone. One of these were at codon 293 (GCT-->ACT) in katG, one with dual mutation at 279 (GGC-->ACC) in katG and at 15th C-->T in inhA gene, one at 15th C-->T in inhA gene and one at 279 (GGC-->ACC) in katG gene region. DNA membrane strip assay can be a use ful tool for the rapid detection of resistant M. tuberculosis complex isolates and therefore

  13. Detection by GenoType MTBDRsl test of complex mechanisms of resistance to second-line drugs and ethambutol in multidrug-resistant Mycobacterium tuberculosis complex isolates.

    Science.gov (United States)

    Brossier, Florence; Veziris, Nicolas; Aubry, Alexandra; Jarlier, Vincent; Sougakoff, Wladimir

    2010-05-01

    The GenoType MTBDRsl test rapidly detects resistance to ethambutol, fluoroquinolones, and second-line aminoglycosides (amikacin and kanamycin) and cyclic peptide (capreomycin) in Mycobacterium tuberculosis. A set of 41 multidrug-resistant (MDR) M. tuberculosis strains, 8 extensively drug-resistant (XDR) M. tuberculosis strains, and 3 non-MDR M. tuberculosis strains were tested by the MTBDRsl test and by DNA sequencing of the resistance-determining regions in gyrA and gyrB (fluoroquinolones [FQ]), rpsL (streptomycin), rrs and tlyA (aminoglycosides and/or cyclic peptide), and embB (ethambutol). The sensitivity and specificity of the MTBDRsl test were as follows: 87% and 96%, respectively, for fluoroquinolones; 100% for both for amikacin; 77% and 100%, respectively, for kanamycin, 80% and 98%, respectively, for capreomycin; and 57% and 92%, respectively, for ethambutol. Analysis of the discrepant results indicated that three FQ-resistant strains (including one XDR strain) with mutations in gyrB were missed by the MTBDRsl test and that one FQ-susceptible strain, identified as resistant by the MTBDRsl test, had a double mutation (T80A-A90G) in GyrA that did not confer resistance to FQ. Five strains (including two XDR strains) without mutations in rrs were monoresistant to aminoglycosides or cyclic peptide and were missed by the MTBDRsl test. Finally, 12/28 ethambutol-resistant strains had no mutation at codon 306 in embB, while 2/24 ethambutol-susceptible strains had such a mutation. In conclusion, the MTBDRsl test efficiently detects the most common mutations involved in resistance to fluoroquinolones, aminoglycosides/cyclic peptide, and ethambutol and accurately assesses susceptibility to amikacin. However, due to mutations not included in the test (particularly in gyrB) or resistance mechanisms not yet characterized (particularly those related to ethambutol resistance and to monoresistance to aminoglycosides or cyclic peptide), the wild-type results yielded by the

  14. Mycobacterium tuberculosis monoarthritis in a child

    Directory of Open Access Journals (Sweden)

    Rosenberg Alan M

    2008-09-01

    Full Text Available Abstract A child with isolated Mycobacterium tuberculosis monoarthritis, with features initially suggesting oligoarthritis subtype of juvenile idiopathic arthritis, is presented. This patient illustrates the need to consider the possibility of tuberculosis as the cause of oligoarthritis in high-risk pediatric populations even in the absence of a tuberculosis contact history and without evidence of overt pulmonary disease.

  15. Copper Homeostasis in Mycobacterium tuberculosis

    Science.gov (United States)

    Shi, Xiaoshan; Darwin, K. Heran

    2015-01-01

    Copper (Cu) is a trace element essential for the growth and development of almost all organisms, including bacteria. However, Cu overload in most systems is toxic. Studies show Cu accumulates in macrophage phagosomes infected with bacteria, suggesting Cu provides an innate immune mechanism to combat invading pathogens. To counteract the host-supplied Cu, increasing evidence suggests that bacteria have evolved Cu resistance mechanisms to facilitate their pathogenesis. In particular, Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, has evolved multiple pathways to respond to Cu. Here, we summarize what is currently known about Cu homeostasis in Mtb and discuss potential sources of Cu encountered by this and other pathogens in a mammalian host. PMID:25614981

  16. [Drug sensitivity in Mycobacterium tuberculosis versus its viability, cytotoxicity, genotype, and the course of the process in patients with pulmonary tuberculosis].

    Science.gov (United States)

    Manicheva, O A; Lasunskaia, E B; Zhuravlev, V Iu; Otten, T F; Barnaulov, A O; Mokrousov, I V; Pavlova, M V; Vishnevskiĭ, B I; Narvskaia, O V

    2008-01-01

    The authors studied drug sensitivity, mutations in the katG, in-hA, alpC, rpoB genes, virulence via the cytotoxicity test on THP-1 cells, and the viability and genetic affiliation of 53 clinical M. tuberculosis isolates versus data on the form and dynamics of a process. Sensitive and resistant strains did not significantly differ in viability and cytotoxicity. The highest death of infected macrophages was observed was seen with infection of M. tuberculosis of the Beijing B0 genotype, the least one seen with that of LAM with the similar rate of multiple drug resistance. There was a correlation of the changes in the count of lymphocytes in patients with the genetic affiliation of a causative agent. The severest course of the tuberculous process was observed in baseline lymphopenia (before treatment) in combination with multidrug resistance of mycobacteria, high and moderate cytotoxicity and high viability. Ser-Leu 531 mutation resulted in cross resistance to rifampicin and mycobutin in most cases.

  17. Diversity of Mycobacterium tuberculosis lineages in French Polynesia.

    Science.gov (United States)

    Osman, Djaltou Aboubaker; Phelippeau, Michael; Drancourt, Michel; Musso, Didier

    2017-04-01

    French Polynesia is an overseas territory located in the South Pacific. The incidence of tuberculosis in French Polynesia has been stable since 2000 with an average of 20 cases/y/100,000 inhabitants. Molecular epidemiology of Mycobacterium tuberculosis in French Polynesia is unknown because M. tuberculosis isolates have not been routinely genotyped. From 2009 to 2012, 34 isolates collected from 32 French Polynesian patients were identified as M. tuberculosis by probe hybridization. These isolates were genotyped using spoligotyping and 24-loci mycobacterial interspersed repetitive units (MIRUs)-variable number of tandem repeat (VNTR). Spoligotype patterns obtained using commercial kits were compared with the online international database SITVIT. MIRU-VNTR genotyping was performed using an in-house protocol based on capillary electrophoresis sizing for 24-loci MIRU-VNTR genotyping. The results of the spoligotyping method revealed that 25 isolates grouped into six previously described spoligotypes [H1, H3, U likely (S), T1, Manu, and Beijing] and nine isolates grouped into six new spoligotypes. Comparison with the international database MIRU-VNTRplus distributed 30 isolates into five lineages (Haarlem, Latin American Mediterranean, S, X, and Beijing) and four as unassigned isolates. Genotyping identified four phylogenetic lineages belonging to the modern Euro-American subgroup, one Beijing genotype responsible for worldwide pandemics, including remote islands in the South Pacific, and one Manu genotype of the ancestral lineage of M. tuberculosis. Copyright © 2015. Published by Elsevier B.V.

  18. Comparison of antibody responses against Mycobacterium tuberculosis antigen Rv0679c in tuberculosis patients from the endemic and non-endemic regions of the Beijing genotype: a case control study.

    Science.gov (United States)

    Zhao, Jingge; Matsuba, Takashi; Zhang, Xiaoyan; Leano, Susan; Nakajima, Chie; Chagan-Yasutan, Haorile; Telan, Elizabeth Freda; Suzuki, Yasuhiko; Hattori, Toshio

    2017-05-15

    Strains of the Beijing genotype of Mycobacterium tuberculosis (MTB) are reportedly associated with the virulence of tuberculosis (TB) infection, unfavorable outcomes of anti-TB treatment, and the global TB pandemic. Rv0679c, a hypothetical membrane protein related to host cell invasion, has a Beijing genotype-specific mutation at residue 142 (Asn142Lys). Antigenicity differences between Rv0679c-Asn142 (N-type) and Rv0679c-Lys142 (K-type) have been previously observed in mice antigen-antibody responses. However, the immune response to Rv0679c in humans remains unknown. Therefore, we aimed to investigate the anti-Rv0679c immune response in TB patients from the endemic and non-endemic regions of the Beijing MTB genotype. We analyzed the Rv0679c-specific antibody responses in 84 subjects from the endemic region of the Beijing genotype MTB in China, including 45 pulmonary TB patients (C-PTB) and 39 healthy controls (C-HC), and 81 subjects from the Philippines (the endemic region of the non-Beijing genotype), including 51 pulmonary TB patients (P-PTB) and 30 healthy controls (P-HC). Anti-tuberculous-glycolipid (TBGL) antigen was used as the control antibody. TBGL IgG titers were higher in both C-PTB and P-PTB than those in their corresponding HC (C-PTB median 4.2, P-PTB median 11.2; C-PTB vs. P-PTB, p > 0.05), suggesting immune response comparability in PTB from two different countries. C-PTB showed a higher response compared to C-HC for anti-K-type IgG (53.3%) than anti-N-type IgG (6.67%); this response was not observed in P-PTB (both N-type and K-type 9.80%). Dimorphic antigen Rv0679c was found to be associated with distinct immune response patterns, indicating the role of Beijing/non-Beijing genotype of MTB in stimulating specific responses in TB patients from the endemic region of Beijing MTB. Meanwhile, reactions to Rv0679c in patients and HC from non-endemic regions of the Beijing MTB may be caused by the response to the common epitope of Rv0679c N/K-type.

  19. Polymorphisms of twenty regulatory proteins between Mycobacterium tuberculosis and Mycobacterium bovis

    Science.gov (United States)

    Mycobacterium tuberculosis and Mycobacterium bovis are responsible for tuberculosis in humans or animals, respectively. Both species are closely related and belong to the Mycobacterium tuberculosis complex (MTC). M. tuberculosis is the most ancient species from which M. bovis and the other members o...

  20. Osteoarticular manifestations of Mycobacterium tuberculosis infection.

    Science.gov (United States)

    Zychowicz, Michael E

    2010-01-01

    Mycobacterium tuberculosis has affected humans for much of our existence. The incidence of global tuberculosis infection continues to rise, especially in concert with HIV coinfection. Many disease processes, such as diabetes, increase the likelihood of tuberculosis infection. Tuberculosis bacteria can infect any bone, joint, tendon, or bursa; however, the most common musculoskeletal site for infection includes the spine and weight-bearing joints of the hip and knee. Many patients who present with osteoarticular tuberculosis infection will have a gradual onset of pain at the site of infection. Many patients who develop a musculoskeletal tuberculosis infection will have no evidence of a pulmonary tuberculosis infection on x-ray film and many will have very mild symptoms with the initial infection. Healthcare providers must remember that many patients who develop tuberculosis infection do not progress to active tuberculosis disease; however, the latent infection may become active with immune compromise.

  1. Peritoneal tuberculosis due to Mycobacterium caprae

    Directory of Open Access Journals (Sweden)

    T. Nebreda

    2016-01-01

    Full Text Available The incidence of tuberculosis in humans due to Mycobacterium caprae is very low and is almost confined to Europe. We report a case of a previously healthy 41-year-old Moroccan with a 6 month history of abdominal pain, weight loss, fatigue and diarrhea. A diagnosis of peritoneal tuberculosis due to M. caprae was made.

  2. Using Approximate Bayesian Computation to Estimate Tuberculosis Transmission Parameters From Genotype Data

    OpenAIRE

    2006-01-01

    Tuberculosis can be studied at the population level by genotyping strains of Mycobacterium tuberculosis isolated from patients. We use an approximate Bayesian computational method in combination with a stochastic model of tuberculosis transmission and mutation of a molecular marker to estimate the net transmission rate, the doubling time, and the reproductive value of the pathogen. This method is applied to a published data set from San Francisco of tuberculosis genotypes based on the marker ...

  3. Animal-adapted members of the Mycobacterium tuberculosis complex endemic to the southern African subregion

    Directory of Open Access Journals (Sweden)

    Charlene Clarke

    2016-02-01

    Full Text Available Members of the Mycobacterium tuberculosis complex (MTC cause tuberculosis (TB in both animals and humans. In this article, three animal-adapted MTC strains that are endemic to the southern African subregion – that is, Mycobacterium suricattae, Mycobacterium mungi, and the dassie bacillus – are reviewed with a focus on clinical and pathological presentations, geographic distribution, genotyping methods, diagnostic tools and evolution. Moreover, factors influencing the transmission and establishment of TB pathogens in novel host populations, including ecological, immunological and genetic factors of both the host and pathogen, are discussed. The risks associated with these infections are currently unknown and further studies will be required for greater understanding of this disease in the context of the southern African ecosystem.Keywords: dassie bacillus; ecology; evolution; host jump; Mycobacterium mungi; Mycobacterium suricattae; Mycobacterium tuberculosis complex; phylogeny

  4. Dormancy models for Mycobacterium tuberculosis: A minireview

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    Amani M. Alnimr

    2015-09-01

    Full Text Available Dormancy models for Mycobacterium tuberculosis play important roles in understanding various aspects of tuberculosis pathogenesis and in the testing of novel therapeutic regimens. By simulating the latent tuberculosis infection, in which the bacteria exist in a non-replicative state, the models demonstrate reduced susceptibility to antimycobacterial agents. This minireview outlines the models available for simulating latent tuberculosis both in vitro and in several animal species. Additionally, this minireview discusses the advantages and disadvantages of these models for investigating the bacterial subpopulations and susceptibilities to sterilization by various antituberculosis drugs.

  5. VNTR位点对结核分枝杆菌基因分型的研究%A study on genotyping of mycobacterium tuberculosis by variable number tandem repeat analysis method

    Institute of Scientific and Technical Information of China (English)

    张思潮; 董正全; 杨中荣

    2014-01-01

    Objective To preliminarily evaluate the ability of 15 -loci variable number tandem repeats on the genotyping of clinical isolates of mycobacterium tuberculosis,and explore the suitable loci for genotyping of Mycobacterium tuberculosis in Huzhou City.Methods From 201 1 January to 201 1 April,the clinical isolates of mycobacterium tuberculosis were selected from Huzhou area to test the polymorphism of every loci by using MLVA detection method and analyze hunter-gaston discriminatory index of individual locus as well as combinations of 15 locus.Results There were two locus of Mtub-21 and Mtub-39 which had better degree of polymorphism;seven locus of ETR-A,ETR-C,MIRU-10,MIRU-23,MIRU-27,MIRU-40 and Mtub-30 had moderate degree of polymorphism;six locus of ETR-B,ETR-D,ETR-E,MIRU-16,MIRU-26 and MIRU-39 had poor degree of polymorphism.By calculation,the result showed that HGDI was 0.99 by using 15 locus to genotype,which indicated the ability to distinguish the genotype was good.Conclusion We will continue to look for other rich VNTR polymorphism locus to fit the local region fast genotyping of mycobacterium tuberculosis for higher resolution and cost effectiveness of mycobacterium tuberculosis control and prevention.%目的:初步评价15个可变串联重复序列(VNTR)位点对结核分枝杆菌临床分离株的分型能力,寻找适合湖州地区结核分枝杆菌基因分型的位点。方法2011年1-4月连续收集湖州地区结核分枝杆菌临床分离株,采用多位点数目多位点串联系复序列(MLVA)分析方法,对15个VNTR位点进行基因多态性检测,分析单个位点以及15个位点组合的基因分型能力(Hunter-gaston指数,HGDI)。结果分辨率较好的多态性位点有2个:Mtub-21和Mtub -39;分辨率中等的多态性位点共7个:ETR-A,ETR-C,MIRU -10,MIRU -23, MIRU-27,MIRU-40和Mtub-30;分辨率较差的多态性位点有6个:ETR-B,ETR-D,ETR-E,MIRU-16和MIRU-26和MIRU-39。通

  6. [Resistance to first-line drugs and major genotypic lineages of Mycobacterium tuberculosis in the 3 French Department of the Americas: Profiles, evolution, and trends (1995-2011)].

    Science.gov (United States)

    Millet, J; Berchel, M; Prudenté, F; Streit, E; Bomer, A-G; Schuster, F; Vanhomwegen, J; Paasch, D; Galbert, I; Valery, E; Aga, R; Rastogi, N

    2014-05-01

    This is the first overview on resistant and multidrug resistant isolates of Mycobacterium tuberculosis circulating in the French Department of the Americas (Guadeloupe, Martinique, and French Guiana) over 17 years (January 1995-December 2011). A total of 1,239 cases were studied: 1,199 new cases (primary and multidrug resistance of 11.8 and 1.6% respectively), and 40 persistent (defined as cases with a previous history of positive culture over 6 months interval and whose spoligotypes remain unchanged), in which significantly higher proportions of resistance to at least isoniazid (22.5%, P = 0.002), rifampicin (20.0%, P French Guiana, department in which one may also observe the presence of strains of M. tuberculosis phylogeographically associated to Guiana and Suriname (SIT 131 and SIT 1340).

  7. Genotyping of Mycobacterium tuberculosis Isolates from Hormozgan Province of Iran Based on 15-Locus MIRU-VNTR and Spoligotyping

    Science.gov (United States)

    Zamani, Samin; Haeili, Mehri

    2016-01-01

    Background. Considering that Hormozgan province in Iran (southern part of Iran on the Persian Gulf) is among the areas with high prevalence of MDR-MTB and attracts so many sailors and tourists, genetic diversity of MTB isolates circulating in this part of Iran was evaluated. Pattern of TB transmission was also examined. Methods and Material. A total of 38 isolates of MTB were cultured from TB patients from Hormozgan province of Iran and standard MIRU-VNTR typing and spoligotyping were applied to genotype these isolates. Drug susceptibility testing was performed using proportion method. Results. There were 28 VNTR profiles comprising 5 clusters and 23 unique isolates compared to 21 spoligotyping profiles, which contained 9 clusters and 12 unique isolates. Latin American-Mediterranean (n = 9, 23.6%) was found to be the most predominant lineage. MIRU-VNTR analysis, with an HGDI of 0.975, was more discriminating than spoligotyping, which had an HGDI of 0.955. The estimated proportion of TB cases due to recent transmission was 26.3% and 44.7% by MIRU-VNTR and spoligotyping, respectively. The rates of monodrug resistance and MDR were 15.8% and 7.9%, respectively. Two of 3 MDR strains were found to be related to MIRU-VNTR and belonged to the same spoligotyping cluster characterized with T1/SIT53 genotype. Conclusions. The high genetic diversity among MTB isolates suggests that transmission occurred from different sources to this area. Reactivation of a priori, latent MTB infection was found to contribute mainly to TB cases in this geographic region. PMID:27819023

  8. Early detection of multi-drug resistance and common mutations in Mycobacterium tuberculosis isolates from Delhi using GenoType MTBDRplus assay

    OpenAIRE

    2015-01-01

    Purpose: There is scarcity of prevalence data of multi-drug-resistant tuberculosis (MDR-TB) data and common mutations responsible in North India. This study aimed to detect MDR-TB among MDR-TB suspects from Delhi and mutation patterns using GenoType MTBDRplus assay. Materials and Methods: All MDR suspects in five districts of New Delhi were referred to the laboratory from 1 st October 2011 to 31 st December 2012 as per criterion defined by Programmatic Management of Drug Resistant Tuberculosi...

  9. Diterpene production in Mycobacterium tuberculosis

    Science.gov (United States)

    Prach, Lisa; Kirby, James; Keasling, Jay D.; Alber, Tom

    2011-01-01

    Diterpenes are a structurally diverse class of molecules common in plants, although they are very rarely found in bacteria. We report the identification in Mycobacterium tuberculosis (Mtb) of three diterpenes proposed to promote phagolysosome maturation arrest. MS analysis reveals that these diterpenes are novel compounds not previously identified in other organisms. The diterpene with highest abundance in Mtb has a mass fragmentation pattern identical to edaxadiene, which is produced in vitro from geranylgeranyl diphosphate by the enzymes Rv3377c and Rv3378c [Mann FM et al. (2009) J Am Chem Soc 131, 17526–17527]. A second diterpene found in Mtb has a similar mass spectrum, and is always observed in the same proportion relative to edaxadiene, indicating that it is a side product of the Rv3378c reaction in vivo. We name this second diterpene olefin edaxadiene B. The least abundant of the three diterpenes in Mtb extracts is tuberculosinol, a dephosphorylated side-product of the edaxadiene pathway intermediate produced by Rv3377c [Nakano C et al. (2009) Chembiochem 10, 2060–2071; Nakano C et al. (2005) Chem Commun (Camb) 8, 1016–1018]. A frameshift in Rv3377c in Mtb completely eliminates diterpene production, whereas expression of Rv3377c and Rv3378c in the nonpathogenic M. smegmatis is sufficient to produce edaxadiene and edaxadiene B. These studies define the pathway of edaxadiene and edaxadiene B biosynthesis in vivo. Rv3377c and Rv3378c are unique to Mtb and M. bovis, making them candidates for selective therapeutics and diagnostics. PMID:20670276

  10. Performance assessment of the GenoType MTBDRsl test and DNA sequencing for detection of second-line and ethambutol drug resistance among patients infected with multidrug-resistant Mycobacterium tuberculosis.

    Science.gov (United States)

    Huang, Wei-Lun; Chi, Ting-Lin; Wu, Mei-Hua; Jou, Ruwen

    2011-07-01

    The GenoType MTBDRsl test and DNA sequencing were used to rapidly detect second-line drug- and ethambutol (EMB)-resistant Mycobacterium tuberculosis. The ability of these two assays to detect the presence of mutations associated with resistance to fluoroquinolones (FLQ), aminoglycosides/cyclic peptide (AG/CP), and EMB in the gyrA, rrs, and embB genes (for the GenoType MTBDRsl test) and gyrA, gyrB, rrs, eis, embC, embA, embB, and embR genes (for DNA sequencing) was compared to that of conventional agar proportion drug susceptibility testing (DST). We evaluated 234 multidrug-resistant (MDR) M. tuberculosis isolates. The two molecular methods had high levels of specificity (95.8 to 100%). The sensitivities for FLQ resistance detection for both methods were 85.1%. For AG (kanamycin [KM] and amikacin [AM]) and CP (capreomycin CAP]), the sensitivities of resistance detection using the GenoType MTBDRsl test were 43.2%, 84.2%, and 71.4%, respectively, while with the inclusion of an extra gene, eis, in sequencing, the sensitivity reached 70.3% for detection of KM resistance. The sensitivities of EMB resistance detection were 56.2% and 90.7% with the GenoType MTBDRsl test and sequencing, respectively. We found that the GenoType MTBDRsl test can rapidly detect resistance to FLQ, CAP, and AM. The accuracy of the GenoType MTBDRsl test for the detection of FLQ and AM resistance was comparable to that of conventional DST; however, the test was less accurate for the detection of KM and EMB resistance and demonstrated a poor predictive value for CAP resistance. We recommend including new alleles consisting of the eis promoter and embB genes in molecular analysis. However, conventional DST is necessary to rule out false-negative results from molecular assays.

  11. Chemical engineering of Mycobacterium tuberculosis dodecin hybrids.

    Science.gov (United States)

    Vinzenz, Xenia; Grosse, Wolfgang; Linne, Uwe; Meissner, Britta; Essen, Lars-Oliver

    2011-10-21

    The suitability for chemical engineering of the highly symmetrical Mycobacterium tuberculosis dodecin was investigated, its inner cavity providing a large compartment shields introduced compounds from bulk solvent. Hybrids were obtained by S-alkylation of cysteine mutants and characterized by spectroscopic methods, including the crystal structures of wild type and biohybrid dodecins.

  12. [Mycobacterium tuberculosis infection following organ transplantation].

    Science.gov (United States)

    Haas, Charles; Le Jeunne, Claire

    2006-11-01

    In transplant recipients, immunosuppressive treatment affects cell-mediated immunity and increases the risk of tuberculosis. Tuberculosis may be transmitted by the donor organ or occur de novo, but such cases are rare. The vast majority of cases of active tuberculosis in transplant recipients result from reactivation of latent Mycobacterium tuberculosis infection. The incidence varies from one region of the globe to another, from 0.5-1.0% in North America, to 0.36-5.5% in Europe and 7.0-11.8% in India. The incidence of tuberculosis among transplant recipients is much higher than in the general population. Diabetes mellitus, renal impairment, systemic lupus erythematosus, chronic liver disease and AIDS all increase the risk of post-transplant tuberculosis. Extrapulmonary and disseminated forms are frequent in this setting. The diagnosis of tuberculosis in transplant recipients is often difficult, and treatment is frequently delayed. Tuberculosis can be life-threatening in such cases. Treatment is difficult because rifampicin is a cytochrome P450 inducer (leading to reduced levels of cyclosporine), and because the hepatotoxicity of isoniazid, rifampin and pyrazinamide is frequently increased in transplant recipients. Treatment of latent tuberculosis before transplantation markedly reduces the risk of developing active tuberculosis after transplantation.

  13. Mechanisms of fluoroquinolone resistance in Mycobacterium tuberculosis.

    Science.gov (United States)

    Yujiao, Zhang; Xiaojing, Li; Kaixia, Mi

    2016-10-20

    Tuberculosis, caused by the pathogen Mycobacterium tuberculosis, is one of the world's deadliest bacterial infectious disease. It is still a global-health threat, particularly because of the drug-resistant forms. Fluoroquinolones, with target of gyrase, are among the drugs used to treat tuberculosis. However, their widespread use has led to bacterial resistance. The molecular mechanisms of fluoroquinolone resistance in mycobacterium tuberculosis have been reported, such as DNA gyrase mutations, drug efflux pumps system, bacterial cell wall thickness and pentapeptide proteins (MfpA) mediated regulation of gyrase. Mutations in gyrase conferring quinolone resistance play important roles and have been extensively studied. Recent studies have shown that the regulation of DNA gyrase affects mycobacterial drug resistance, but the mechanisms, especially by post-translational modification and regulatory proteins, are poorly understood. In this review, we summarize the fluoroquinolone drug development, and the molecular genetics of fluoroquinolone resistance in mycobacteria. Comprehensive understanding of the mechanisms of fluoroquinolone resistance in Mycobacterium tuberculosis will open a new view on understanding drug resistance in mycobacteria and lead to novel strategies to develop new accurate diagnosis methods.

  14. Cytokinins beyond plants: synthesis by Mycobacterium tuberculosis

    Science.gov (United States)

    Samanovic, Marie I.; Darwin, K. H.

    2015-01-01

    Mycobacterium tuberculosis (M. tuberculosis) resides mainly inside macrophages, which produce nitric oxide (NO) to combat microbial infections. Earlier studies revealed that proteasome-associated genes are required for M. tuberculosis to resist NO via a previously uncharacterized mechanism. Twelve years later, we elucidated the link between proteasome function and NO resistance in M. tuberculosis in Molecular Cell, 57 (2015), pp. 984-994. In a proteasome degradation-defective mutant, Rv1205, a homologue of the plant enzyme LONELY GUY (LOG) that is involved in the synthesis of phytohormones called cytokinins, accumulates and as a consequence results in the overproduction of cytokinins. Cytokinins break down into aldehydes that kill mycobacteria in the presence of NO. Importantly, this new discovery reveals for the first time that a mammalian bacterial pathogen produces cytokinins and leaves us with the question: why is M. tuberculosis, an exclusively human pathogen, producing cytokinins?

  15. Controlling strategy of dormant Mycobacterium tuberculosis

    Institute of Scientific and Technical Information of China (English)

    Gan Yiling; Guo Shuliang

    2014-01-01

    Objective This study aimed to review the available literatures on control of latent tuberculosis (TB) infection and propose a new control strategy to shorten the course of TB chemotherapy.Data sources The data used in this review were mainly obtained from articles listed in PubMed.The search terms were "therapy (treatment) of tuberculosis," "therapy (treatment) of latent TB infection," and "vaccine of TB."Study selection Articles regarding treatment and vaccine of TB were selected and reviewed.Results The most crucial reason causing the prolonged course of TB chemotherapy is the dormant state of Mycobacterium tuberculosis (M.tuberculosis).Nevertheless,there are,to date,no effective drugs that can directly kill the dormant cells of M.tuberculosis in clinical therapy.In accordance with the growth cycle of dormant M.tuberculosis in the body,the methods for controlling dormant M.tuberculosis include direct killing with drugs,prevention of dormant M.tuberculosis resuscitation with vaccines,and resuscitating dormant M.tuberculosis with preparations or drugs and then thoroughly killing these resuscitated M.tuberculosis by using anti-TB therapy.Conclusions The comprehensive analysis of the above three methods suggests that the drugs directly killing dormant cells are in clinical trials,TMC207 is the most beneficial for controlling TB.Because the side effect of vaccines is less and their action period is long,prevention of dormant cells resuscitation with vaccines is promising.The last control method makes it probable that when a huge number of active cells of M.tuberculosis have been killed and eradicated after 1-month short chemotherapy,only a strong short-term subsequent chemotherapy can completely kill and eradicate the remaining M.tuberculosis.This control strategy is expected to significantly shorten the course of TB chemotherapy and bring a new change and breakthrough in TB treatment.

  16. Parallel reaction monitoring of clinical Mycobacterium tuberculosis lineages reveals pre-existent markers of rifampicin tolerance in the emerging Beijing lineage

    NARCIS (Netherlands)

    Keijzer, J. de; Mulder, A.; Ru, A.H. de; Soolingen, D. van; Veelen, P.A. van

    2017-01-01

    The spread of multidrug resistant Mycobacterium tuberculosis is one of the major challenges in tuberculosis control. In Eurasia, the spread of multidrug resistant tuberculosis is driven by the M. tuberculosis Beijing genotype. In this study, we examined whether selective advantages are present in th

  17. Parallel reaction monitoring of clinical Mycobacterium tuberculosis lineages reveals pre-existent markers of rifampicin tolerance in the emerging Beijing lineage

    NARCIS (Netherlands)

    Keijzer, J. de; Mulder, A.; Ru, A.H. de; Soolingen, D. van; Veelen, P.A. van

    2017-01-01

    The spread of multidrug resistant Mycobacterium tuberculosis is one of the major challenges in tuberculosis control. In Eurasia, the spread of multidrug resistant tuberculosis is driven by the M. tuberculosis Beijing genotype. In this study, we examined whether selective advantages are present in

  18. Genotypic Analysis of Genes Associated with Independent Resistance and Cross-Resistance to Isoniazid and Ethionamide in Mycobacterium tuberculosis Clinical Isolates.

    Science.gov (United States)

    Rueda, Johana; Realpe, Teresa; Mejia, Gloria Isabel; Zapata, Elsa; Rozo, Juan Carlos; Ferro, Beatriz Eugenia; Robledo, Jaime

    2015-12-01

    Ethionamide (ETH) is an antibiotic used for the treatment of multidrug-resistant (MDR) tuberculosis (TB) (MDR-TB), and its use may be limited with the emergence of resistance in the Mycobacterium tuberculosis population. ETH resistance in M. tuberculosis is phenomenon independent or cross related when accompanied with isoniazid (INH) resistance. In most cases, resistance to INH and ETH is explained by mutations in the inhA promoter and in the following genes: katG, ethA, ethR, mshA, ndh, and inhA. We sequenced the above genes in 64 M. tuberculosis isolates (n = 57 ETH-resistant MDR-TB isolates; n = 3 ETH-susceptible MDR-TB isolates; and n = 4 fully susceptible isolates). Each isolate was tested for susceptibility to first- and second-line drugs using the agar proportion method. Mutations were observed in ETH-resistant MDR-TB isolates at the following rates: 100% in katG, 72% in ethA, 45.6% in mshA, 8.7% in ndh, and 33.3% in inhA or its promoter. Of the three ETH-susceptible MDR-TB isolates, all showed mutations in katG; one had a mutation in ethA, and another, in mshA and inhA. Finally, of the four fully susceptible isolates, two showed no detectable mutation in the studied genes, and two had mutations in mshA gene unrelated to the resistance. Mutations not previously reported were found in the ethA, mshA, katG, and ndh genes. The concordance between the phenotypic susceptibility testing to INH and ETH and the sequencing was 1 and 0.45, respectively. Among isolates exhibiting INH resistance, the high frequency of independent resistance and cross-resistance with ETH in the M. tuberculosis isolates suggests the need to confirm the susceptibility to ETH before considering it in the treatment of patients with MDR-TB.

  19. Mycobacterium tuberculosis L-forms

    OpenAIRE

    2011-01-01

    M. tuberculosis L-forms researches done in China during the recent years were reviewed in this article. M. tuberculosis L-forms could be produced spontaneously or induced by isoniazid or D-cyclic serine, that influenced the synthesis of cell walls. Among the acid-fast organisms isolated from the patients, more L-forms were found than vegetative forms. In extrapulmonary tuberculosis, M. tuberculosis disseminated through the blood mainly in L-forms, L-forms could adhere on the surface or harbor...

  20. Tuberculosis genotyping information management system: enhancing tuberculosis surveillance in the United States.

    Science.gov (United States)

    Ghosh, Smita; Moonan, Patrick K; Cowan, Lauren; Grant, Juliana; Kammerer, Steve; Navin, Thomas R

    2012-06-01

    Molecular characterization of Mycobacterium tuberculosis complex isolates (genotyping) can be used by public health programs to more readily identify tuberculosis (TB) transmission. The Centers for Disease Control and Prevention's National Tuberculosis Genotyping Service has offered M. tuberculosis genotyping for every culture-confirmed case in the United States since 2004. The TB Genotyping Information Management System (TB GIMS), launched in March 2010, is a secure online database containing genotype results linked with case characteristics from the national TB registry for state and local TB programs to access, manage and analyze these data. As of September 2011, TB GIMS contains genotype results for 89% of all culture-positive TB cases for 2010. Over 400 users can generate local and national reports and maps using TB GIMS. Automated alerts on geospatially concentrated cases with matching genotypes that may represent outbreaks are also generated by TB GIMS. TB genotyping results are available to enhance national TB surveillance and apply genotyping results to conduct TB control activities in the United States.

  1. The non-clonality of drug resistance in Beijing-genotype isolates of Mycobacterium tuberculosis from the Western Cape of South Africa

    Directory of Open Access Journals (Sweden)

    Van Helden Paul D

    2010-11-01

    Full Text Available Abstract Background The Beijing genotype of M. tuberculosis is a virulent strain that is disseminating worldwide and has a strong association with drug resistance. In the Western Cape of South Africa, epidemiological studies have identified the R220 cluster of the Beijing genotype as a major contributor to a recent outbreak of drug-resistant tuberculosis. Although the outbreak is considered to be due to clonal transmission, the relationship among drug resistant isolates has not yet been established. Results To better understand the evolution of drug resistance among these strains, 14 drug-resistant clinical isolates of the Beijing genotype were sequenced by whole-genome sequencing, including eight from R220 and six from a more ancestral Beijing cluster, R86, for comparison. While each cluster shares a distinct resistance mutation for isoniazid, mapping of other drug-resistance mutations onto a phylogenetic tree constructed from single nucleotide polymorphisms shows that resistance mutations to many drugs have arisen multiple times independently within each cluster of isolates. Thus, drug resistance among these isolates appears to be acquired, not clonally derived. This observation suggests that, although the Beijing genotype as a whole might have selective advantages enabling its rapid dissemination, the XDR isolates are relatively less fit and do not propagate well. Although it has been hypothesized that the increased frequency of drug resistance in some Beijing lineages might be caused by a mutator phenotype, no significant shift in synonymous substitution patterns is observed in the genomes. Conclusion While MDR-TB is spreading by transmission in the Western Cape, our data suggests that further drug resistance (i.e. XDR-TB at this stage is acquired.

  2. Mycobacterium tuberculosis effectors interfering host apoptosis signaling.

    Science.gov (United States)

    Liu, Minqiang; Li, Wu; Xiang, Xiaohong; Xie, Jianping

    2015-07-01

    Tuberculosis remains a serious human public health concern. The coevolution between its pathogen Mycobacterium tuberculosis and human host complicated the way to prevent and cure TB. Apoptosis plays subtle role in this interaction. The pathogen endeavors to manipulate the apoptosis via diverse effectors targeting key signaling nodes. In this paper, we summarized the effectors pathogen used to subvert the apoptosis, such as LpqH, ESAT-6/CFP-10, LAMs. The interplay between different forms of cell deaths, such as apoptosis, autophagy, necrosis, is also discussed with a focus on the modes of action of effectors, and implications for better TB control.

  3. Molecular Detection of Mixed Infections of Mycobacterium tuberculosis Strains in Sputum Samples from Patients in Karonga District, Malawi ▿ †

    OpenAIRE

    Mallard, Kim; McNerney, Ruth; Crampin, Amelia C; Houben, Rein; Ndlovu, Richard; Munthali, Lumbani; Warren, Robin M.; French, Neil; Judith R Glynn

    2010-01-01

    The occurrence of mixed infections of Mycobacterium tuberculosis is no longer disputed. However, their frequency, and the impact they may have on our understanding of tuberculosis (TB) pathogenesis and epidemiology, remains undetermined. Most previous studies of frequency applied genotyping techniques to cultured M. tuberculosis isolates and found mixed infections to be rare. PCR-based techniques may be more sensitive for detecting multiple M. tuberculosis strains and can be applied to sputum...

  4. Molecular epidemiology of Mycobacterium tuberculosis in the United States-Affiliated Pacific Islands.

    Science.gov (United States)

    Bamrah, Sapna; Desmond, Edward; Ghosh, Smita; France, Anne Marie; Kammerer, J Steve; Cowan, Lauren S; Heetderks, Andrew; Forbes, Alstead; Moonan, Patrick K

    2014-01-01

    The United States-Affiliated Pacific Islands (USAPI) are part of the US National Tuberculosis (TB) Surveillance System and use laboratory services contracted through a cooperative agreement with the Centers for Disease Control and Prevention (CDC). In 2004, the CDC established the National Tuberculosis Genotyping Service, a system to genotype 1 isolate from each culture-confirmed case of TB. To describe the molecular epidemiology of TB in the region, we examined all Mycobacterium tuberculosis isolates submitted for genotyping from January 1, 2004, to December 31, 2008. Over this time period, the USAPI jurisdictions reported 1339 verified TB cases to the National Tuberculosis Surveillance System. Among 419 (31%) reported culture-confirmed TB cases, 352 (84%) had complete genotype results. Routine TB genotyping allowed, for the first time, an exploration of the molecular epidemiology of TB in the USAPI.

  5. 基因型分析法快速检测耐药结核分枝杆菌%Rapid genotype assay for detection of drug-resistant Mycobacterium tuberculosis

    Institute of Scientific and Technical Information of China (English)

    石瑞如; 高飞絮; 沈青玉; 黄祖峰; 秦殊; 王伟; 张国龙

    2014-01-01

    Objective To explore the value of the genotype assays detection of the resistance of Mycobacterium tuberculosis to isoniazid and rifampin.Methods Seventy-eight clinical isolates susceptible of harboring Mycobacterium tuberculosis were subject to the mutation assay of katG gene S315T,inhA gene C-15Tand rpoB genes D516V,H526Y,H526D,S531L by using multiplex polymerase chain reaction-based linear probe membrane hybridization for analysis of isoniazid and rifampin resistance.These results were compared with the L-J solid medium culture,the gold standard,and MGIT BACTEC 960 drug sensitivity test.Results Genotype assay was associated with shorter time for the detection compared with routine drug sensitivity test (6 hours vs 3 months).The genotype assay yielded the sensitivity and specificity of 89% (16/18) and 99% (77/78) for isoniazid and 100% and 100% (13/13 and 78/78) for rifampin.Conclusion Genotype assay of Mycobacterium tuberculosis offers a rapid and accurate approach for the early diagnosis and treatment of drug-resistant tuberculosis,rendering it a promising technique to be extensively applied in clinical laboratories.%目的 探讨基因型分析法检测结核分枝杆菌异烟肼和利福平耐药性的价值.方法 采用多重PCR和线性探针反向膜杂交法来检测异烟肼耐药基因katG S315T和inhA C-15T突变以及利福平耐药基因rpoB D516V,H526Y,H526D,S531L突变来判断78株结核分枝杆菌临床分离株的耐药性,并与金标准L-J固体培养基药敏法以及BACTEC960液体培养药敏法进行对比分析.结果 基因型分析法在6h内可以完成;结核分枝杆菌常规药敏检测需要3个月.与后者相比,基因型分析法检测异烟肼耐药的敏感性和特异性分别为89% (16/18)和99% (77/78);检测利福平耐药的敏感性和特异性均为100%(13/13,78/78).结论 基因型分析法检测结核菌耐药性快速准确,对耐药结核病的早期诊断和治疗很有帮助,有望在临床实验室广泛开展.

  6. Early detection of multi-drug resistance and common mutations in Mycobacterium tuberculosis isolates from Delhi using GenoType MTBDRplus assay

    Directory of Open Access Journals (Sweden)

    R Singhal

    2015-01-01

    Full Text Available Purpose: There is scarcity of prevalence data of multi-drug-resistant tuberculosis (MDR-TB data and common mutations responsible in North India. This study aimed to detect MDR-TB among MDR-TB suspects from Delhi and mutation patterns using GenoType MTBDRplus assay. Materials and Methods: All MDR suspects in five districts of New Delhi were referred to the laboratory from 1 st October 2011 to 31 st December 2012 as per criterion defined by Programmatic Management of Drug Resistant Tuberculosis (PMDT. GenoType MTBDRplus assay was performed on 2182 samples or cultures and mutations in the rpoB gene for rifampicin (RIF and katG and inhA genes for isoniazid (INH were analyzed. Results: A total of 366 (16.8% MDR-TB cases were diagnosed. MDR rate was found to be 32%, 16.6% and 10.2% during criterion A, B and C respectively. The most common mutation detected for RIF was S531L (59.0% and for INH was S315T1 (88.3%. Mutations S531L and S315T1 occurred significantly higher in MDR strains as compared to RIF mono-resistant and INH mono-resistant strains, respectively. Average laboratory turn-around time (TAT for dispatch of result to districts for test conducted on samples was 4.4 days. Conclusion: GenoType MTBDRplus is a useful assay for rapid detection of MDR-TB. The common mutations for RIF and INH were similar to those seen in other regions. However, mutations determining MDR strains and mono-resistant strains differed significantly for both RIF and INH.

  7. Erythema nodosum and Mycobacterium Tuberculosis

    Directory of Open Access Journals (Sweden)

    Ovgu Kul

    2016-01-01

    Full Text Available Erythema nodosum[EN] is a common form of panniculitis and inflammatory disease of the subcutaneous fat tissue.EN occurs in a variety of disorders including tuberculosis, sarcoidosis, Behcet%u2019s disease, inflammatory bowel disease and streptococcal infection but idiopathic cases account for 55%. A 14 years old patient was diagnosed as Erythema nodosum[EN] with prolonged fever and red subcutaneous nodules in pretibial location. With tuberculin skin and Quantiferon positivity and tree in bud sign in chest x ray she was diagnosed as tuberculosis. On the same dates a 11 years old patient was admitted with EN. She was also diagnosed as tuberculosis due to tuberculin skin and Quantiferon positivity and hilar lymphadenopathy in thorax CT. The other causes were excluded in both cases. We present these two adolescents cases to keep in mind the close relationship between tuberculosis and erythema nodosum.

  8. Spoligotype patterns of Mycobacterium tuberculosis isolated from extra pulmonary tuberculosis patients in Puducherry, India

    Directory of Open Access Journals (Sweden)

    G Kandhakumari

    2015-01-01

    Full Text Available Purpose: Genotyping studies like spoligotyping are valuable tools in understanding the genetic diversity and epidemiology of Mycobacterium tuberculosis. Though there are reports of spoligotyping of M. tuberculosis isolates from pulmonary specimens from different parts of India, spoligotyping of extra pulmonary tuberculosis isolates are very few. Puducherry has not yet recorded spoligopatterns of M. tuberculosis from either pulmonary or extra pulmonary (EPTB specimens. The aim of this study is to analyze the spoligotype patterns of EPTB strains circulating in Puducherry and neighboring districts of Tamil Nadu. Materials and Methods: During June 2011 to December 2013, 570 EPTB specimens were processed by culturing on to Lowenstein Jensen (LJ medium and automated Mycobacterium Growth Indicator Tube system (MGIT960. Identification of M. tuberculosis was carried out as per standard procedures, and MPT 64 antigen positivity in a commercial immunochromatography kit. Spoligotyping was carried out at National Institute of Research in Tuberculosis (ICMR, Chennai. Results: M. tuberculosis was isolated from 67 single EPTB specimens (11.8% like pus/cold abscess (34, TB spine (10, pleural fluid (10, urine (5, tissue bit (2, lymph nodes (2, ascitic fluid (2, synovial fluid (1 and endometrial curetting (1. Among 67 isolates with 41 spoligopatterns, EAI lineage with 28 isolates (41.8% predominated followed by 18 orphans (26.9%, 10 Beijing (14.9% and 8 U (11.9%. BOVIS1_BCG (ST482, T1-T2 (ST78 and H3 (ST50 were represented by one strain each (1.5%. C onclusions: Spoligotyping plays a significant role in the epidemiology of tuberculosis. Three spoligotypes, T1-T2 (ST78, EAI6 (ST292 and U (ST1429 are reported for the first time in India.

  9. High Persister Mutants in Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Heather L Torrey

    Full Text Available Mycobacterium tuberculosis forms drug-tolerant persister cells that are the probable cause of its recalcitrance to antibiotic therapy. While genetically identical to the rest of the population, persisters are dormant, which protects them from killing by bactericidal antibiotics. The mechanism of persister formation in M. tuberculosis is not well understood. In this study, we selected for high persister (hip mutants and characterized them by whole genome sequencing and transcriptome analysis. In parallel, we identified and characterized clinical isolates that naturally produce high levels of persisters. We compared the hip mutants obtained in vitro with clinical isolates to identify candidate persister genes. Genes involved in lipid biosynthesis, carbon metabolism, toxin-antitoxin systems, and transcriptional regulators were among those identified. We also found that clinical hip isolates exhibited greater ex vivo survival than the low persister isolates. Our data suggest that M. tuberculosis persister formation involves multiple pathways, and hip mutants may contribute to the recalcitrance of the infection.

  10. Risk factors for Mycobacterium tuberculosis infection among children in Greenland

    DEFF Research Database (Denmark)

    Søborg, Bolette; Andersen, Aase Bengaard; Melbye, Mads

    2011-01-01

    To examine the risk factors for Mycobacterium tuberculosis infection (MTI) among Greenlandic children for the purpose of identifying those at highest risk of infection.......To examine the risk factors for Mycobacterium tuberculosis infection (MTI) among Greenlandic children for the purpose of identifying those at highest risk of infection....

  11. Cryopreservation of Mycobacterium tuberculosis complex cells.

    Science.gov (United States)

    Shu, Zhiquan; Weigel, Kris M; Soelberg, Scott D; Lakey, Annie; Cangelosi, Gerard A; Lee, Kyong-Hoon; Chung, Jae-Hyun; Gao, Dayong

    2012-11-01

    Successful long-term preservation of Mycobacterium tuberculosis cells is important for sample transport, research, biobanking, and the development of new drugs, vaccines, biomarkers, and diagnostics. In this report, Mycobacterium bovis bacillus Calmette-Guérin and M. tuberculosis H37Ra were used as models of M. tuberculosis complex strains to study cryopreservation of M. tuberculosis complex cells in diverse sample matrices at different cooling rates. Cells were cryopreserved in diverse sample matrices, namely, phosphate-buffered saline (PBS), Middlebrook 7H9 medium with or without added glycerol, and human sputum. The efficacy of cryopreservation was quantified by microbiological culture and microscopy with BacLight LIVE/DEAD staining. In all sample matrices examined, the microbiological culture results showed that the cooling rate was the most critical factor influencing cell viability. Slow cooling (a few degrees Celsius per minute) resulted in much higher M. tuberculosis complex recovery rates than rapid cooling (direct immersion in liquid nitrogen) (P tuberculosis complex cells in 7H9 broth for 20 h before culture on solid Middlebrook 7H10 plates did not help the recovery of the cells from cryoinjury (P = 0.14 to 0.71). The BacLight LIVE/DEAD staining kit, based on Syto 9 and propidium iodide (PI), was also applied to assess cell envelope integrity after cryopreservation. Using the kit, similar percentages of "live" cells with intact envelopes were observed for samples cryopreserved under different conditions, which was inconsistent with the microbiological culture results. This implies that suboptimal cryopreservation might not cause severe damage to the cell wall and/or membrane but instead cause intracellular injury, which leads to the loss of cell viability.

  12. Occupational Tuberculosis in Denmark through 21 Years Analysed by Nationwide Genotyping

    DEFF Research Database (Denmark)

    Pedersen, Mathias Klok; Andersen, Aase Bengaard; Andersen, Peter Henrik;

    2016-01-01

    Tuberculosis (TB) is a well-known occupational hazard. Based on more than two decades (1992-2012) of centralized nationwide genotyping of all Mycobacterium tuberculosis culture-positive TB patients in Denmark, we compared M. tuberculosis genotypes from all cases notified as presumed occupational (N...... = 130) with M. tuberculosis genotypes from all TB cases present in the country (N = 7,127). From 1992 through 2006, the IS6110 Restriction Fragment Length Polymorphism (RFLP) method was used for genotyping, whereas from 2005 to present, the 24-locus-based Mycobacterial Interspersed Repetitive Unit......-Variable Number of Tandem Repeat (MIRU-VNTR) was used. An occupational TB case was classified as clustered if the genotype was 100% identical to at least one other genotype. Subsequently, based on genotype, time period, smear positivity, geography, susceptibility pattern, and any reported epidemiological links...

  13. Beta-lactamases of Mycobacterium tuberculosis and Mycobacterium kansasii.

    Science.gov (United States)

    Segura, C; Salvadó, M

    1997-09-01

    Re-emergence of infectious diseases caused by mycobacteria as well as the emergence of multiresistant strains of Mycobacterium has promoted the research on the use of beta-lactames in the treatment of such diseases. Mycobacteria produce beta-lactamases: M. tuberculosis produces a wide-spectrum beta-lactamase whose behaviour mimicks those of Gram-negative bacteria. M. kansasii produces also beta-lactamase which can be inhibited by clavulanic acid. An overview on beta-lactamases from both species is reported.

  14. Molecular epidemiology of Mycobacterium tuberculosis in Gansu province of China

    Institute of Scientific and Technical Information of China (English)

    TIAN Li-li; ZHU Bing-dong; SI Hong-yan; MU Tao-jun; FAN Wen-bing; WANG Jing; JIANG Wei-min; LI Qing; YANG Biao; ZHANG Ying

    2012-01-01

    Background Mycobacterial interspersed repetitive units-variable number tandem repeat (MIRU-VNTR) and Beijing family typing based on detecting the deletion of RD105 sequence are two common genotyping methods used to study the molecular epidemiologic characteristics of Mycobacterium (M.) tuberculosis.We collected 218 strains of M.tuberculosis between 2004 and 2006 in the Linxia Hui Autonomous Prefecture of Gansu province in Northwest China.Methods MIRU-VNTR analysis and Beijing family typing based on detecting the deletion of RD105 sequence were used to type the 218 strains,and their typing power was evaluated to look for practical and efficient genotyping methods suitable for the region.Results The MIRU typing yielded 115 distinct genotypes,including 98 unique isolates and 17 different clusters containing 120 isolates (55.05%); the cluster rate was 47.25%.By detecting the deletion of RD105 sequence,188 of 218 (86.23%) isolates belonged to Beijing family.Combination of Beijing family typing and MIRU typing yielded 118 distinct patterns,including 101 unique isolates and 17 clusters containing 117 isolates (54.13%).The largest cluster contained 58 strains with MIRU genotype of 223325173533 which contained 50 strains belonging to Beijing family and 8 strains belonging to non-Beijing family.Conclusions The Beijing family strains occupied a large proportion and the Beijing family MIRU genotype 223325173533 is a dominant strain in Linxia of Gansu.Combining detecting the deletion of RD105 and MIRU typing together provides a simple,fast,and effective method which is low in cost and might be practical and suitable for M.tuberculosis genotyping in China.

  15. Characteristics of multidrug-resistant Mycobacterium tuberculosis in southern Brazil.

    Science.gov (United States)

    Perizzolo, Paulo F; Dalla Costa, Elis R; Ribeiro, Andrezza W; Spies, Fernanda S; Ribeiro, Marta O; Dias, Cláudia F; Unis, Gisela; Almeida da Silva, Pedro; Gomes, Harrison M; Suffys, Philip N; Rossetti, Maria Lucia R

    2012-01-01

    A major threat to tuberculosis (TB) control programs is the emergence of drug resistant Mycobacterium tuberculosis strains that cause TB that cannot be cured by standard anti-TB drug regimens. Because few data exist on MDR-TB in this region of the country, we performed an epidemiologic study that combined conventional and molecular analysis of MDR-TB cases from Rio Grande do Sul (RS) that were diagnosed in this period and included cases that were under treatment with second line drug schemes. Included were 121 MDR cases and sequencing of rpoB and katG showed that 106 (87.6%) strains were mutated in rpoB and 97 (80.2%) in katG. Spoligotyping demonstrated that the LAM genotype was predominant (n = 70, 57.8%) and included the largest group composed by 22 (18.1%) strains with the LAM5 ST93 genotype. Other main genotypes belonged to the families T (n = 22, 18.2%), U family (n = 16, 13.2%), Haarlem (n = 5, 4.1%) and X (n = 1, 0.8%). Genotyping by IS6110-RFLP analysis showed 51 distinct fingerprints, 38 (31.4%) of these observed only once and the other 13 patterns being shared among the rest of the isolates (n = 83, 68.6%). Among the 22 strains that were LAM5 ST93, only two had different IS6110-RFLP genotypes. In conclusion, there exists a high degree of M. Tuberculosis genotype clustering among MDR-TB cases in Rio Grande do Sul. Moreover, we observed a large MDR-TB outbreak.

  16. Implication of the RD(Rio) Mycobacterium tuberculosis sublineage in multidrug resistant tuberculosis in Portugal.

    Science.gov (United States)

    David, Susana; Duarte, Elsa L; Leite, Clarice Queico Fugimura; Ribeiro, João-Nuno; Maio, José-Nuno; Paixão, Eleonora; Portugal, Clara; Sancho, Luísa; Germano de Sousa, José

    2012-10-01

    Multidrug and extensively drug resistant Mycobacterium tuberculosis are a threat to tuberculosis control programs. Genotyping methods, such as spoligotyping and MIRU-VNTR typing (Mycobacterial Interspersed Repetitive Units), are useful in monitoring potentially epidemic strains and estimating strain phylogenetic lineages and/or genotypic families. M. tuberculosis Latin American Mediterranean (LAM) family is a major worldwide contributor to tuberculosis (TB). LAM specific molecular markers, Ag85C(103) single nucleotide polymorphism (SNP) and RD(Rio) long-sequence polymorphism (LSP), were used to characterize spoligotype signatures from 859 patient isolates from Portugal. LAM strains were found responsible for 57.7% of all tuberculosis cases. Strains with the RD(Rio) deletion (referred to as RD(Rio)) were estimated to represent 1/3 of all the strains and over 60% of the multidrug resistant (MDR) strains. The major spoligotype signature SIT20 belonging to the LAM1 RD(Rio) sublineage, represented close to 1/5th of all the strains, over 20% of which were MDR. Analysis of published datasets according to stipulated 12loci MIRU-VNTR RD(Rio) signatures revealed that 96.3% (129/134) of MDR and extensively drug resistant (XDR) clusters were RD(Rio). This is the first report associating the LAM RD(Rio) sublineage with MDR. These results are an important contribution to the monitoring of these strains with heightened transmission for future endeavors to arrest MDR-TB and XDR-TB.

  17. Predominance of modern Mycobacterium tuberculosis strains and active transmission of Beijing sublineage in Jayapura, Indonesia Papua.

    Science.gov (United States)

    Chaidir, Lidya; Sengstake, Sarah; de Beer, Jessica; Oktavian, Antonius; Krismawati, Hana; Muhapril, Erfin; Kusumadewi, Inri; Annisa, Jessi; Anthony, Richard; van Soolingen, Dick; Achmad, Tri Hanggono; Marzuki, Sangkot; Alisjahbana, Bachti; van Crevel, Reinout

    2016-04-01

    Mycobacterium tuberculosis genotype distribution is different between West and Central Indonesia, but there are no data on the most Eastern part, Papua. We aimed to identify the predominant genotypes of M. tuberculosis responsible for tuberculosis in coastal Papua, their transmission, and the association with patient characteristics. A total of 199 M. tuberculosis isolates were collected. Spoligotyping was applied to describe the population structure of M. tuberculosis, lineage identification was performed using a combination of lineage-specific markers, and genotypic clusters were identified using a combination of 24-locus-MIRU-VNTR and spoligotyping. A high degree of genetic diversity was observed among isolates based on their spoligopatterns. Strains from modern lineage 4 made up almost half of strains (46.9%), being more abundant than the ancient lineage 1 (33.7%), and modern lineage 2 (19.4%). Thirty-five percent of strains belonged to genotypic clusters, especially strains in the Beijing genotype. Previous TB treatment and mutations associated with drug resistance were more common in patients infected with strains of the Beijing genotype. Papua shows a different distribution of M. tuberculosis genotypes compared to other parts of Indonesia. Clustering and drug resistance of modern strains recently introduced to Papua may contribute to the high tuberculosis burden in this region.

  18. Application of genotype MTBDRplus in rapid detection of the Mycobacterium tuberculosis complex as well as its resistance to isoniazid and rifampin in a high volume laboratory in Southern China.

    Science.gov (United States)

    Zhang, Lei; Ye, Yuanxing; Duo, Lina; Wang, Tingting; Song, Xingbo; Lu, Xiaojun; Ying, Binwu; Wang, Lanlan

    2011-03-01

    The alarmingly worsening epidemics of drug-resistant tuberculosis (TB) call urgent need for a simple method for the rapid detection of drug-resistant TB in clinical settings. In an attempt to establish a rapid procedure for laboratory diagnosis of TB and investigate the local TB epidemiology, molecular line probe assay of the Genotype MTBDRplus was used to identify Mycobacterium tuberculosis complex (MTBC) and detect mutations conferring resistance to two most active first-line drugs against TB: Rifampin and Isoniazid. 96 acid-fast bacillus (AFB) smear- positive sputums and 18 PCR-positive non-sputum specimens have been determined for the MTBC and resistance to Rifampin and Isoniazid. The MTBC detection rates in two sources of specimens were 93.8% (90/96) and 77.8% (14/18) respectively. The overall drug resistance (Rifampin or Isoniazid) occurred in 34.6% (36/104). Resistance to rifampin (RMP) was 28.8% (30/104) and 25% (26/104) was to Isoniazid (INH), in which high level drug resistance accounted for 88.5% (23/26) and low level drug resistance accounted for 7.7% (2/26). Multidrug resistance (MDR), defined as resistant to both RMP and INH, was found in 19.2% (20/104) of clinical samples, which was double that of official statistics. In addition, 63.3% (19/30) RMP-resistant mutations were identified in the region of RopB 530-533 and 57.9% (11/19) were the S531L mutation. 84.6% (22/26) of resistance to INH was mediated by Kat S315T1 mutations which conferred the high-level resistance to INH. The Genotype MTBDRplus line probe assay is a suitable and applicable method for establishing the rapidness in detection of drug-resistant TB in clinical laboratory. It will be a valuable addition to the conventional TB diagnostic approaches.

  19. Comparison of Xpert MTB/RIF Assay and GenoType MTBDRplus DNA Probes for Detection of Mutations Associated with Rifampicin Resistance in Mycobacterium tuberculosis.

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    Arfatur Rahman

    Full Text Available GeneXpert MTB/RIF (Xpert and Genotype MTBDRplus (DRplus are two World Health Organization (WHO endorsed probe based molecular drug susceptibility testing (DST methods for rapid diagnosis of drug resistant tuberculosis. Both methods target the same 81 bp Rifampicin Resistance Determining Region (RRDR of bacterial RNA polymerase β subunit (rpoB for detection of Rifampicin (RIF resistance associated mutations using DNA probes. So there is a correspondence of the probes of each other and expected similarity of probe binding.We analyzed 92 sputum specimens by Xpert, DRplus and LJ proportion method (LJ-DST. We compared molecular DSTs with gold standard LJ-DST. We wanted to see the agreement level of two molecular methods for detection of RIF resistance associated mutations. The 81bp RRDR region of rpoB gene of discrepant cases between the two molecular methods was sequenced by Sanger sequencing.The agreement of Xpert and DRplus with LJ-DST for detection of RIF susceptibility was found to be 93.5% and 92.4%, respectively. We also found 92.4% overall agreement of two molecular methods for the detection of RIF susceptibility. A total of 84 out of 92 samples (91.3% had agreement on the molecular locus of RRDR mutation by DRplus and Xpert. Sanger sequencing of 81bp RRDR revealed that Xpert probes detected seven of eight discrepant cases correctly and DRplus was erroneous in all the eight cases.Although the overall concordance with LJ-DST was similar for both Xpert and DRplus assay, Xpert demonstrated more accuracy in the detection of RIF susceptibility for discrepant isolates compared with DRplus. This observation would be helpful for the improvement of probe based detection of drug resistance associated mutations especially rpoB mutation in M. tuberculosis.

  20. Detection of multi-drug resistance & characterization of mutations in Mycobacterium tuberculosis isolates from North- Eastern States of India using GenoType MTBDRplus assay

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    Ritu Singhal

    2014-01-01

    Full Text Available Background & objectives: Information on drug resistance tuberculosis is sparse from North-East (N-E States of India. We undertook this study to detect multi-drug resistant tuberculosis (MDR-TB among MDR-TB suspects, and common mutations among MDR-TB cases using GenoType MTBDRplus. Methods: All MDR suspect patients deposited sputum samples to peripheral designated microscopy centres (DMC in North-East States. The district TB officers (DTOs facilitated the transport of samples collected during January 2012 to August 2012 to our laboratory. The line probe assay to detect common mutations in the rpoB gene for rifampicin (RIF and katG and inhA genes for isoniazid (INH, respectively was performed on 339 samples or cultures. Results: A total of 553 sputum samples from MDR suspects were received of which, 181 (32.7% isolates were found to be multi-drug resistant. Missing WT8 along with mutation in codon S531L was commonest pattern for rifampicin resistant isolates (65.1% and missing WT along with mutations in codon S315T1 of katG gene was commonest pattern for isoniazid resistant isolates (86.2%. Average turn-around time for dispatch of LPA result to these States from cultures and samples was 23.4 and 5.2 days, respectively. Interpretations & conclusions: The MDR-TB among MDR-TB suspects in North-Eastern States of India was found to be 32.7 per cent. The common mutations obtained for RIF and INH in the region were mostly similar to those reported earlier.

  1. First molecular epidemiology study of Mycobacterium tuberculosis in Kiribati.

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    Eman Aleksic

    Full Text Available Tuberculosis incidence rates in Kiribati are among the highest in the Western Pacific Region, however the genetic diversity of circulating Mycobacterium tuberculosis complex strains (MTBC and transmission dynamics are unknown. Here, we analysed MTBC strains isolated from culture positive pulmonary tuberculosis (TB cases from the main TB referral centre between November 2007 and October 2009. Strain genotyping (IS6110 typing, spoligotyping, 24-loci MIRU-VNTR and SNP typing was performed and demographic information collected. Among 73 MTBC strains analysed, we identified seven phylogenetic lineages, dominated by Beijing strains (49%. Beijing strains were further differentiated in two main branches, Beijing-A (n = 8 and -B (n = 28, that show distinct genotyping patterns and are characterized by specific deletion profiles (Beijing A: only RD105, RD207 deleted; Beijing B: RD150 and RD181 additionally deleted. Many Kiribati strains (59% based on IS6110 typing of all strains occurred in clusters, suggesting ongoing local transmission. Beijing-B strains and over-crowded living conditions were associated with strain clustering (likely recent transmission, however little evidence of anti-tuberculous drug resistance was observed. We suggest enhanced case finding amongst close contacts and continued supervised treatment of all identified cases using standard first-line drugs to reduce TB burden in Kiribati. Beijing strains can be subdivided in different principle branches that might be associated with differential spreading patterns in the population.

  2. Immunological consequences of strain variation within the Mycobacterium tuberculosis complex.

    Science.gov (United States)

    Tientcheu, Leopold D; Koch, Anastasia; Ndengane, Mthawelenga; Andoseh, Genevieve; Kampmann, Beate; Wilkinson, Robert J

    2017-03-01

    In 2015, there were an estimated 10.4 million new cases of tuberculosis (TB) globally, making it one of the leading causes of death due to an infectious disease. TB is caused by members of the Mycobacterium tuberculosis complex (MTBC), with human disease resulting from infection by M. tuberculosis sensu stricto and M. africanum. Recent progress in genotyping techniques, in particular the increasing availability of whole genome sequence data, has revealed previously under appreciated levels of genetic diversity within the MTBC. Several studies have shown that this genetic diversity may translate into differences in TB transmission, clinical manifestations of disease, and host immune responses. This suggests the existence of MTBC genotype-dependent host-pathogen interactions which may influence the outcome of infection and progression of disease. In this review, we highlight the studies demonstrating differences in innate and adaptive immunological outcomes consequent on MTBC genetic diversity, and discuss how these differences in immune response might influence the development of TB vaccines, diagnostics and new therapies. © 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. PET/CT imaging of Mycobacterium tuberculosis infection.

    NARCIS (Netherlands)

    Ankrah, Alfred; Werf, van der Tjip; de Vries, Erik; Dierckx, Rudi; Sathekge, M. M.; Glaudemans, Andor W.J.M.

    Tuberculosis has a high morbidity and mortality worldwide. Mycobacterium tuberculosis (Mtb) has a complex pathophysiology; it is an aerobic bacillus capable of surviving in anaerobic conditions in a latent state for a very long time before reactivation to active disease. In the latent tuberculosis

  4. Radiometric studies of mycobacterium tuberculosis

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    Edwaldo E. Camargo

    1987-02-01

    Full Text Available An in vitro assay system that included automated radiometric quantification of 14CO2 released as a result of oxidation of 14C- substrates was applied for studying the metabolic activity of M. tuberculosis under various experimental conditions. These experiments included the study of a mtabolic pathways, b detection times for various inoculum sizes, c effect of filtration on reproducibility of results, d influence of stress environment e minimal inhibitory concentrations for isoniazid, streptomycin, ethambutol and rifampin, and f generation times of M. tuberculosis and M. bovis. These organisms were found to metabolize 14C-for-mate, (U-14C acetate, (U-14C glycerol, (1-14C palmitic acid, 1-14C lauric acid, (U-14C L-malic acid, (U-14C D-glucose, and (U-14C D-glucose, but not (1-14C L-glucose, (U-14C glycine, or (U-14C pyruvate to 14CO2. By using either 14C-for-mate, (1-14C palmitic acid, or (1-14C lauric acid, 10(7 organisms/vial could be detected within 24 48 hours and as few as 10 organisms/vial within 16-20 days. Reproducible results could be obtained without filtering the bacterial suspension, provided that the organisms were grown in liquid 7H9 medium with 0.05% polysorbate 80 and homogenized prior to the study. Drugs that block protein synthesis were found to have lower minimal inhibitory concentrations with the radiometric method when compared to the conventional agar dilution method. The mean generation time obtained for M. bovis and different strains of M. tuberculosis with various substrates was 9 ± 1 hours.

  5. Genetic diversity of Mycobacterium tuberculosis isolated from tuberculosis patients in the Serengeti ecosystem in Tanzania.

    Science.gov (United States)

    Mbugi, Erasto V; Katale, Bugwesa Z; Siame, Keith K; Keyyu, Julius D; Kendall, Sharon L; Dockrell, Hazel M; Streicher, Elizabeth M; Michel, Anita L; Rweyemamu, Mark M; Warren, Robin M; Matee, Mecky I; van Helden, Paul D

    2015-03-01

    This study was part of a larger cross-sectional survey that was evaluating tuberculosis (TB) infection in humans, livestock and wildlife in the Serengeti ecosystem in Tanzania. The study aimed at evaluating the genetic diversity of Mycobacterium tuberculosis isolates from TB patients attending health facilities in the Serengeti ecosystem. DNA was extracted from 214 sputum cultures obtained from consecutively enrolled newly diagnosed untreated TB patients aged ≥18 years. Spacer oligonucleotide typing (spoligotyping) and Mycobacterium Interspersed Repetitive Units and Variable Number Tandem Repeat (MIRU-VNTR) were used to genotype M. tuberculosis to establish the circulating lineages. Of the214 M. tuberculosis isolates genotyped, 55 (25.7%) belonged to the Central Asian (CAS) family, 52 (24.3%) were T family (an ill-defined family), 38 (17.8%) belonged to the Latin American Mediterranean (LAM) family, 25 (11.7%) to the East-African Indian (EAI) family, 25 (11.7%) comprised of different unassigned ('Serengeti') strain families, while 8 (3.7%) belonged to the Beijing family. A minority group that included Haarlem, X, U and S altogether accounted for 11 (5.2%) of all genotypes. MIRU-VNTR typing produced diverse patterns within and between families indicative of unlinked transmission chains. We conclude that, in the Serengeti ecosystem only a few successful families predominate namely CAS, T, LAM and EAI families. Other types found in lower prevalence are Beijing, Haarlem, X, S and MANU. The Haarlem, EAI_Somalia, LAM3 and S/convergent and X2 subfamilies found in this study were not reported in previous studies in Tanzania.

  6. Genetic diversity of Mycobacterium tuberculosis isolated from tuberculosis patients in the Serengeti ecosystem in Tanzania

    Science.gov (United States)

    Mbugi, Erasto V.; Katale, Bugwesa Z.; Siame, Keith K.; Keyyu, Julius D.; Kendall, Sharon L.; Dockrell, Hazel M.; Streicher, Elizabeth M.; Michel, Anita L.; Rweyemamu, Mark M.; Warren, Robin M.; Matee, Mecky I.; van Helden, Paul D.

    2015-01-01

    Summary This study was part of a larger cross-sectional survey that was evaluating tuberculosis (TB) infection in humans, livestock and wildlife in the Serengeti ecosystem in Tanzania. The study aimed at evaluating the genetic diversity of Mycobacterium tuberculosis isolates from TB patients attending health facilities in the Serengeti ecosystem. DNA was extracted from 214 sputum cultures obtained from consecutively enrolled newly diagnosed untreated TB patients aged ≥18 years. Spacer oligonucleotide typing (spoligotyping) and Mycobacterium Interspersed Repetitive Units and Variable Number Tandem Repeat (MIRU-VNTR) were used to genotype M. tuberculosis to establish the circulating lineages. Of the214 M. tuberculosis isolates genotyped, 55 (25.7%) belonged to the Central Asian (CAS) family, 52 (24.3%) were T family (an ill-defined family), 38 (17.8%) belonged to the Latin American Mediterranean (LAM) family, 25 (11.7%) to the East-African Indian (EAI) family, 25 (11.7%) comprised of different unassigned (‘Serengeti’) strain families, while 8 (3.7%) belonged to the Beijing family. A minority group that included Haarlem, X, U and S altogether accounted for 11 (5.2%) of all genotypes. MIRU-VNTR typing produced diverse patterns within and between families indicative of unlinked transmission chains. We conclude that, in the Serengeti ecosystem only a few successful families predominate namely CAS, T, LAM and EAI families. Other types found in lower prevalence are Beijing, Haarlem, X, S and MANU. The Haarlem, EAI_Somalia, LAM3 and S/convergent and X2 subfamilies found in this study were not reported in previous studies in Tanzania. PMID:25522841

  7. Tuberculosis Caused by Mycobacterium africanum, United States, 2004-2013.

    Science.gov (United States)

    Sharma, Aditya; Bloss, Emily; Heilig, Charles M; Click, Eleanor S

    2016-03-01

    Mycobacterium africanum is endemic to West Africa and causes tuberculosis (TB). We reviewed reported cases of TB in the United States during 2004-2013 that had lineage assigned by genotype (spoligotype and mycobacterial interspersed repetitive unit variable number tandem repeats). M. africanum caused 315 (0.4%) of 73,290 TB cases with lineage assigned by genotype. TB caused by M. africanum was associated more with persons from West Africa (adjusted odds ratio [aOR] 253.8, 95% CI 59.9-1,076.1) and US-born black persons (aOR 5.7, 95% CI 1.2-25.9) than with US-born white persons. TB caused by M. africanum did not show differences in clinical characteristics when compared with TB caused by M. tuberculosis. Clustered cases defined as >2 cases in a county with identical 24-locus mycobacterial interspersed repetitive unit genotypes, were less likely for M. africanum (aOR 0.1, 95% CI 0.1-0.4), which suggests that M. africanum is not commonly transmitted in the United States.

  8. Pros and cons of direct genotyping on tuberculosis clinical samples.

    Science.gov (United States)

    Sadegh, Hamidreza; Kargarpour Kamakoli, Mansour; Farmanfarmaei, Ghazaleh; Masoumi, Morteza; Abdolrahimi, Farid; Fateh, Abolfazl; Ebrahimzadeh, Nayereh; Rahimi Jamnani, Fatemeh; Vaziri, Farzam; Siadat, Seyed Davar

    2017-02-01

    Prompt genotyping of Mycobacterium tuberculosis (M. tuberculosis) is crucial for improving molecular epidemiological investigation of tuberculosis (TB). We performed a retrospective study to evaluate the use of 24 loci MIRU-VNTR (mycobacterial interspersed repetitive unit-variable number of tandem-repeat) directly on 135 clinical samples from 84 TB patients. There was a direct correlation between genotyping on clinical samples by MIRU-VNTR and bacterial load (P = 0.001). VNTR loci were amplified successfully for 41.5% of the clinical samples (19-24 loci), 32.6% (13-18 loci), 23.7% (7-12 loci) and 2.2% (1-6 loci). Loci of 2401, 577, 2996 and 154 had the highest power to show the mixed strains infection in clinical samples. Direct MIRU-VNTR is partially successful in complete genotyping of M. tuberculosis strains. On the other hand, detection of polyclonal infection is undoubtedly reliable based on the direct MIRU-VNTR. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Inositol monophosphate phosphatase genes of Mycobacterium tuberculosis

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    Parish Tanya

    2010-02-01

    Full Text Available Abstract Background Mycobacteria use inositol in phosphatidylinositol, for anchoring lipoarabinomannan (LAM, lipomannan (LM and phosphatidylinosotol mannosides (PIMs in the cell envelope, and for the production of mycothiol, which maintains the redox balance of the cell. Inositol is synthesized by conversion of glucose-6-phosphate to inositol-1-phosphate, followed by dephosphorylation by inositol monophosphate phosphatases (IMPases to form myo-inositol. To gain insight into how Mycobacterium tuberculosis synthesises inositol we carried out genetic analysis of the four IMPase homologues that are present in the Mycobacterium tuberculosis genome. Results Mutants lacking either impA (Rv1604 or suhB (Rv2701c were isolated in the absence of exogenous inositol, and no differences in levels of PIMs, LM, LAM or mycothiol were observed. Mutagenesis of cysQ (Rv2131c was initially unsuccessful, but was possible when a porin-like gene of Mycobacterium smegmatis was expressed, and also by gene switching in the merodiploid strain. In contrast, we could only obtain mutations in impC (Rv3137 when a second functional copy was provided in trans, even when exogenous inositol was provided. Experiments to obtain a mutant in the presence of a second copy of impC containing an active-site mutation, in the presence of porin-like gene of M. smegmatis, or in the absence of inositol 1-phosphate synthase activity, were also unsuccessful. We showed that all four genes are expressed, although at different levels, and levels of inositol phosphatase activity did not fall significantly in any of the mutants obtained. Conclusions We have shown that neither impA, suhB nor cysQ is solely responsible for inositol synthesis. In contrast, we show that impC is essential for mycobacterial growth under the conditions we used, and suggest it may be required in the early stages of mycothiol synthesis.

  10. Mycobacterium tuberculosis replicates within necrotic human macrophages

    Science.gov (United States)

    Lerner, Thomas R.; Repnik, Urska; Herbst, Susanne; Collinson, Lucy M.; Griffiths, Gareth

    2017-01-01

    Mycobacterium tuberculosis modulation of macrophage cell death is a well-documented phenomenon, but its role during bacterial replication is less characterized. In this study, we investigate the impact of plasma membrane (PM) integrity on bacterial replication in different functional populations of human primary macrophages. We discovered that IFN-γ enhanced bacterial replication in macrophage colony-stimulating factor–differentiated macrophages more than in granulocyte–macrophage colony-stimulating factor–differentiated macrophages. We show that permissiveness in the different populations of macrophages to bacterial growth is the result of a differential ability to preserve PM integrity. By combining live-cell imaging, correlative light electron microscopy, and single-cell analysis, we found that after infection, a population of macrophages became necrotic, providing a niche for M. tuberculosis replication before escaping into the extracellular milieu. Thus, in addition to bacterial dissemination, necrotic cells provide first a niche for bacterial replication. Our results are relevant to understanding the environment of M. tuberculosis replication in the host. PMID:28242744

  11. Complex multifractal nature in Mycobacterium tuberculosis genome

    Science.gov (United States)

    Mandal, Saurav; Roychowdhury, Tanmoy; Chirom, Keilash; Bhattacharya, Alok; Brojen Singh, R. K.

    2017-04-01

    The mutifractal and long range correlation (C(r)) properties of strings, such as nucleotide sequence can be a useful parameter for identification of underlying patterns and variations. In this study C(r) and multifractal singularity function f(α) have been used to study variations in the genomes of a pathogenic bacteria Mycobacterium tuberculosis. Genomic sequences of M. tuberculosis isolates displayed significant variations in C(r) and f(α) reflecting inherent differences in sequences among isolates. M. tuberculosis isolates can be categorised into different subgroups based on sensitivity to drugs, these are DS (drug sensitive isolates), MDR (multi-drug resistant isolates) and XDR (extremely drug resistant isolates). C(r) follows significantly different scaling rules in different subgroups of isolates, but all the isolates follow one parameter scaling law. The richness in complexity of each subgroup can be quantified by the measures of multifractal parameters displaying a pattern in which XDR isolates have highest value and lowest for drug sensitive isolates. Therefore C(r) and multifractal functions can be useful parameters for analysis of genomic sequences.

  12. Drug resistance of Mycobacterium tuberculosis strains in southern Brazil

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    Laynara Katize Grutzmacher

    2012-02-01

    Full Text Available INTRODUCTION: The aim of this work was to evaluate the prevalence of Mycobacterium tuberculosis (MT strains with mutations that could result in resistance to the main drugs used in treatment in a region with one of the highest numbers of tuberculosis (TB cases in southern Brazil. METHODS: Deoxyribonucleic acid (DNA from 120 sputum samples from different patients suspicious of pulmonary tuberculosis who attended the Municipal Public Laboratory for Mycobacterium sp. diagnosis was directly amplified and analyzed by PCR-SSCP. The DNA was amplified in known hotspot mutation regions of the genes rpoB, ahpC, embB, katG, inhA, and pncA. RESULTS: The percentage of samples positive by culture was 9.2% (11/120; 5% (6/120 were positive by bacilloscopy and MT-PCR, and DNA fragments of the aforementioned resistance genes could be amplified from seven (7 of the eleven (11 samples with positive results, either by culture or PCR/bacilloscopy. All presented a SSCP pattern similar to a native, nonresistant genotype, with the ATCC strain 25177 as control, except for one sample (0.01%, which presented a SSCP profile demonstrating mutation at the embB gene. CONCLUSIONS: These results are consistent with the empirical observations by physicians treating TB patients in our region of a low occurrence of cases that are refractory to conventional treatment schemes, in contrast to other parts of the country. Continued surveillance, especially molecular, is essential to detect and monitor the outbreak of MT-resistant strains.

  13. Molecular epidemiology of Mycobacterium tuberculosis clinical isolates in Southwest Ireland.

    LENUS (Irish Health Repository)

    Ojo, Olabisi O

    2010-10-01

    Tuberculosis has had significant effects on Ireland over the past two centuries, causing persistently higher morbidity and mortality than in neighbouring countries until the last decade. This study describes the results of genotyping and drug susceptibility testing of 171 strains of Mycobacterium tuberculosis complex isolated between January 2004 and December 2006 in a region of Ireland centred on the city of Cork. Spoligotype comparisons were made with the SpolDB4 database and clustered 130 strains in 23 groups, forty-one strains showed unique Spoligotyping patterns. The commonest spoligotypes detected were ST0137 (X2) (16.9%), and ST0351 (15.8%) (\\'U\\' clade). The major spoligotype clades were X (26.2%), U (19.3%), T (15.2%), Beijing (5.9%), Haarlem (4.7%), LAM (4.1%), BOVIS (1.75%), with 12.9% unassigned strains. A 24-locus VNTR genotyping produced 15 clusters containing 49 isolates, with high discrimination index (HGDI>0.99). A combination of Spoligotyping and VNTR reduced the number of clustered isolates to 47 in 15 clusters (27.5%). This study identified ST351 as common among Irish nationals, and found a low rate of drug resistance with little evidence of transmission of drug resistant strains. Strain clustering was significantly associated with age under 55 years and Irish nationality. Only strains of Euro-American lineage formed clusters. Molecular typing did not completely coincide with the results of contact investigations.

  14. Molecular epidemiology of Mycobacterium tuberculosis clinical isolates in Southwest Ireland.

    Science.gov (United States)

    Ojo, Olabisi O; Sheehan, Stella; Corcoran, Daniel G; Nikolayevsky, Vladyslav; Brown, Timothy; O'Sullivan, Margaret; O'Sullivan, Kathleen; Gordon, Stephen V; Drobniewski, Francis; Prentice, Michael B

    2010-10-01

    Tuberculosis has had significant effects on Ireland over the past two centuries, causing persistently higher morbidity and mortality than in neighbouring countries until the last decade. This study describes the results of genotyping and drug susceptibility testing of 171 strains of Mycobacterium tuberculosis complex isolated between January 2004 and December 2006 in a region of Ireland centred on the city of Cork. Spoligotype comparisons were made with the SpolDB4 database and clustered 130 strains in 23 groups, forty-one strains showed unique Spoligotyping patterns. The commonest spoligotypes detected were ST0137 (X2) (16.9%), and ST0351 (15.8%) ('U' clade). The major spoligotype clades were X (26.2%), U (19.3%), T (15.2%), Beijing (5.9%), Haarlem (4.7%), LAM (4.1%), BOVIS (1.75%), with 12.9% unassigned strains. A 24-locus VNTR genotyping produced 15 clusters containing 49 isolates, with high discrimination index (HGDI>0.99). A combination of Spoligotyping and VNTR reduced the number of clustered isolates to 47 in 15 clusters (27.5%). This study identified ST351 as common among Irish nationals, and found a low rate of drug resistance with little evidence of transmission of drug resistant strains. Strain clustering was significantly associated with age under 55 years and Irish nationality. Only strains of Euro-American lineage formed clusters. Molecular typing did not completely coincide with the results of contact investigations.

  15. Animal-adapted members of the Mycobacterium tuberculosis complex endemic to the southern African subregion.

    Science.gov (United States)

    Clarke, Charlene; Van Helden, Paul; Miller, Michele; Parsons, Sven

    2016-04-26

    Members of the Mycobacterium tuberculosis complex (MTC) cause tuberculosis (TB) in both animals and humans. In this article, three animal-adapted MTC strains that are endemic to the southern African subregion - that is, Mycobacterium suricattae, Mycobacterium mungi, and the dassie bacillus - are reviewed with a focus on clinical and pathological presentations, geographic distribution, genotyping methods, diagnostic tools and evolution. Moreover, factors influencing the transmission and establishment of TB pathogens in novel host populations, including ecological, immunological and genetic factors of both the host and pathogen, are discussed. The risks associated with these infections are currently unknown and further studies will be required for greater understanding of this disease in the context of the southern African ecosystem.

  16. Rapid confirmation of drug susceptibility in Mycobacterium tuberculosis using MPT 64 Ag based test

    Directory of Open Access Journals (Sweden)

    Ernest Afu Ochang

    2013-06-01

    Full Text Available Objective: To evaluate the possible use of MPT64 based rapid test to detect multi-drug resistant Mycobacterium tuberculosis (M. tuberculosis in antibiotic broth dilution cultures. Methods: Twenty five isolates of M. tuberculosis whose susceptibility pattern had previously been identified by HAIN Genotype MTBDRplus (HainLifecience, Herhen, Germany were processed and cultured according to the microscopic-observation drug susceptibility technique. These included 20 susceptible, two multi-drug resistant M. tuberculosis and three isoniazid mono-resistant isolates. After 10-day incubation, aspirates from each well were tested with the MPT64 rapid test. Results: The rapid test correctly identified all 25 isolates and detected rifampicin and isoniazid resistance in all but one isoniazid mono-resistant isolate. Conclusions: MPT64 rapid test could be useful in detecting M. tuberculosis and drug resistance from Middlebrook 7H9 antimicrobial broth dilution in resource poor settings without an inverted microscope.

  17. Use of Genotype MTBDRplus Assay for Diagnosis of Multidrug-Resistant Tuberculosis in Nepal

    Directory of Open Access Journals (Sweden)

    Elina Maharjan

    2017-01-01

    Full Text Available The main aims of this study were to study the patterns of mutations in rpoB, katG, and inhA genes in Mycobacterium tuberculosis strains isolated from patients from Nepal and to evaluate the performance of genotype MTBDRplus assay, taking conventional drug susceptibility testing as gold standard for diagnosis of MDR-TB. A total of 69 Mycobacterium tuberculosis strains isolated from 73 smear positive sputum samples from patients suspected of suffering from multidrug-resistant tuberculosis were used in our study. The drug susceptibility pattern of Mycobacterium tuberculosis isolated from these sputum specimens was determined by using genotype MTBDRplus assay taking conventional drug susceptibility testing as reference. The sensitivity and specificity of the genotype MTBDRplus assay for the detection of MDR-TB were found to be 88.7% and 100%, respectively. 88.7% of the rifampicin resistant isolates had mutations in rpoB gene. Similarly, 79.7% and 9.4% of isoniazid resistant isolates had mutations in katG and inhA genes, respectively. Genotype MTBDRplus assay was found to be very rapid and highly sensitive and specific method for diagnosis of MDR-TB and will be very helpful for early diagnosis of MDR-TB in high tuberculosis burden countries.

  18. Heteroresistance in Mycobacteria tuberculosis is an important factor for the inconsistency between the results of phenotype and genotype drug susceptibility tests

    Institute of Scientific and Technical Information of China (English)

    高旭

    2014-01-01

    Objective To investigate the impact of heteroresistance on the results of genotype drug susceptibility test for Mycobacterium tuberculosis(M.tuberculosis).Methods A total of 80 phenotype ofloxacin-resistant M.tuberculosis isolates obtained from Shanghai Municipal Centers for Disease Control and Prevention were included in the study.The mutations of gyrA and gyrB in each isolate

  19. Adaptation and evolution of drug-resistant Mycobacterium tuberculosis

    NARCIS (Netherlands)

    Bergval, I.L.

    2013-01-01

    Many studies have been conducted on drug resistance and the evolution of Mycobacterium tuberculosis. Notwithstanding, many molecular mechanisms facilitating the emergence, adaptation and spread of drug-resistant tuberculosis have yet to be discovered. This thesis reports studies of the adaptive mech

  20. Exposure of dentists to Mycobacterium tuberculosis, Ibadan, Nigeria

    NARCIS (Netherlands)

    Cadmus, S.I.; Okoje, V.N.; Taiwo, B.O.; Soolingen, D. van

    2010-01-01

    To determine the prevalence of Mycobacterium tuberculosis infection among dental patients and to assess dentists' risk for exposure, we conducted a study among dental patients at a large tertiary hospital in Nigeria, a country where tuberculosis is endemic. Ten (13%) of 78 sputum samples obtained we

  1. Mycobacterium tuberculosis in Wild Asian Elephants, Southern India.

    Science.gov (United States)

    Zachariah, Arun; Pandiyan, Jeganathan; Madhavilatha, G K; Mundayoor, Sathish; Chandramohan, Bathrachalam; Sajesh, P K; Santhosh, Sam; Mikota, Susan K

    2017-03-01

    We tested 3 ild Asian elephants (Elephas maximus) in southern India and confirmed infection in 3 animals with Mycobacterium tuberculosis, an obligate human pathogen, by PCR and genetic sequencing. Our results indicate that tuberculosis may be spilling over from humans (reverse zoonosis) and emerging in wild elephants.

  2. Molecular epidemiology of Mycobacterium tuberculosis, Buenos Aires, Argentina.

    Science.gov (United States)

    Gonzalo, Ximena; Ambroggi, Marta; Cordova, Ezequiel; Brown, Tim; Poggi, Susana; Drobniewski, Francis

    2011-03-01

    To analyze the molecular epidemiology of Mycobacterium tuberculosis strains at a hospital in Buenos Aires, Argentina, and mutations related to multidrug-resistant and extensively drug-resistant tuberculosis, we conducted a prospective case-control study. Our findings reinforce the value of incorporating already standardized molecular methods for rapidly detecting resistance.

  3. Adaptation and evolution of drug-resistant Mycobacterium tuberculosis

    NARCIS (Netherlands)

    Bergval, I.L.

    2013-01-01

    Many studies have been conducted on drug resistance and the evolution of Mycobacterium tuberculosis. Notwithstanding, many molecular mechanisms facilitating the emergence, adaptation and spread of drug-resistant tuberculosis have yet to be discovered. This thesis reports studies of the adaptive

  4. Benzothiazinones kill Mycobacterium tuberculosis by blocking arabinan synthesis

    DEFF Research Database (Denmark)

    Makarov, Vadim; Manina, Giulia; Mikusova, Katarina

    2009-01-01

    New drugs are required to counter the tuberculosis (TB) pandemic. Here, we describe the synthesis and characterization of 1,3-benzothiazin-4-ones (BTZs), a new class of antimycobacterial agents that kill Mycobacterium tuberculosis in vitro, ex vivo, and in mouse models of TB. Using genetics...

  5. Molecular diversity of Mycobacterium tuberculosis isolates from patients with tuberculosis in Honduras

    Directory of Open Access Journals (Sweden)

    Ghebremichael Solomon

    2010-08-01

    Full Text Available Abstract Background Tuberculosis persists as a public health problem in Honduras. A better knowledge of the molecular characteristics of Mycobacterium tuberculosis strains will contribute to understand the transmission dynamics of the disease within the country. The aim of this study was to provide an insight of the genetic biodiversity of M. tuberculosis clinical isolates collected in Honduras between 1994 and 2002. Genotyping was performed using spoligotyping and RFLP. The spoligotypes obtained were compared with the SITVIT2 proprietary database of the Pasteur Institute of Guadeloupe. Results Spoligotyping grouped 84% of the isolates into 27 clusters (2 to 43 strains per cluster. Of the 44 shared international types (SITs identified among the Honduran stains, 8 SITs were newly identified either within the present study or after match with an orphan type previously identified in the SITVIT2 database. In addition, 16 patterns corresponded to orphan, previously unreported isolates. The Latin American Mediterranean (LAM lineage was the most common in this study; 55% of the strains belonged to this family. Other genotypes found were Haarlem (16%, T (16%, X-clade (6%, Unknown signature (5% and S (1%. Only one Beijing strain was identified (0.5%. We observed a high degree of diversity after characterizing the 43 isolates belonging to the main spoligotyping cluster (SIT 33, LAM3 with IS6110-RFLP. A total of 35 different RFLP-fingerprints were detected, of which 6 patterns corresponded to the same number of clusters comprising 14 strains. Conclusions The findings obtained in this study show that tuberculosis transmission in Honduras is due to modern M. tuberculosis lineages with high level of biodiversity.

  6. Whole genome analysis of Mycobacterium tuberculosis isolates from recurrent episodes of tuberculosis, Finland, 1995-2013.

    Science.gov (United States)

    Korhonen, V; Smit, P W; Haanperä, M; Casali, N; Ruutu, P; Vasankari, T; Soini, H

    2016-06-01

    Recurrent tuberculosis (TB) is caused by an endogenous re-activation of the same strain of Mycobacterium tuberculosis (relapse) or exogenous infection with a new strain (re-infection). Recurrence of TB in Finland was analysed in a population-based, 19-year study, and genotyping was used to define relapse and re-infection. The M. tuberculosis isolates from patients with suspected relapse were further analysed by whole genome sequencing (WGS) to determine the number and type of mutations occurring in the bacterial genome between the first and second disease episodes. In addition, publicly available tools (PhyResSE and SpolPred) were used to predict drug resistance and spoligotype profile from the WGS data. Of the 8299 notified TB cases, 48 (0.6%) patients had episodes classified as recurrent. Forty-two patients had more than one culture-confirmed TB episode, and isolates from two episodes in 21 patients were available for genotyping. In 18 patients, the M. tuberculosis isolates obtained from the first and second TB episodes had identical spoligotypes. The WGS analysis of the 36 M. tuberculosis isolates from the 18 suspected relapse patients (average time between isolates 2.8 years) revealed 0 to 38 single nucleotide polymorphisms (median 1, mean 3.78) between the first and second isolate. There seemed to be no direct relation between the number of years between the two isolates, or treatment outcome, and the number of single nucleotide polymorphisms. The results suggest that the mutation rate may depend on multiple host-, strain- and treatment-related factors.

  7. Autophagy in Mycobacterium tuberculosis and HIV infections

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    Lucile eEspert

    2015-06-01

    Full Text Available Human Immunodeficiency Virus (HIV and Mycobacterium tuberculosis (M.tb are among the most lethal human pathogens worldwide, each being responsible for around 1.5 million deaths annually. Moreover, synergy between acquired immune deficiency syndrome (AIDS and tuberculosis (TB has turned HIV/M.tb co-infection into a major public health threat in developing countries. In the past decade, autophagy, a lysosomal catabolic process, has emerged as a major host immune defense mechanism against infectious agents like M.tb and HIV. Nevertheless, in some instances, autophagy machinery appears to be instrumental for HIV infection. Finally, there is mounting evidence that both pathogens deploy various countermeasures to thwart autophagy. This mini-review proposes an overview of the roles and regulations of autophagy in HIV and M.tb infections with an emphasis on microbial factors. We also discuss the role of autophagy manipulation in the context of HIV/M.tb co-infection. In future, a comprehensive understanding of autophagy interaction with these pathogens will be critical for development of autophagy-based prophylactic and therapeutic interventions for AIDS and TB.

  8. Human Lung Immunity against Mycobacterium tuberculosis

    Science.gov (United States)

    Schwander, Stephan; Dheda, Keertan

    2011-01-01

    The study of human pulmonary immunity against Mycobacterium tuberculosis (M.tb) provides a unique window into the biological interactions between the human host and M.tb within the broncho-alveolar microenvironment, the site of natural infection. Studies of bronchoalveolar cells (BACs) and lung tissue evaluate innate, adaptive, and regulatory immune mechanisms that collectively contribute to immunological protection or its failure. In aerogenically M.tb–exposed healthy persons lung immune responses reflect early host pathogen interactions that may contribute to sterilization, the development of latent M.tb infection, or progression to active disease. Studies in these persons may allow the identification of biomarkers of protective immunity before the initiation of inflammatory and disease-associated immunopathological changes. In healthy close contacts of patients with tuberculosis (TB) and during active pulmonary TB, immune responses are compartmentalized to the lungs and characterized by an exuberant helper T-cell type 1 response, which as suggested by recent evidence is counteracted by local suppressive immune mechanisms. Here we discuss how exploring human lung immunity may provide insights into disease progression and mechanisms of failure of immunological protection at the site of the initial host–pathogen interaction. These findings may also aid in the identification of new biomarkers of protective immunity that are urgently needed for the development of new and the improvement of current TB vaccines, adjuvant immunotherapies, and diagnostic technologies. To facilitate further work in this area, methodological and procedural approaches for bronchoalveolar lavage studies and their limitations are also discussed. PMID:21075901

  9. SPECIFIC GYRB SEQUENCE OF MYCOBACTERIUM TUBERCULOSIS CLINICAL ISOLATED FROM SPUTUM OF PULMONARY TUBERCULOSIS PATIENTS IN INDONESIA

    Directory of Open Access Journals (Sweden)

    N. M. Mertaniasih

    2014-12-01

    Full Text Available Background: Indonesia have many different geographic areas which could be various on the variant strains of Mycobacterium tuberculosis. The gyrB gene codes GyrB protein as sub unit compound of Gyrase enzyme that functioning in multiplication of bacteria. Detection of gyrB gene could be a marker of active multiplication of viable bacteria in the specimen from patients; and some of the DNA sequence regions were conserved and specific in the strain of Mycobacterium tuberculosis that would be a marker for identification. This research aims to analyze the sequence of gyrB gene of Mycobacterium tuberculosis clinical isolates from sputum of pulmonary TB patients in Indonesia, and determine the specific region. Method: Mycobacterium tuberculosis clinical isolates have been collected from sputum of the patients with pulmonary TB that live in some area in Indonesia. Isolation and identification of Mycobacterium tuberculosis clinical isolates using standard culture method; sequence analysis using PCR-direct sequencing of the part bases region of gyrB. Results: this study revealed that nucleotide sequence on a fragment 764 bases of gyrB gene Mycobacterium tuberculosis strains among clinical isolates almost identically to a wild type strain Mycobacterium tuberculosis H37Rv and subspecies member of Mycobacterium tuberculosis complex (MTBC, with a little difference of SNPs; there are many difference nucleotide sequence with MOTT and Gram positive or negative bacteria, except Corynebacterium diphtheriae identically with MTBC. Conclusion: the gyrB sequence in Mycobacterium tuberculosis strains among these clinical isolates from sputum of pulmonary TB patients in Indonesia have the conserved specific DNA region that almost identically with wild type strain H37Rv and MTBC.

  10. Standartization of broth microdilution method for Mycobacterium tuberculosis

    OpenAIRE

    Clarice Queico Fujimura Leite; Ana Laura Remédio Zeni Beretta; Ivone Shizuko Anno; Maria Alice da Silva Telles

    2000-01-01

    Indirect drug susceptibility tests of Mycobacterium tuberculosis was done to investigate the accuracy and feasibility of a broth microdilution method (BMM) for determining minimal inhibitory concentrations of conventional drugs against M. tuberculosis. Test drugs included isoniazid (H), rifampicin (R), ethambutol (E), streptomycin (S) and pyrazinamide (Z). Fifty isolates of M. tuberculosis from patients who had never received drug therapy, and H37Rv strain for control, were evaluated in the s...

  11. Multidrug-Resistant Mycobacterium tuberculosis of the Latin American Mediterranean Lineage, Wrongly Identified as Mycobacterium pinnipedii (Spoligotype International Type 863 [SIT863]), Causing Active Tuberculosis in South Brazil

    KAUST Repository

    Dalla Costa, Elis R.

    2015-09-23

    We recently detected the spoligotype patterns of strains of Mycobacterium pinnipedii, a species of the Mycobacterium tuberculosis complex, in sputum samples from nine cases with pulmonary tuberculosis residing in Porto Alegre, South Brazil. Because this species is rarely encountered in humans, we further characterized these nine isolates by additional genotyping techniques, including 24-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) typing, verification of the loci TbD1, RD9, pks15/1, RDRio, and fbpC, the insertion of IS6110 at a site specific to the M. tuberculosis Latin American Mediterranean (LAM) lineage, and whole-genome sequencing. The combined analysis of these markers revealed that the isolates are in fact M. tuberculosis and more specifically belong to the LAM genotype. Most of these isolates (n = 8) were shown to be multidrug resistant (MDR), which prompted us to perform partial sequencing of the rpoA, rpoB, rpoC, katG, and inhA genes. Seven isolates (77.8%) carried the S315T mutation in katG, and one of these (11%) also presented the C(−17)T single-nucleotide polymorphism (SNP) in inhA. Interestingly, six of the MDR isolates also presented an undescribed insertion of 12 nucleotides (CCA GAA CAA CCC) in codon 516 of rpoB. No putative compensatory mutation was found in either rpoA or rpoC. This is the first report of an M. tuberculosis LAM family strain with a convergent M. pinnipedii spoligotype. These spoligotypes are observed in genotype databases at a modest frequency, highlighting that care must be taken when identifying isolates in the M. tuberculosis complex on the basis of single genetic markers.

  12. Study of Mycobacterium tuberculosis Complex Genotypic Diversity in Malaysia Reveals a Predominance of Ancestral East-African-Indian Lineage with a Malaysia-Specific Signature: e114832

    National Research Council Canada - National Science Library

    Fazli Ismail; David Couvin; Izzah Farakhin; Zaidah Abdul Rahman; Nalin Rastogi; Siti Suraiya

    2014-01-01

    .... Objectives This study intended a first assessment of spoligotyping-based MTBC genotypic diversity in Malaysia followed by a comparison of strains with those prevailing in neighboring countries...

  13. pncA Mutations in Pyrazinamide-Resistant Mycobacterium tuberculosis Isolates from Northwestern Russia

    OpenAIRE

    1999-01-01

    Thirty-six pyrazinamide-resistant and eight pyrazinamide-susceptible Mycobacterium tuberculosis isolates from Russia were analyzed for their pncA mutations. Thirty-one (86.1%) of the resistant isolates had a mutation either in pncA or upstream of the gene. Twenty of the 23 different mutations found in this study had not been described earlier. pncA genotype correlated well with pyrazinamidase activity and BACTEC 460 susceptibility test results.

  14. Assessment of the probability of introducing Mycobacterium tuberculosis into Danish cattle herds

    DEFF Research Database (Denmark)

    Foddai, Alessandro; Nielsen, Liza Rosenbaum; Krogh, Kaspar

    2015-01-01

    Tuberculosis is a zoonosis caused by Mycobacterium spp. International trade in cattle is regulated with respect to Mycobacterium bovis (M. bovis) but not Mycobacterium tuberculosis (M. tuberculosis), despite that cattle can become infected with both species. In this study we estimated the annual...

  15. Mycobacterium tuberculosis volatiles for diagnosis of tuberculosis by Cricetomys rats.

    Science.gov (United States)

    Mgode, Georgies F; Weetjens, Bart J; Nawrath, Thorben; Lazar, Doris; Cox, Christophe; Jubitana, Maureen; Mahoney, Amanda; Kuipers, Dian; Machang'u, Robert S; Weiner, January; Schulz, Stefan; Kaufmann, Stefan H E

    2012-11-01

    Tuberculosis (TB) diagnosis in regions with limited resources depends on microscopy with insufficient sensitivity. Rapid diagnostic tests of low cost but high sensitivity and specificity are needed for better point-of-care management of TB. Trained African giant pouched rats (Cricetomys sp.) can diagnose pulmonary TB in sputum but the relevant Mycobacterium tuberculosis (Mtb)-specific volatile compounds remain unknown. We investigated the odour volatiles of Mtb detected by rats in reference Mtb, nontuberculous mycobacteria, Nocardia sp., Streptomyces sp., Rhodococcus sp., and other respiratory tract microorganisms spiked into Mtb-negative sputum. Thirteen compounds were specific to Mtb and 13 were shared with other microorganisms. Rats discriminated a blend of Mtb-specific volatiles from individual, and blends of shared, compounds (P = 0.001). The rats' sensitivity for typical TB-positive sputa was 99.15% with 92.23% specificity and 93.14% accuracy. These findings underline the potential of trained Cricetomys rats for rapid TB diagnosis in resource-limited settings, particularly in Africa where Cricetomys rats occur widely and the burden of TB is high. Copyright © 2012 Elsevier Ltd. All rights reserved.

  16. At Baltic crossroads: a molecular snapshot of Mycobacterium tuberculosis population diversity in Kaliningrad, Russia.

    Science.gov (United States)

    Mokrousov, Igor; Otten, Tatiana; Zozio, Thierry; Turkin, Eugeni; Nazemtseva, Vera; Sheremet, Aleksandra; Vishnevsky, Boris; Narvskaya, Olga; Rastogi, Nalin

    2009-01-01

    The Kaliningrad region is the westernmost part of the Russian Federation; it includes an enclave on the Baltic Sea inside the European Union separated from mainland Russia by Lithuania and Poland. The incidence of tuberculosis in Kaliningrad has shown a steady and dramatic increase from 83/100,000 in 2000 to 134/100,000 in 2006; the rate of multidrug-resistant tuberculosis (MDR-tuberculosis) in the Kaliningrad region was reported to be 30.5% among newly diagnosed tuberculosis patients. This study presents a first molecular snapshot of the population diversity of Mycobacterium tuberculosis in this region. A total of 90 drug-resistant and susceptible M. tuberculosis strains from Kaliningrad were subjected to spoligotyping, 12-locus MIRU typing and mutation analysis of the drug resistance genes rpoB and katG. A comparison with international databases showed that the M. tuberculosis population in this region shares a joint pool of strains with the European part of Russia, and also exhibits a certain affinity with those of its northern European neighbours, such as Poland and Germany. Comparison of the genotyping and drug resistance data emphasized that the high prevalence of the MDR Beijing genotype strains is a major cause of the adverse epidemiological situation of MDR-tuberculosis in the Kaliningrad region.

  17. Mycobacterium marinum: a potential immunotherapy for Mycobacterium tuberculosis infection

    Directory of Open Access Journals (Sweden)

    Tian WW

    2013-07-01

    Full Text Available Wei-wei Tian,1 Qian-qiu Wang,1 Wei-da Liu,2 Jian-ping Shen,1 Hong-sheng Wang11Laboratory of Mycobacterial Disease, Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs, Nanjing, Jiangsu, People’s Republic of China; 2Department of Mycology, Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs, Nanjing, Jiangsu, People’s Republic of ChinaPurpose: The aim of the present study was to investigate the immune response induced by Mycobacterium marinum infection in vitro and the potential of M. marinum as an immunotherapy for M. tuberculosis infection.Methods: The potential human immune response to certain bacillus infections was investigated in an immune cell–bacillus coculture system in vitro. As a potential novel immunotherapy, M. marinum was studied and compared with two other bacilli, Bacillus Calmette-Guérin (BCG and live attenuated M. tuberculosis. We examined the changes in both the bacilli and immune cells, especially the time course of the viability of mycobacteria in the coculture system and host immune responses including multinuclear giant cell formation by Wright–Giemsa modified staining, macrophage polarization by cell surface antigen expression, and cytokines/chemokine production by both mRNA expression and protein secretion.Results: The M. marinum stimulated coculture group showed more expression of CD209, CD68, CD80, and CD86 than the BCG and M. tuberculosis (an attenuated strain, H37Ra groups, although the differences were not statistically significant. Moreover, the M. marinum group expressed more interleukin (IL-1B and IL-12p40 on day 3 (IL-1B: P = 0.003 and 0.004, respectively; IL-12p40: P = 0.001 and 0.011, respectively, a higher level of CXCL10 on day 1 (P = 0.006 and 0.026, respectively, and

  18. Mixed Infections and Rifampin Heteroresistance among Mycobacterium tuberculosis Clinical Isolates.

    Science.gov (United States)

    Zheng, Chao; Li, Song; Luo, Zhongyue; Pi, Rui; Sun, Honghu; He, Qingxia; Tang, Ke; Luo, Mei; Li, Yuqing; Couvin, David; Rastogi, Nalin; Sun, Qun

    2015-07-01

    Mixed infections and heteroresistance of Mycobacterium tuberculosis contribute to the difficulty of diagnosis, treatment, and control of tuberculosis. However, there is still no proper solution for these issues. This study aimed to investigate the potential relationship between mixed infections and heteroresistance and to determine the high-risk groups related to these factors. A total of 499 resistant and susceptible isolates were subjected to spoligotyping and 24-locus variable-number tandem repeat methods to analyze their genotypic lineages and the occurrence of mixed infections. Two hundred ninety-two randomly selected isolates were sequenced on their rpoB gene to examine mutations and heteroresistance. The results showed that 12 patients had mixed infections, and the corresponding isolates belonged to Manu2 (n = 8), Beijing (n = 2), T (n = 1), and unknown (n = 1) lineages. Manu2 was found to be significantly associated with mixed infections (odds ratio, 47.72; confidence interval, 9.68 to 235.23; P mutation in the rpoB gene were significantly associated with mixed infections (χ(2), 56.78; P mutation in the rpoB gene to become rifampin resistant. Further studies should focus on this lineage to clarify its relevance to mixed infections.

  19. Study of Mycobacterium tuberculosis complex genotypic diversity in Malaysia reveals a predominance of ancestral East-African-Indian lineage with a Malaysia-specific signature

    National Research Council Canada - National Science Library

    Ismail, Fazli; Couvin, David; Farakhin, Izzah; Abdul Rahman, Zaidah; Rastogi, Nalin; Suraiya, Siti

    2014-01-01

    .... This study intended a first assessment of spoligotyping-based MTBC genotypic diversity in Malaysia followed by a comparison of strains with those prevailing in neighboring countries by comparison...

  20. Genetic diversity of Mycobacterium tuberculosis isolates from Tochigi prefecture, a local region of Japan.

    Science.gov (United States)

    Mizukoshi, Fuminori; Miyoshi-Akiyama, Tohru; Iwai, Hiroki; Suzuki, Takako; Kiritani, Reiko; Kirikae, Teruo; Funatogawa, Keiji

    2017-05-25

    Foreign-born patients with tuberculosis (TB) may introduce globally disseminated isolates of Mycobacterium tuberculosis into large cities in Japan. The risk of dissemination of these isolates into local regions, however, has not been determined. This study analyzed the molecular epidemiology of M. tuberculosis isolates obtained from TB patients living in a local region of Japan. Whole genome sequences of 169 M. tuberculosis isolates, obtained from 148 Japanese-born and 21 foreign-born patients living in Tochigi, Japan, were analyzed using the Comprehensive analysis server for the Mycobacterium t u b erculosis complex (CASTB). The 169 isolates were clustered into four clades; Lineage 2 (111 isolates 65.7%), Lineage 4 (43 isolates, 25.4%), Lineage 1 (13 isolates, 7.7%), and Lineage 3 (2 isolates, 1.2%). Of the 111 isolates belonging to Lineage 2, 79 (71.2%) were of the atypical Beijing sub-genotype. Of the 13 Lineage 1 isolates, nine (69.2%) were from foreign-born patients. The isolates belonging to Lineage 4 were further clustered into three clades, two containing isolates shared by both Japanese- and foreign-born patients. The two isolates belonging to Lineage 3 were obtained from foreign-born patients. The genotypic diversity of M. tuberculosis in a local region of Japan is increased primarily by the presence of isolates obtained from foreign-born patients.

  1. Molecular diversity of Mycobacterium tuberculosis isolates from patients with pulmonary tuberculosis in Mozambique

    Directory of Open Access Journals (Sweden)

    Hill Véronique

    2010-07-01

    Full Text Available Abstract Background Mozambique is one of the countries with the highest burden of tuberculosis (TB in Sub-Saharan Africa, and information on the predominant genotypes of Mycobacterium tuberculosis circulating in the country are important to better understand the epidemic. This study determined the predominant strain lineages that cause TB in Mozambique. Results A total of 445 M. tuberculosis isolates from seven different provinces of Mozambique were characterized by spoligotyping and resulting profiles were compared with the international spoligotyping database SITVIT2. The four most predominant lineages observed were: the Latin-American Mediterranean (LAM, n = 165 or 37%; the East African-Indian (EAI, n = 132 or 29.7%; an evolutionary recent but yet ill-defined T clade, (n = 52 or 11.6%; and the globally-emerging Beijing clone, (n = 31 or 7%. A high spoligotype diversity was found for the EAI, LAM and T lineages. Conclusions The TB epidemic in Mozambique is caused by a wide diversity of spoligotypes with predominance of LAM, EAI, T and Beijing lineages.

  2. The road to tuberculosis (Mycobacterium tuberculosis elimination in Arkansas; a re-examination of risk groups.

    Directory of Open Access Journals (Sweden)

    Anna Berzkalns

    Full Text Available OBJECTIVES: This study was conducted to generate knowledge useful for developing public health interventions for more effective tuberculosis control in Arkansas. METHODS: The study population included 429 culture-confirmed reported cases (January 1, 2004-December 31, 2010. Mycobacterium tuberculosis genotyping data were used to identify cases likely due to recent transmission (clustered versus reactivation (non-clustered. Poisson regression models estimated average decline rate in incidence over time and assessed the significance of differences between subpopulations. A multinomial logistic model examined differences between clustered and non-clustered incidence. RESULTS: A significant average annual percent decline was found for the overall incidence of culture-confirmed (9%; 95% CI: 5.5%, 16.9%, clustered (6%; 95% CI: 0.5%, 11.6%, and non-clustered tuberculosis cases (12%; 95% CI: 7.6%, 15.9%. However, declines varied among demographic groups. Significant declines in clustered incidence were only observed in males, non-Hispanic blacks, 65 years and older, and the rural population. CONCLUSIONS: These findings suggest that the Arkansas tuberculosis control program must target both traditional and non-traditional risk groups for successful tuberculosis elimination. The present study also demonstrates that a thorough analysis of TB trends in different population subgroups of a given geographic region or state can lead to the identification of non-traditional risk factors for TB transmission. Similar studies in other low incidence populations would provide beneficial data for how to control and eventually eliminate TB in the U.S.

  3. [MOLECULAR CHARACTERISTICS OF THE MULTIDRUG-RESISTANT MYCOBACTERIUM TUBERCULOSIS STRAINS IN THE NORTHWEST RUSSIA].

    Science.gov (United States)

    Vyazovaya, A A; Mokrousov, I V; Zhuravlev, V Yu; Solovieva, N S; Otten, T F; Manicheva, O A; Vishnevsky, B I; Narvskaya, O V

    2016-01-01

    The goal of this work was to study the genotypic characteristics of the multidrug-resistant (MDR, i.e., resistant to at least rifampicine and isoniazid) Mycobacterium tuberculosis strains isolated in 2011-2012 from tuberculosis (TB) patients in the Northwest Russia. Spoligotyping of 195 M. tuberculosis isolates identified 14 different spoligotypes and assigned isolates to the genetic families Beijing (n = 162, 83%), LAM (n = 15), H3/URAL (n = 14), as well as T, Haarlem and X. Spoligotypes SIT1 (Beijing), SIT42 (LAM) and SIT262 (H3/URAL) were the most prevalent. Irrespective to the genotype, all the isolates were resistant to streptomycin. The multidrug resistance was accompanied by the resistance to ethionamide (56%), amikacin (31%), kanamycin (40%), and capreomycin (33%). The ethambutol resistance was found in 71% (n = 115) and 42% (n = 14) of the Beijing and non-Beijing strains, respectively (p Russia continues to be dominated by the Beijing family strains.

  4. Escape of Mycobacterium tuberculosis from oxidative killing by neutrophils.

    Science.gov (United States)

    Corleis, Björn; Korbel, Daniel; Wilson, Robert; Bylund, Johan; Chee, Ronnie; Schaible, Ulrich E

    2012-07-01

    Neutrophils enter sites of infection, where they can eliminate pathogenic bacteria in an oxidative manner. Despite their predominance in active tuberculosis lesions, the function of neutrophils in this important human infection is still highly controversial. We observed that virulent Mycobacterium tuberculosis survived inside human neutrophils despite prompt activation of these defence cells' microbicidal effectors. Survival of M. tuberculosis was accompanied by necrotic cell death of infected neutrophils. Necrotic cell death entirely depended on radical oxygen species production since chronic granulomatous disease neutrophils were protected from M. tuberculosis-triggered necrosis. More, importantly, the M. tuberculosis ΔRD1 mutant failed to induce neutrophil necrosis rendering this strain susceptible to radical oxygen species-mediated killing. We conclude that this virulence function is instrumental for M. tuberculosis to escape killing by neutrophils and contributes to pathogenesis in tuberculosis.

  5. Genotypic analysis of isoniazid-resistant Mycobacterium tuberculosis isolates recovered from Wuhan%武汉地区结核分枝杆菌异烟肼耐药性的基因分析

    Institute of Scientific and Technical Information of China (English)

    孙战强; 陈高瞻; 余晓丽

    2015-01-01

    探讨武汉地区结核分枝杆菌临床分离株异烟肼耐药相关基因突变特征。通过PCR直接序列分析法对异烟肼耐药相关基因katG基因及mabA-inhA启动子进行序列分析。20株异烟肼敏感株未检测到突变,49株耐药株中有40株(81.6%)存在突变,其中katG的S315突变率占主导优势,突变率为73.5%(36/49),mabA-inhA启动子区核苷酸置换率为8.2%(4/49),其中一株同时检测到了katG和mabA-inhA启动子区的突变。此外,还检测到了一种国内尚未报道的katG突变类型P232S(CCG→TCG)。研究证实katG基因和mabA-inhA启动子的突变与异烟肼耐药相关,为进一步研究结核分枝杆菌异烟肼耐药性奠定了基础。%To evaluate the characteristic of genes associated with isoniazid-resistant Mycobacterium tuberculosis clini-cal isolates collected from Wuhan , China .Methods The genotypes of katG and mabA-inhA promoter that associated with isoniazid-resistance were analyzed with PCR direct sequencing .Results Of the 49 isoniazid-resistant clinical i-solates, 43 strains(81.6%) carried mutations in the target genes , and no mutations was found in the 20 isoniazid-sensitive strains.The mutations in katG gene at codon 315 was the most frequent mutations up to 73.5%(36/49) in all.The nucleotide replacement rate of mabA-inhA promoter was 8.2%(4/49).One isolate strain was detected mutations both in katG mabA-inhA promoter.Moreover, we found a new mutant type P232S(CCG→TCG).Con-clusions This study confirmed that mutations in the katG and mabA-inhA promoter related with isoniazid-resistance , and provided clues to explore the mechanism of isoniazid -resistant Mycobacterium tuberculosis.

  6. Strain Diversity of Mycobacterium tuberculosis Isolates from Pulmonary Tuberculosis Patients in Afar Pastoral Region of Ethiopia

    Directory of Open Access Journals (Sweden)

    Mulugeta Belay

    2014-01-01

    Full Text Available Data on genotypic diversity of Mycobacterium tuberculosis complex (MTBC is important to understand its epidemiology, human adaptation, clinical phenotypes, and drug resistance. This study aimed to characterize MTBC clinical isolates circulating in a predominantly pastoralist area in Ethiopia, a country where tuberculosis is the second leading cause of mortality. Culture of sputum samples collected from a total of 325 pulmonary TB suspects was done to isolate MTBC. Spoligotyping was used to characterize 105 isolates from culture positive slopes and the result was compared with an international database. Forty-four spoligotype patterns were observed to correspond to 35 shared-types (SITs containing 96 isolates and 9 orphan patterns; 27 SITs containing 83 isolates matched a preexisting shared-type in the database, whereas 8 SITs (n=13 isolates were newly created. A total of 19 SITs containing 80 isolates were clustered within this study (overall clustering of 76.19%. Three dominant lineages (T, CAS, and Manu accounted for 76.19% of the isolates. SIT149/T3-ETH was one of the two most dominant sublineages. Unlike previous reports, we show that Manu lineage strains not only constitute a dominant lineage, but are also associated with HIV infection in Afar region of Ethiopia. The high level of clustering suggests the presence of recent transmission that should be further studied using additional genotyping markers.

  7. Pulmonary disease due to Mycobacterium tuberculosis in a horse: zoonotic concerns and limitations of antemortem testing

    Science.gov (United States)

    A case of pulmonary tuberculosis caused by Mycobacterium tuberculosis was diagnosed in a horse. Clinical evaluation performed prior to euthanasia did not suggest tuberculosis, but postmortem examination provided pathological and bacteriological evidence of disease. In the lungs, multiple tuberculoid...

  8. Molecular epidemiology of Mycobacterium tuberculosis in a South African community with high HIV prevalence.

    Science.gov (United States)

    Middelkoop, Keren; Bekker, Linda-Gail; Mathema, Barun; Shashkina, Elena; Kurepina, Natalia; Whitelaw, Andrew; Fallows, Dorothy; Morrow, Carl; Kreiswirth, Barry; Kaplan, Gilla; Wood, Robin

    2009-10-15

    To explore the relationship between human immunodeficiency virus (HIV) and Mycobacterium tuberculosis genotypes, we performed IS6110-based restriction fragment-length polymorphism analysis on M. tuberculosis culture specimens from patients with smear-positive tuberculosis in a periurban community in South Africa from 2001 through 2005. Among 151 isolates, 95 strains were identified within 26 families, with 54% clustering. HIV status was associated with W-Beijing strains (P = .009) but not with clustering per se. The high frequency of clustering suggests ongoing transmission in both HIV-negative and HIV-positive individuals in this community. The strong association between W-Beijing and HIV infection may have important implications for tuberculosis control.

  9. Molecular epidemiology of Mycobacterium tuberculosis strains circulating in the penitentiary system of Kazakhstan.

    Science.gov (United States)

    Ibrayeva, A; Kozhamkulov, U; Raiymbek, D; Alenova, A; Igilikova, S; Zholdybayeva, E; Abildaev, T; Momynaliev, K

    2014-03-01

    A total of 60 Mycobacterium tuberculosis isolates collected from patients in prisons in Kazakhstan and 125 from the civilian sector were examined using mycobacterial interspersed repetitive units-variable number of tandem repeat analysis in 2012. The proportion of tuberculosis strains with unique genotypes isolated from the civilian patients was 50.4%, while that in the prison patients was 31.7%. This difference was statistically significant (χ(2) 4.42, P 0.035), and may reflect a low genetic diversity of M. tuberculosis strains isolated from prison patients. The frequencies of mutations in the rpoB531 and katG315 genes of the M. tuberculosis strains isolated from the civilians and in the penitentiary system were not significantly different (rpoB531: 82.4% vs. 88.3%, and katG315: 98.4% vs. 100%, respectively).

  10. Integration of published information into a resistance-associated mutation database for Mycobacterium tuberculosis.

    Science.gov (United States)

    Salamon, Hugh; Yamaguchi, Ken D; Cirillo, Daniela M; Miotto, Paolo; Schito, Marco; Posey, James; Starks, Angela M; Niemann, Stefan; Alland, David; Hanna, Debra; Aviles, Enrique; Perkins, Mark D; Dolinger, David L

    2015-04-01

    Tuberculosis remains a major global public health challenge. Although incidence is decreasing, the proportion of drug-resistant cases is increasing. Technical and operational complexities prevent Mycobacterium tuberculosis drug susceptibility phenotyping in the vast majority of new and retreatment cases. The advent of molecular technologies provides an opportunity to obtain results rapidly as compared to phenotypic culture. However, correlations between genetic mutations and resistance to multiple drugs have not been systematically evaluated. Molecular testing of M. tuberculosis sampled from a typical patient continues to provide a partial picture of drug resistance. A database of phenotypic and genotypic testing results, especially where prospectively collected, could document statistically significant associations and may reveal new, predictive molecular patterns. We examine the feasibility of integrating existing molecular and phenotypic drug susceptibility data to identify associations observed across multiple studies and demonstrate potential for well-integrated M. tuberculosis mutation data to reveal actionable findings.

  11. Molecular characterization of Mycobacterium tuberculosis isolates from elephants of Nepal.

    Science.gov (United States)

    Paudel, Sarad; Mikota, Susan K; Nakajima, Chie; Gairhe, Kamal P; Maharjan, Bhagwan; Thapa, Jeewan; Poudel, Ajay; Shimozuru, Michito; Suzuki, Yasuhiko; Tsubota, Toshio

    2014-05-01

    Mycobacterium tuberculosis was cultured from the lung tissues of 3 captive elephants in Nepal that died with extensive lung lesions. Spoligotyping, TbD1 detection and multi-locus variable number of tandem repeat analysis (MLVA) results suggested 3 isolates belonged to a specific lineage of Indo-Oceanic clade, EAI5 SIT 138. One of the elephant isolates had a new synonymous single nucleotide polymorphism (SNP) T231C in the gyrA sequence, and the same SNP was also found in human isolates in Nepal. MLVA results and transfer history of the elephants suggested that 2 of them might be infected with M. tuberculosis from the same source. These findings indicated the source of M. tuberculosis infection of those elephants were local residents, presumably their handlers. Further investigation including detailed genotyping of elephant and human isolates is needed to clarify the infection route and eventually prevent the transmission of tuberculosis to susceptible hosts.

  12. Osteomielite esternal por Mycobacterium tuberculosis Sternal osteomyelitis caused by infection with Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Diego Michelon De Carli

    2009-07-01

    Full Text Available Descrevemos o caso de um paciente de 74 anos, masculino, com dor torácica na porção superior do esterno com um ano de evolução associada a eritema, edema e fístula com drenagem de material purulento. Paciente HIV negativo e sem história prévia de contato com TB. A TC de tórax evidenciou lesão osteolítica esternal, e o material de biópsia revelou granuloma caseoso negativo para fungos e bacilos álcool-ácido resistentes no exame microbiológico direto. O diagnóstico de osteomielite esternal por Mycobacterium tuberculosis foi realizado por PCR.We report the case of a 74-year-old male patient with a one-year history of chest pain in the suprasternal notch associated with erythema, edema and drainage of purulent material from a fistulous lesion. The patient was HIV-negative with no history of TB. A CT scan of the chest showed an osteolytic lesion in the sternum, and a biopsy revealed caseous granuloma, which, in the microbiological evaluation, was negative for fungi and acid-fast bacilli. The diagnosis of sternal osteomyelitis caused by Mycobacterium tuberculosis was confirmed using PCR.

  13. Immune responses to the Mycobacterium tuberculosis-specific antigen ESAT-6 signal subclinical infection among contacts of tuberculosis patients

    DEFF Research Database (Denmark)

    Doherty, T Mark; Demissie, Abebech; Olobo, Joseph;

    2002-01-01

    Diagnosis of latent Mycobacterium tuberculosis infection is considered essential for tuberculosis control but is hampered by the lack of specific reagents. We report that strong recognition of tuberculosis complex-specific antigen ESAT-6 by healthy household contacts of tuberculosis patients...... correlates with the subsequent development of active tuberculosis during a 2-year follow-up period....

  14. Phylogenetic assignment of Mycobacterium tuberculosis Beijing clinical isolates in Japan by maximum a posteriori estimation.

    Science.gov (United States)

    Seto, Junji; Wada, Takayuki; Iwamoto, Tomotada; Tamaru, Aki; Maeda, Shinji; Yamamoto, Kaori; Hase, Atsushi; Murakami, Koichi; Maeda, Eriko; Oishi, Akira; Migita, Yuji; Yamamoto, Taro; Ahiko, Tadayuki

    2015-10-01

    Intra-species phylogeny of Mycobacterium tuberculosis has been regarded as a clue to estimate its potential risk to develop drug-resistance and various epidemiological tendencies. Genotypic characterization of variable number of tandem repeats (VNTR), a standard tool to ascertain transmission routes, has been improving as a public health effort, but determining phylogenetic information from those efforts alone is difficult. We present a platform based on maximum a posteriori (MAP) estimation to estimate phylogenetic information for M. tuberculosis clinical isolates from individual profiles of VNTR types. This study used 1245 M. tuberculosis clinical isolates obtained throughout Japan for construction of an MAP estimation formula. Two MAP estimation formulae, classification of Beijing family and other lineages, and classification of five Beijing sublineages (ST11/26, STK, ST3, and ST25/19 belonging to the ancient Beijing subfamily and modern Beijing subfamily), were created based on 24 loci VNTR (24Beijing-VNTR) profiles and phylogenetic information of the isolates. Recursive estimation based on the formulae showed high concordance with their authentic phylogeny by multi-locus sequence typing (MLST) of the isolates. The formulae might further support phylogenetic estimation of the Beijing lineage M. tuberculosis from the VNTR genotype with various geographic backgrounds. These results suggest that MAP estimation can function as a reliable probabilistic process to append phylogenetic information to VNTR genotypes of M. tuberculosis independently, which might improve the usage of genotyping data for control, understanding, prevention, and treatment of TB.

  15. Molecular Strain Typing of Mycobacterium tuberculosis: a Review of Frequently Used Methods.

    Science.gov (United States)

    Ei, Phyu Win; Aung, Wah Wah; Lee, Jong Seok; Choi, Go Eun; Chang, Chulhun L

    2016-11-01

    Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, remains one of the most serious global health problems. Molecular typing of M. tuberculosis has been used for various epidemiologic purposes as well as for clinical management. Currently, many techniques are available to type M. tuberculosis. Choosing the most appropriate technique in accordance with the existing laboratory conditions and the specific features of the geographic region is important. Insertion sequence IS6110-based restriction fragment length polymorphism (RFLP) analysis is considered the gold standard for the molecular epidemiologic investigations of tuberculosis. However, other polymerase chain reaction-based methods such as spacer oligonucleotide typing (spoligotyping), which detects 43 spacer sequence-interspersing direct repeats (DRs) in the genomic DR region; mycobacterial interspersed repetitive units-variable number tandem repeats, (MIRU-VNTR), which determines the number and size of tandem repetitive DNA sequences; repetitive-sequence-based PCR (rep-PCR), which provides high-throughput genotypic fingerprinting of multiple Mycobacterium species; and the recently developed genome-based whole genome sequencing methods demonstrate similar discriminatory power and greater convenience. This review focuses on techniques frequently used for the molecular typing of M. tuberculosis and discusses their general aspects and applications.

  16. Line probe assay for differentiation within Mycobacterium tuberculosis complex. Evaluation on clinical specimens and isolates including Mycobacterium pinnipedii

    DEFF Research Database (Denmark)

    Kjeldsen, Marianne Kirstine; Bek, Dorte; Rasmussen, Erik Michael;

    2009-01-01

    A line probe assay (GenoType MTBC) was evaluated for species differentiation within the Mycobacterium tuberculosis complex (MTBC). We included 387 MTBC isolates, 43 IS6110 low-copy MTBC isolates, 28 clinical specimens with varying microscopy grade, and 30 isolates of non-tuberculous mycobacteria....... The assay was 100% specific and identified all 387 isolates and 98% of all IS6110 low-copy strains in concordance with the gold standard. The 2% discrepancy was caused by 1 isolate showing a faint restriction fragment length polymorphism (RFLP) pattern. The assay could provide specifies identification in 13...

  17. Tuberculosis:an experience from Mycobacterium smears and culture analysis

    Institute of Scientific and Technical Information of China (English)

    Zeehaida M; Siti Asma H; Siti Hawa H; Zaidah AR; Norbanee TH

    2009-01-01

    Objective:Simple tests like direct smear of the acid fast bacilli (AFB)and Mycobacterium culture could assist the diagnosis of tuberculosis.This study is aimed at reviewing the outcome of smears,culture results and con-tamination rate among specimens requested for AFB smear and Mycobacterium culture.Methods:Retrospec-tive laboratory data analysis requesting for Mycobacterium culture from January 2005 till December 2006 was done in a tertiary teaching hospital of Universiti Sains Malaysia,Kubang Kerian,Kelantan,Malaysia.Re-sults:Four hundred and sixty seven (36.6%)isolates grew from 1 277 specimens.Of these isolates,314 (67.2%)grew Mycobacterium tuberculosis,23 (4.9%)grew Mycobacterium other than tuberculosis and 38 (8.1%)grew contaminants.Among the M.tuberculosis cultures,165 (52.5%)had growth of more than 100 confluent colonies,whereas 39 cultures (12.4%)had growth of less than 19 colonies.Direct smear for AFB among smear positive cases showed presence of more than 50 bacilli /line in 231 (49.5%)cases and smear negative cases accounted for 63 (13.5%).Among smear positive cases,291 (94.5%)cultures grew Myco-bacterium species and another 17 (5.5%)cultures grew contaminants.In smear negative cases,32 (62.7%) cultures grew Mycobacterium species and 19 (37.3%)cultures grew contaminants.Conclusion:The results from data analysis of the Mycobacterium cultures should be critically utilized in order to review the laboratory performance and to improve its services in the future.Some of the data is also useful to the administrators of the hospital in terms of estimating the risk of occupational hazard faced by the health care workers.

  18. Phenotypic and genotypic analysis of multidrug-resistant tuberculosis in Ethiopia.

    NARCIS (Netherlands)

    Agonafir, M.; Lemma, E.; Wolde-Meskel, D.; Goshu, S.; Santhanam, A.; Girmachew, F.; Demissie, D.; Getahun, M.; Gebeyehu, M.; Soolingen, D. van

    2010-01-01

    SETTING: National Tuberculosis Reference Laboratory, Addis Ababa, Ethiopia. OBJECTIVES: To determine the drug susceptibility pattern of Mycobacterium tuberculosis isolates and to genetically characterise multidrug-resistant tuberculosis (MDR-TB) isolates. DESIGN: A total of 107 M. tuberculosis isola

  19. A Case of False-Positive Mycobacterium tuberculosis Caused by Mycobacterium celatum

    Directory of Open Access Journals (Sweden)

    Edward Gildeh

    2016-01-01

    Full Text Available Mycobacterium celatum is a nontuberculous mycobacterium shown to cause symptoms similar to pulmonary M. tuberculosis. Certain strains have been shown to cross-react with the probes used to detect M. tuberculosis, making this a diagnostic challenge. We present a 56-year-old gentleman who developed signs and symptoms of lung infection with computed tomography scan of the chest showing right lung apex cavitation. Serial sputum samples were positive for acid-fast bacilli and nucleic acid amplification testing identified M. tuberculosis ribosomal RNA, resulting in treatment initiation. Further testing with high performance liquid chromatography showed a pattern consistent with M. celatum. This case illustrates the potential for M. celatum to mimic M. tuberculosis in both its clinical history and laboratory testing due to the identical oligonucleotide sequence contained in both. An increasing number of case reports suggest that early reliable differentiation could reduce unnecessary treatment and public health intervention associated with misdiagnosed tuberculosis.

  20. Novel multiplex real-time PCR diagnostic assay for identification and differentiation of Mycobacterium tuberculosis, Mycobacterium canettii, and Mycobacterium tuberculosis complex strains.

    NARCIS (Netherlands)

    Reddington, K.; O'Grady, J.; Dorai-Raj, S.; Maher, M.; Soolingen, D. van; Barry, T.

    2011-01-01

    Tuberculosis (TB) in humans is caused by members of the Mycobacterium tuberculosis complex (MTC). Rapid detection of the MTC is necessary for the timely initiation of antibiotic treatment, while differentiation between members of the complex may be important to guide the appropriate antibiotic treat

  1. Sub-speciation of Mycobacterium tuberculosis complex from tuberculosis patients in Japan.

    Science.gov (United States)

    Ueyama, Masako; Chikamatsu, Kinuyo; Aono, Akio; Murase, Yoshiro; Kuse, Naoyuki; Morimoto, Kozo; Okumura, Masao; Yoshiyama, Takashi; Ogata, Hideo; Yoshimori, Kozo; Kudoh, Shoji; Azuma, Arata; Gemma, Akihiko; Mitarai, Satoshi

    2014-01-01

    Mycobacterium tuberculosis is the major causative agent of tuberculosis in humans. It is well known that Mycobacterium bovis and other species in the M. tuberculosis complex (MTC) can cause respiratory diseases as zoonosis. We analyzed the MTC isolates collected from tuberculosis patients from Japan in 2002 using a multiplex PCR system that detected cfp32, RD9 and RD12. A total of 970 MTC isolates that were representative of the tuberculosis cases throughout Japan, were examined using this method. As a result, 966 (99.6%) M. tuberculosis, two Mycobacterium africanum and two Mycobacterium canettii were identified using a multiplex PCR system, while no M. bovis was detected. Two isolates that lacked RD9 were initially considered to be M. canettii, but further analysis of the hsp65 sequence revealed them to be M. tuberculosis. Also two M. africanum were identified as M. tuberculosis using the -215 narG nucleotide polymorphism. Though PCR-linked methods have been used for a rapid differentiation of MTC and NTM, from our cases we suggest careful interpretation of RD based identification.

  2. Evaluation of susceptibility to antimycobacterial drugs in Mycobacterium tuberculosis complex strains isolated from cattle in Poland

    Directory of Open Access Journals (Sweden)

    Krajewska-Wędzina Monika

    2017-03-01

    Full Text Available Introduction: Tuberculosis is a highly infectious disease affecting humans and animals. It is caused by the Mycobacterium tuberculosis complex (MTBC – Mycobacterium bovis and Mycobacterium caprae, which are aetiological factors of bovine tuberculosis (bTB. In Poland, the bTB eradication programme exists. Animals diagnosed with tuberculosis are in the majority of cases not treated, but removed from their herd and then sanitary slaughtered.

  3. Siderocalin inhibits the intracellular replication of Mycobacterium tuberculosis in macrophages

    DEFF Research Database (Denmark)

    Johnson, Erin E; Srikanth, Chittur V; Sandgren, Andreas;

    2010-01-01

    Siderocalin is a secreted protein that binds to siderophores to prevent bacterial iron acquisition. While it has been shown to inhibit the growth of Mycobacterium tuberculosis (M.tb) in extracellular cultures, its effect on this pathogen within macrophages is not clear. Here, we show...... findings are consistent with an important role for siderocalin in protection against M.tb infection and suggest that exogenously administered siderocalin may have therapeutic applications in tuberculosis....

  4. Dramatic reduction of culture time of Mycobacterium tuberculosis

    Science.gov (United States)

    Ghodbane, Ramzi; Raoult, Didier; Drancourt, Michel

    2014-02-01

    Mycobacterium tuberculosis culture, a critical technique for routine diagnosis of tuberculosis, takes more than two weeks. Here, step-by-step improvements in the protocol including a new medium, microaerophlic atmosphere or ascorbic-acid supplement and autofluorescence detection dramatically shortened this delay. In the best case, primary culture and rifampicin susceptibility testing were achieved in 72 hours when specimens were inoculated directly on the medium supplemented by antibiotic at the beginning of the culture.

  5. Protective immunity against tuberculosis induced by vaccination with major extracellular proteins of Mycobacterium tuberculosis.

    OpenAIRE

    1995-01-01

    Tuberculosis, caused by the intracellular pathogen Mycobacterium tuberculosis, is the world's leading cause of death in humans from a single infectious agent. A safe and effective vaccine against this scourge is urgently needed. This study demonstrates that immunization with the 30-kDa major secretory protein, alone or in combination with other abundant extracellular proteins of M. tuberculosis, induces strong cell-mediated immune responses and substantial protective immunity against aerosol ...

  6. Whole Genome Sequencing Demonstrates Limited Transmission within Identified Mycobacterium tuberculosis Clusters in New South Wales, Australia

    Science.gov (United States)

    Gurjav, Ulziijargal; Outhred, Alexander C.; Jelfs, Peter; McCallum, Nadine; Wang, Qinning; Hill-Cawthorne, Grant A.; Marais, Ben J.; Sintchenko, Vitali

    2016-01-01

    Australia has a low tuberculosis incidence rate with most cases occurring among recent immigrants. Given suboptimal cluster resolution achieved with 24-locus mycobacterium interspersed repetitive unit (MIRU-24) genotyping, the added value of whole genome sequencing was explored. MIRU-24 profiles of all Mycobacterium tuberculosis culture-confirmed tuberculosis cases diagnosed between 2009 and 2013 in New South Wales (NSW), Australia, were examined and clusters identified. The relatedness of cases within the largest MIRU-24 clusters was assessed using whole genome sequencing and phylogenetic analyses. Of 1841 culture-confirmed TB cases, 91.9% (1692/1841) had complete demographic and genotyping data. East-African Indian (474; 28.0%) and Beijing (470; 27.8%) lineage strains predominated. The overall rate of MIRU-24 clustering was 20.1% (340/1692) and was highest among Beijing lineage strains (35.7%; 168/470). One Beijing and three East-African Indian (EAI) clonal complexes were responsible for the majority of observed clusters. Whole genome sequencing of the 4 largest clusters (30 isolates) demonstrated diverse single nucleotide polymorphisms (SNPs) within identified clusters. All sequenced EAI strains and 70% of Beijing lineage strains clustered by MIRU-24 typing demonstrated distinct SNP profiles. The superior resolution provided by whole genome sequencing demonstrated limited M. tuberculosis transmission within NSW, even within identified MIRU-24 clusters. Routine whole genome sequencing could provide valuable public health guidance in low burden settings. PMID:27737005

  7. Simple Multiplex PCR Assay for Identification of Beijing Family Mycobacterium tuberculosis Isolates with a Lineage-Specific Mutation in Rv0679c

    OpenAIRE

    2013-01-01

    The Beijing genotype of Mycobacterium tuberculosis is known to be a worldwide epidemic clade. It is suggested to be a possibly resistant clone against BCG vaccination and is also suggested to be highly pathogenic and prone to becoming drug resistant. Thus, monitoring the prevalence of this lineage seems to be important for the proper control of tuberculosis. The Rv0679c protein of M. tuberculosis has been predicted to be one of the outer membrane proteins and is suggested to contribute to hos...

  8. Structural annotation of Mycobacterium tuberculosis proteome.

    Directory of Open Access Journals (Sweden)

    Praveen Anand

    Full Text Available Of the ∼4000 ORFs identified through the genome sequence of Mycobacterium tuberculosis (TB H37Rv, experimentally determined structures are available for 312. Since knowledge of protein structures is essential to obtain a high-resolution understanding of the underlying biology, we seek to obtain a structural annotation for the genome, using computational methods. Structural models were obtained and validated for ∼2877 ORFs, covering ∼70% of the genome. Functional annotation of each protein was based on fold-based functional assignments and a novel binding site based ligand association. New algorithms for binding site detection and genome scale binding site comparison at the structural level, recently reported from the laboratory, were utilized. Besides these, the annotation covers detection of various sequence and sub-structural motifs and quaternary structure predictions based on the corresponding templates. The study provides an opportunity to obtain a global perspective of the fold distribution in the genome. The annotation indicates that cellular metabolism can be achieved with only 219 folds. New insights about the folds that predominate in the genome, as well as the fold-combinations that make up multi-domain proteins are also obtained. 1728 binding pockets have been associated with ligands through binding site identification and sub-structure similarity analyses. The resource (http://proline.physics.iisc.ernet.in/Tbstructuralannotation, being one of the first to be based on structure-derived functional annotations at a genome scale, is expected to be useful for better understanding of TB and for application in drug discovery. The reported annotation pipeline is fairly generic and can be applied to other genomes as well.

  9. Preventing Transmission of Mycobacterium tuberculosis in Health Care Settings.

    Science.gov (United States)

    Punjabi, Chitra D; Perloff, Sarah R; Zuckerman, Jerry M

    2016-12-01

    Patients with tuberculosis (TB) pose a risk to other patients and health care workers, and outbreaks in health care settings occur when appropriate infection control measures are not used. In this article, we discuss strategies to prevent transmission of Mycobacterium tuberculosis within health care settings. All health care facilities should have an operational TB infection control plan that emphasizes the use of a hierarchy of controls (administrative, environmental, and personal respiratory protection). We also discuss resources available to clinicians who work in the prevention and investigation of nosocomial transmission of M tuberculosis. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Deciphering the biology of Mycobacterium tuberculosis from thecomplete genome sequence

    DEFF Research Database (Denmark)

    Cole, S.T.; Krogh, Anders Stærmose

    1998-01-01

    Countless millions of people have died from tuberculosis, a chronic infectious disease caused by the tubercle bacillus. The complete genome sequence of the best-characterized strain of Mycobacterium tuberculosis, H37Rv, has been determined and analysed in order to improve our understanding....... tuberculosis differs radically from other bacteria in that a very large portion of its coding capacity is devoted to the production of enzymes involved in lipogenesis and lipolysis, and to two new families of glycine-rich proteins with a repetitive structure that may represent a source of antigenic variation....

  11. A strip array for spoligotyping of Mycobacterium tuberculosis complex isolates.

    Science.gov (United States)

    Tu, Yiling; Zeng, Xiaohong; Li, Hui; Zheng, Rongrong; Xu, Ye; Li, Qingge

    2016-03-01

    A novel strip array was developed for a nine-spacer spoligotyping scheme of Mycobacterium tuberculosis complex (MTBC). The new method was evaluated using 211 MTBC isolates and the results were fully concordant with the traditional spoligotyping approach. The strip array proved to be rapid and convenient for spoligotyping of MTBC.

  12. Mechanism of phagolysosome biogenesis block by viable Mycobacterium tuberculosis

    OpenAIRE

    Vergne, Isabelle; Chua, Jennifer; Lee, Hwang-Ho; Lucas, Megan; Belisle, John; Deretic, Vojo

    2005-01-01

    Live Mycobacterium tuberculosis persists in macrophage phagosomes by interfering with phagolysosome biogenesis. Here, using four-dimensional microscopy and in vitro assays, we report the principal difference between phagosomes containing live and dead mycobacteria. Phosphatidylinositol 3-phosphate (PI3P), a membrane trafficking regulatory lipid essential for phagosomal acquisition of lysosomal constituents, is retained on phagosomes harboring dead mycobacteria but is continuously eliminated f...

  13. Autophagy modulates the Mycobacterium tuberculosis-induced cytokine response

    NARCIS (Netherlands)

    Kleinnijenhuis, J.; Oosting, M.; Plantinga, T.S.; Meer, J.W.M. van der; Joosten, L.A.B.; Crevel, R. van; Netea, M.G.

    2011-01-01

    Both autophagy and pro-inflammatory cytokines are involved in the host defence against mycobacteria, but little is known regarding the effect of autophagy on Mycobacterium tuberculosis (MTB)-induced cytokine production. In the present study, we assessed the effect of autophagy on production of monoc

  14. The draft genome of Mycobacterium aurum, a potential model organism for investigating drugs against Mycobacterium tuberculosis and Mycobacterium leprae

    KAUST Repository

    Phelan, Jody

    2015-06-04

    Mycobacterium aurum (M. aurum) is an environmental mycobacteria that has previously been used in studies of anti-mycobacterial drugs due to its fast growth rate and low pathogenicity. The M. aurum genome has been sequenced and assembled into 46 contigs, with a total length of 6.02 Mb containing 5684 annotated protein-coding genes. A phylogenetic analysis using whole genome alignments positioned M. aurum close to Mycobacterium vaccae and Mycobacterium vanbaalenii, within a clade related to fast-growing mycobacteria. Large-scale genomic rearrangements were identified by comparing the M. aurum genome to those of Mycobacterium tuberculosis and Mycobacterium leprae. M. aurum orthologous genes implicated in resistance to anti-tuberculosis drugs in M. tuberculosis were observed. The sequence identity at the DNA level varied from 68.6% for pncA (pyrazinamide drug-related) to 96.2% for rrs (streptomycin, capreomycin). We observed two homologous genes encoding the catalase-peroxidase enzyme (katG) that is associated with resistance to isoniazid. Similarly, two embB homologues were identified in the M. aurum genome. In addition to describing for the first time the genome of M. aurum, this work provides a resource to aid the use of M. aurum in studies to develop improved drugs for the pathogenic mycobacteria M. tuberculosis and M. leprae.

  15. Antibacterial Activity of Medicinal Aqueous Plant Extracts against Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Muna Mohammed Buzayan

    2012-09-01

    Full Text Available Tuberculosis (TB remains a serious health problem in many regions of the world, and the development of resistance to antibiotics by this microbe created the need for new drugs to replace those which have lost effectiveness. This study assesses the medicinal anti-Mycobacterium tuberculosis properties of natural products obtained from plants collected from Eastern Libya. In this study aqueous extracts of nine different plants were assayed for their Mycobacterium tuberculosis inhibitory activity using the BACTEC MGIT960 susceptibility test method. The aqueous extracts of Ceratonia siliqua L, Helichrysum stoechas (L. Moench and Thymus algeriensis did not show any activity against M. tuberculosis in different concentrations. The aqueous extract of Marrubium vulgare L. from Syria showed high activity against M. tuberculosis. Marrubium alysson L., Marrubium vulgare L., Pistacia lentiscus L, Quercus coccifera L, Thymus capitatus (L. Hoffm. & Link, showed varying degrees of activity against M. tuberculosis. The results of this study show that aqueous extracts from six different medicinal plants have different effects against M. tuberculosis in vitro.

  16. Mycobacterium tuberculosis drug-resistance testing: challenges, recent developments and perspectives.

    Science.gov (United States)

    Schön, T; Miotto, P; Köser, C U; Viveiros, M; Böttger, E; Cambau, E

    2017-03-01

    Drug-resistance testing, or antimicrobial susceptibility testing (AST), is mandatory for Mycobacterium tuberculosis in cases of failure on standard therapy. We reviewed the different methods and techniques of phenotypic and genotypic approaches. Although multiresistant and extensively drug-resistant (MDR/XDR) tuberculosis is present worldwide, AST for M. tuberculosis (AST-MTB) is still mainly performed according to the resources available rather than the drug-resistance rates. Phenotypic methods, i.e. culture-based AST, are commonly used in high-income countries to confirm susceptibility of new cases of tuberculosis. They are also used to detect resistance in tuberculosis cases with risk factors, in combination with genotypic tests. In low-income countries, genotypic methods screening hot-spot mutations known to confer resistance were found to be easier to perform because they avoid the culture and biosafety constraint. Given that genotypic tests can rapidly detect the prominent mechanisms of resistance, such as the rpoB mutation for rifampicin resistance, we are facing new challenges with the observation of false-resistance (mutations not conferring resistance) and false-susceptibility (mutations different from the common mechanism) results. Phenotypic and genotypic approaches are therefore complementary for obtaining a high sensitivity and specificity for detecting drug resistances and susceptibilities to accurately predict MDR/XDR cure and to gather relevant data for resistance surveillance. Although AST-MTB was established in the 1960s, there is no consensus reference method for MIC determination against which the numerous AST-MTB techniques can be compared. This information is necessary for assessing in vitro activity and setting breakpoints for future anti-tuberculosis agents. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  17. High Throughput Phenotypic Analysis of Mycobacterium tuberculosis and Mycobacterium bovis Strains' Metabolism Using Biolog Phenotype Microarrays

    OpenAIRE

    Khatri, Bhagwati; Fielder, Mark; Jones, Gareth; Newell, William; Abu-Oun, Manal; Wheeler, Paul R.

    2013-01-01

    Tuberculosis is a major human and animal disease of major importance worldwide. Genetically, the closely related strains within the Mycobacterium tuberculosis complex which cause disease are well-characterized but there is an urgent need better to understand their phenotypes. To search rapidly for metabolic differences, a working method using Biolog Phenotype MicroArray analysis was developed. Of 380 substrates surveyed, 71 permitted tetrazolium dye reduction, the readout over 7 days in the m...

  18. Insights into redox sensing metalloproteins in Mycobacterium tuberculosis.

    Science.gov (United States)

    Chim, Nicholas; Johnson, Parker M; Goulding, Celia W

    2014-04-01

    Mycobacterium tuberculosis, the pathogen that causes tuberculosis, has evolved sophisticated mechanisms for evading assault by the human host. This review focuses on M. tuberculosis regulatory metalloproteins that are sensitive to exogenous stresses attributed to changes in the levels of gaseous molecules (i.e., molecular oxygen, carbon monoxide and nitric oxide) to elicit an intracellular response. In particular, we highlight recent developments on the subfamily of Whi proteins, redox sensing WhiB-like proteins that contain iron-sulfur clusters, sigma factors and their cognate anti-sigma factors of which some are zinc-regulated, and the dormancy survival regulon DosS/DosT-DosR heme sensory system. Mounting experimental evidence suggests that these systems contribute to a highly complex and interrelated regulatory network that controls M. tuberculosis biology. This review concludes with a discussion of strategies that M. tuberculosis has developed to maintain redox homeostasis, including mechanisms to regulate endogenous nitric oxide and carbon monoxide levels.

  19. Comparative genomics of archived pyrazinamide resistant Mycobacterium tuberculosis complex isolates from Uganda

    Science.gov (United States)

    Bovine tuberculosis is a ‘neglected zoonosis’ and its contribution to the proportion of Mycobacterium tuberculosis complex infections in humans is unknown. A retrospective study on archived Mycobacterium tuberculosis complex (MTC) isolates from a reference laboratory in Uganda was undertaken to iden...

  20. Siderocalin inhibits the intracellular replication of Mycobacterium tuberculosis in macrophages

    DEFF Research Database (Denmark)

    Johnson, Erin E; Srikanth, Chittur V; Sandgren, Andreas

    2010-01-01

    Siderocalin is a secreted protein that binds to siderophores to prevent bacterial iron acquisition. While it has been shown to inhibit the growth of Mycobacterium tuberculosis (M.tb) in extracellular cultures, its effect on this pathogen within macrophages is not clear. Here, we show that sideroc......Siderocalin is a secreted protein that binds to siderophores to prevent bacterial iron acquisition. While it has been shown to inhibit the growth of Mycobacterium tuberculosis (M.tb) in extracellular cultures, its effect on this pathogen within macrophages is not clear. Here, we show...... findings are consistent with an important role for siderocalin in protection against M.tb infection and suggest that exogenously administered siderocalin may have therapeutic applications in tuberculosis....

  1. Molecular characterisation of isoniazid- and rifampicin-resistant Mycobacterium tuberculosis in Central Tunisia.

    Science.gov (United States)

    Ben Kahla, I; Marzouk, M; Henry, M; Bedotto, M; Cohen-Bacrie, S; Ben Selma, W; Boukadida, J; Drancourt, M

    2011-12-01

    The aim of our study was to genotypically characterise isoniazid (INH) and rifampicin (RMP) resistant Mycobacterium tuberculosis isolates in Sousse, Central Tunisia, using DNA sequencing and multispacer sequence typing (MST). The results show that 27/28 (96.4%) and 1/28 (3.6%) INH-resistant isolates yielded respectively the kat G S315T and the inh A - 15C → T mutations. Two-thirds of RMP-resistant isolates yielded the rpo B D516V mutation and one sixth yielded either H526D or S531L mutations. Genotyping analysis revealed the multiclonal spread of drug-resistant isolates in Central Tunisia. Data presented here complete the previously published map of resistant M. tuberculosis isolates and highlight their regional disparity in Tunisia.

  2. Rapid diagnosis of Mycobacterium tuberculosis bacteremia by PCR.

    Science.gov (United States)

    Folgueira, L; Delgado, R; Palenque, E; Aguado, J M; Noriega, A R

    1996-01-01

    A method based on DNA amplification and hybridization has been used for the rapid detection of Mycobacterium tuberculosis in blood samples from 38 hospitalized patients (15 human immunodeficiency virus [HIV] positive and 23 HIV negative) in whom localized or disseminated forms of tuberculosis were suspected. In 32 of these patients, the diagnosis of tuberculosis was eventually confirmed by conventional bacteriological or histological procedures. M. tuberculosis DNA was detected with the PCR technique in the peripheral blood mononuclear cells from 9 of 11 (82%) HIV-infected patients and in 7 of 21 (33%) HIV-negative patients (P < 0.01), while M. tuberculosis blood cultures were positive in 1 of 8 (12.5%) and 1 of 18 (5.5%) patients, respectively. PCR was positive in all cases with disseminated disease in both HIV-negative and HIV-positive patients and also in the HIV-positive patients with extrapulmonary tuberculosis. Seven samples from patients with documented illness other than tuberculosis and 12 specimens from healthy volunteers, including seven volunteers with a recent positive purified protein derivative test, were used as controls and had a negative PCR. These results suggest that detection of M. tuberculosis DNA in peripheral blood mononuclear cells may be a useful tool for rapid diagnosis of disseminated and extrapulmonary forms of tuberculosis, especially in an HIV-positive population. PMID:8904404

  3. Mycobacterium tuberculosis resistance to antituberculosis drugs in Mozambique.

    Science.gov (United States)

    Pires, Germano Manuel; Folgosa, Elena; Nquobile, Ndlovu; Gitta, Sheba; Cadir, Nureisha

    2014-01-01

    To determine the drug resistance profile of Mycobacterium tuberculosis in Mozambique. We analyzed secondary data from the National Tuberculosis Referral Laboratory, in the city of Maputo, Mozambique, and from the Beira Regional Tuberculosis Referral Laboratory, in the city of Beira, Mozambique. The data were based on culture-positive samples submitted to first-line drug susceptibility testing (DST) between January and December of 2011. We attempted to determine whether the frequency of DST positivity was associated with patient type or provenance. During the study period, 641 strains were isolated in culture and submitted to DST. We found that 374 (58.3%) were resistant to at least one antituberculosis drug and 280 (43.7%) were resistant to multiple antituberculosis drugs. Of the 280 multidrug-resistant tuberculosis cases, 184 (65.7%) were in previously treated patients, most of whom were from southern Mozambique. Two (0.71%) of the cases of multidrug-resistant tuberculosis were confirmed to be cases of extensively drug-resistant tuberculosis. Multidrug-resistant tuberculosis was most common in males, particularly those in the 21-40 year age bracket. M. tuberculosis resistance to antituberculosis drugs is high in Mozambique, especially in previously treated patients. The frequency of M. tuberculosis strains that were resistant to isoniazid, rifampin, and streptomycin in combination was found to be high, particularly in samples from previously treated patients.

  4. Rapid diagnosis of Mycobacterium tuberculosis bacteremia by PCR.

    Science.gov (United States)

    Folgueira, L; Delgado, R; Palenque, E; Aguado, J M; Noriega, A R

    1996-03-01

    A method based on DNA amplification and hybridization has been used for the rapid detection of Mycobacterium tuberculosis in blood samples from 38 hospitalized patients (15 human immunodeficiency virus [HIV] positive and 23 HIV negative) in whom localized or disseminated forms of tuberculosis were suspected. In 32 of these patients, the diagnosis of tuberculosis was eventually confirmed by conventional bacteriological or histological procedures. M. tuberculosis DNA was detected with the PCR technique in the peripheral blood mononuclear cells from 9 of 11 (82%) HIV-infected patients and in 7 of 21 (33%) HIV-negative patients (P < 0.01), while M. tuberculosis blood cultures were positive in 1 of 8 (12.5%) and 1 of 18 (5.5%) patients, respectively. PCR was positive in all cases with disseminated disease in both HIV-negative and HIV-positive patients and also in the HIV-positive patients with extrapulmonary tuberculosis. Seven samples from patients with documented illness other than tuberculosis and 12 specimens from healthy volunteers, including seven volunteers with a recent positive purified protein derivative test, were used as controls and had a negative PCR. These results suggest that detection of M. tuberculosis DNA in peripheral blood mononuclear cells may be a useful tool for rapid diagnosis of disseminated and extrapulmonary forms of tuberculosis, especially in an HIV-positive population.

  5. Genotyping and drug resistance patterns of M. tuberculosis strains in Pakistan

    Directory of Open Access Journals (Sweden)

    Kanji Akbar

    2008-12-01

    Full Text Available Abstract Background The incidence of tuberculosis in Pakistan is 181/100,000 population. However, information about transmission and geographical prevalence of Mycobacterium tuberculosis strains and their evolutionary genetics as well as drug resistance remains limited. Our objective was to determine the clonal composition, evolutionary genetics and drug resistance of M. tuberculosis isolates from different regions of the country. Methods M. tuberculosis strains isolated (2003–2005 from specimens submitted to the laboratory through collection units nationwide were included. Drug susceptibility was performed and strains were spoligotyped. Results Of 926 M. tuberculosis strains studied, 721(78% were grouped into 59 "shared types", while 205 (22% were identified as "Orphan" spoligotypes. Amongst the predominant genotypes 61% were Central Asian strains (CAS ; including CAS1, CAS sub-families and Orphan Pak clusters, 4% East African-Indian (EAI, 3% Beijing, 2% poorly defined TB strains (T, 2% Haarlem and LAM (0.2. Also TbD1 analysis (M. tuberculosis specific deletion 1 confirmed that CAS1 was of "modern" origin while EAI isolates belonged to "ancestral" strain types. Prevalence of CAS1 clade was significantly higher in Punjab (P Conclusion Our results show variation of prevalent M. tuberculosis strain with greater association of CAS1 with the Punjab province. The fact that the prevalent CAS genotype was not associated with drug resistance is encouraging. It further suggests a more effective treatment and control programme should be successful in reducing the tuberculosis burden in Pakistan.

  6. Extrapulmonary tuberculosis: Mycobacterium tuberculosis strains and host risk factors in a large urban setting in Brazil.

    Directory of Open Access Journals (Sweden)

    Teresa Gomes

    Full Text Available BACKGROUND: Factors related to the development of extrapulmonary forms of tuberculosis (EPTB are still poorly understood, particularly in high-endemic countries like Brazil. The objective of the paper is to determine host and Mycobacterium tuberculosis (MTB strain-related factors associated with the development of EPTB in Espírito Santo state, Brazil. METHODS AND FINDINGS: We conducted a retrospective laboratory-based surveillance study of new tuberculosis (TB cases diagnosed in Espírito Santo state, Brazil between 1998 and 2007. We genotyped 612 isolates of MTB from 606 TB patients using spoligotyping and IS6110-restriction fragment length polymorphism (RFLP typing and compared sociodemographic and clinical characteristics of patients with pulmonary TB (PTB and EPTB. Among 606 patients, 464 (77% had PTB, 79 (13% had EPTB, 51 (8% had both, and 12 (2% had miliary TB. The IS6110 RFLP analysis demonstrated that 250 (41% isolates belonged to clustered RFLP patterns, 27 (11% of which were from EPTB. We identified 73 clusters including 35 (48% composed of 2 isolates each. By spoligotyping, 506 (83% MTB isolates fell into known patterns and 106 (17% fell into patterns with no family assignment; 297 (48% isolates belonged to the Latin-American Mediterranean family. Higher school level (4-7 years OR: 0.16 95% CI 0.34-0.73 and > 8 years of education, OR 0.06 95% CI 0.009-0.50 white ethnicity (OR: 2.54 95% CI 1.03-6.25 and HIV infection (OR: 16.83 95% CI 5.23-54.18 were associated with EPTB. No specific strain lineage or percentage of clustering was associated with EPTB. CONCLUSIONS: These results demonstrate that risk factors for EPTB are related more to host than to MTB strain lineage characteristics.

  7. Evaluation of DNA microarray for detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis isolates

    Institute of Scientific and Technical Information of China (English)

    王峰

    2013-01-01

    Objective To evaluate the performance of DNA microarray for rapid detection resistance to rifampin and isoniazid in Mycobacterium tuberculosis clinical isolates and identify suitable target sites for molecular genetic test. Methods Twenty-four clinical Mycobacterium

  8. Real-time molecular epidemiology of tuberculosis by direct genotyping of smear-positive clinical specimens.

    Science.gov (United States)

    Alonso, María; Herranz, Marta; Martínez Lirola, Miguel; González-Rivera, Milagros; Bouza, Emilio; García de Viedma, Darío

    2012-05-01

    We applied MIRU-VNTR (mycobacterial interspersed repetitive-unit-variable-number tandem-repeat typing) to directly analyze the bacilli present in 61 stain-positive specimens from tuberculosis patients. A complete MIRU type (24 loci) was obtained for all but one (no amplification in one locus) of the specimens (98.4%), and the allelic values fully correlated with those obtained from the corresponding cultures. Our study is the first to demonstrate that real-time genotyping of Mycobacterium tuberculosis can be achieved, fully transforming the way in which molecular epidemiology techniques can be integrated into control programs.

  9. First insights into circulating Mycobacterium tuberculosis complex lineages and drug resistance in Guinea.

    Science.gov (United States)

    Ejo, Mebrat; Gehre, Florian; Barry, Mamadou Dian; Sow, Oumou; Bah, Nene Mamata; Camara, Mory; Bah, Boubacar; Uwizeye, Cecile; Nduwamahoro, Elie; Fissette, Kristina; De Rijk, Pim; Merle, Corinne; Olliaro, Piero; Burgos, Marcos; Lienhardt, Christian; Rigouts, Leen; de Jong, Bouke C

    2015-07-01

    In this study we assessed first-line anti-tuberculosis drug resistance and the genotypic distribution of Mycobacterium tuberculosis complex (MTBC) isolates that had been collected from consecutive new tuberculosis patients enrolled in two clinical trials conducted in Guinea between 2005 and 2010. Among the total 359 MTBC strains that were analyzed in this study, 22.8% were resistant to at least one of the first line anti-tuberculosis drugs, including 2.5% multidrug resistance and 17.5% isoniazid resistance, with or without other drugs. In addition, further characterization of isolates from a subset of the two trials (n = 184) revealed a total of 80 different spoligotype patterns, 29 "orphan" and 51 shared patterns. We identified the six major MTBC lineages of human relevance, with predominance of the Euro-American lineage. In total, 132 (71.7%) of the strains were genotypically clustered, and further analysis (using the DESTUS model) suggesting significantly faster spread of LAM10_CAM family (p = 0.00016). In conclusion, our findings provide a first insight into drug resistance and the population structure of the MTBC in Guinea, with relevance for public health scientists in tuberculosis control programs.

  10. Rapid Detection of Rifampin-resistant Clinical Isolates of Mycobacterium tuberculosis by Reverse Dot Blot Hybridization

    Institute of Scientific and Technical Information of China (English)

    GUO Qian; WAN Kang Lin; YU Yan; ZHU Yan Ling; ZHAO Xiu Qin; LIU Zhi Guang; ZHANG Yuan Yuan; LI Gui Lian; WEI Jian Hao; WU Yi Mou

    2015-01-01

    Objective A PCR-reverse dot blot hybridization (RDBH) assay was developed for rapid detection of rpoB gene mutations in‘hot mutation region’ of Mycobacterium tuberculosis (M. tuberculosis). Methods 12 oligonucleotide probes based on the wild-type and mutant genotype rpoB sequences of M. tuberculosis were designed to screen the most frequent wild-type and mutant genotypes for diagnosing RIF resistance. 300 M. tuberculosis clinical isolates were detected by RDBH, conventional drug-susceptibility testing (DST) and DNA sequencing to evaluate the RDBH assay. Results The sensitivity and specificity of the RDBH assay were 91.2%(165/181) and 98.3%(117/119), respectively, as compared to DST. When compared with DNA sequencing, the accuracy, positive predictive value (PPV) and negative predictive value (NPV) of the RDBH assay were 97.7%(293/300), 98.2%(164/167), and 97.0%(129/133), respectively. Furthermore, the results indicated that the most common mutations were in codons 531 (48.6%), 526 (25.4%), 516 (8.8%), and 511 (6.6%), and the combinative mutation rate was 15 (8.3%). One and two strains of insertion and deletion were found among all strains, respectively. Conclusion Our findings demonstrate that the RDBH assay is a rapid, simple and sensitive method for diagnosing RIF-resistant tuberculosis.

  11. Modeling Synergistic Drug Inhibition of Mycobacterium tuberculosis Growth in Murine Macrophages

    Science.gov (United States)

    2011-01-01

    synergistic drug inhibition of Mycobacterium tuberculosis growth in murine macrophagesw Xin Fang, Anders Wallqvist and Jaques Reifman* Received 15th...inhibition of Mycobacterium tuberculosis in murine macrophage cells. We used it to simulate ex vivo bacterial growth inhibition due to 3-nitropropionate (3...is felt worldwide, with 9.4 million new cases and 1.8 million deaths in 2008.1,2 The causative agent of the disease, Mycobacterium tuberculosis

  12. Determination of Urinary Neopterin/Creatinine Ratio to Distinguish Active Tuberculosis from Latent Mycobacterium tuberculosis Infection

    Directory of Open Access Journals (Sweden)

    Michael Eisenhut

    2016-01-01

    Full Text Available Background. Biomarkers to distinguish latent from active Mycobacterium (M. tuberculosis infection in clinical practice are lacking. The urinary neopterin/creatinine ratio can quantify the systemic interferon-gamma effect in patients with M. tuberculosis infection. Methods. In a prospective observational study, urinary neopterin levels were measured by enzyme linked immunosorbent assay in patients with active tuberculosis, in people with latent M. tuberculosis infection, and in healthy controls and the urinary neopterin/creatinine ratio was calculated. Results. We included a total of 44 patients with M. tuberculosis infection and nine controls. 12 patients had active tuberculosis (8 of them culture-confirmed. The median age was 15 years (range 4.5 to 49. Median urinary neopterin/creatinine ratio in patients with active tuberculosis was 374.1 micromol/mol (129.0 to 1072.3, in patients with latent M. tuberculosis infection it was 142.1 (28.0 to 384.1, and in controls it was 146.0 (40.3 to 200.0, with significantly higher levels in patients with active tuberculosis (p<0.01. The receiver operating characteristics curve had an area under the curve of 0.84 (95% CI 0.70 to 0.97 (p<0.01. Conclusions. Urinary neopterin/creatinine ratios are significantly higher in patients with active tuberculosis compared to patients with latent infection and may be a significant predictor of active tuberculosis in patients with M. tuberculosis infection.

  13. Phospholipase C in Beijing strains of Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    N Faramarzi

    2010-12-01

    Full Text Available Background and Objectives: Phospholipase of Mycobacterium tuberculosis plays an important role in pathogenesis through breaking up phospholipids and production of diacylglycerol. In this study, we examined the Beijing strains of Mycobacterium tuberculosis isolated from Iranian patients for the genes encoding this enzyme."nMaterials and Methods: DNA extraction was performed using CTAB (cetyltrimethylammonium bromide from positive culture specimens in tuberculosis patients. PCR was then used to amplify the plcA, plcB, plcC genes of Beijing strain, and non-Beijing strains were identified by spoligotyping."nResults: Of 200 specimens, 19 (9.5% were Beijing strain and 181 (90.5% were non-Beijing strains. The results of PCR for Beijing strains were as follows: 16 strains (84.2% were positive for plcA, 17 (89.4% were positive for plcB and 17 (89.4% were positive for plcC genes. The standard strain (H37RV was used as control."nConclusion: The majority of Beijing strains have phospholipase C genes which can contribute to their pathogenesis but we need complementary studies to confirm the role of phospholipase C in pathogenecity of Mycobacterium tuberculosis.

  14. Genetic diversity, transmission dynamics and drug resistance of Mycobacterium tuberculosis in Angola

    Science.gov (United States)

    Perdigão, João; Clemente, Sofia; Ramos, Jorge; Masakidi, Pedro; Machado, Diana; Silva, Carla; Couto, Isabel; Viveiros, Miguel; Taveira, Nuno; Portugal, Isabel

    2017-01-01

    Tuberculosis (TB) poses a serious public health problem in Angola. No surveillance data on drug resistance is available and nothing is known regarding the genetic diversity and population structure of circulating Mycobacterium tuberculosis strains. Here, we have genotyped and evaluated drug susceptibility of 89 Mycobacterium tuberculosis clinical isolates from Luanda. Thirty-three different spoligotype profiles corresponding to 24 different Shared International Types (SIT) and 9 orphan profiles were detected. SIT 20 (LAM1) was the most prevalent (n = 16, 18.2%) followed by SIT 42 (LAM9; n = 15, 17.1%). Overall, the M. tuberculosis population structure in this sample was dominated by LAM (64.8%) and T (33.0%) strains. Twenty-four-loci MIRU-VNTR analysis revealed that a total of 13 isolates were grouped in 5 distinct clusters. Drug susceptibility data showed that 22 (24.7%) of the 89 clinical isolates were resistant to one or more antibacillary drugs of which 4 (4.5%) were multidrug resistant. In conclusion, this study demonstrates a high predominance of LAM strains circulating in the Luanda setting and the presence of recent transmission events. The rate and the emergence dynamics of drug resistant TB found in this sample are significant and highlight the need of further studies specifically focused on MDR-TB transmission. PMID:28230095

  15. Asymmetric cell division in Mycobacterium tuberculosis and its unique features.

    Science.gov (United States)

    Vijay, Srinivasan; Nagaraja, Mukkayyan; Sebastian, Jees; Ajitkumar, Parthasarathi

    2014-03-01

    Recently, several reports showed that about 80 % of mid-log phase Mycobacterium smegmatis, Mycobacterium marinum, and Mycobacterium bovis BCG cells divide symmetrically with 5-10 % deviation in the septum position from the median. However, the mode of cell division of the pathogenic mycobacterial species, Mycobacterium tuberculosis, remained unclear. Therefore, in the present study, using electron microscopy, fluorescence microscopy of septum- and nucleoid-stained live and fixed cells, and live cell time-lapse imaging, we show the occurrence of asymmetric cell division with unusually deviated septum/constriction in 20 % of the 15 % septating M. tuberculosis cells in the mid-log phase population. The remaining 80 % of the 15 % septating cells divided symmetrically but with 2-5 % deviation in the septum/constriction position, as reported for M. smegmatis, M. marinum, and M. bovis BCG cells. Both the long and the short portions of the asymmetrically dividing M. tuberculosis cells with unusually deviated septum contained nucleoids, thereby generating viable short and long cells from each asymmetric division. M. tuberculosis short cells were acid fast positive and, like the long cells, further readily underwent growth and division to generate micro-colony, thereby showing that they were neither mini cells, spores nor dormant forms of mycobacteria. The freshly diagnosed pulmonary tuberculosis patients' sputum samples, which are known for the prevalence of oxidative stress conditions, also contained short cells at the same proportion as that in the mid-log phase population. The probable physiological significance of the generation of the short cells through unusually deviated asymmetric cell division is discussed.

  16. Pyrosequencing assay for rapid identification of Mycobacterium tuberculosis complex species

    Directory of Open Access Journals (Sweden)

    Boukadida Jalel

    2011-10-01

    Full Text Available Abstract Background Identification of the Mycobacterium tuberculosis complex organisms to the species level is important for diagnostic, therapeutic and epidemiologic perspectives. Indeed, isolates are routinely identified as belonging to the M. tuberculosis complex without further discrimination in agreement with the high genomic similarity of the M. tuberculosis complex members and the resulting complex available identification tools. Findings We herein develop a pyrosequencing assay analyzing polymorphisms within glpK, pykA and gyrB genes to identify members of the M. tuberculosis complex at the species level. The assay was evaluated with 22 M. tuberculosis, 21 M. bovis, 3 M. caprae, 3 M. microti, 2 M. bovis BCG, 2 M. pinnipedii, 1 M. canettii and 1 M. africanum type I isolates. The resulted pyrograms were consistent with conventional DNA sequencing data and successfully identified all isolates. Additionally, 127 clinical M. tuberculosis complex isolates were analyzed and were unambiguously identified as M. tuberculosis. Conclusion We proposed a pyrosequencing-based scheme for the rapid identification of M. tuberculosis complex isolates at the species level. The assay is robust, specific, rapid and can be easily introduced in the routine activity.

  17. Proteogenomic Investigation of Strain Variation in Clinical Mycobacterium tuberculosis Isolates

    KAUST Repository

    Heunis, Tiaan

    2017-08-18

    Mycobacterium tuberculosis consists of a large number of different strains that display unique virulence characteristics. Whole-genome sequencing has revealed substantial genetic diversity among clinical M. tuberculosis isolates, and elucidating the phenotypic variation encoded by this genetic diversity will be of utmost importance to fully understand M. tuberculosis biology and pathogenicity. In this study we integrated whole-genome sequencing and mass spectrometry (GeLC-MS/MS) to reveal strain-specific characteristics in the proteomes of two clinical M. tuberculosis Latin American-Mediterranean isolates. Using this approach we identified 59 peptides containing single amino acid variants, which covered ~9% of all total coding nonsynonymous single nucleotide variants detected by whole-genome sequencing. Furthermore, we identified 29 distinct peptides that mapped to a hypothetical protein not present in the M. tuberculosis H37Rv reference proteome. Here we provide evidence for the expression of this protein in the clinical M. tuberculosis SAWC3651 isolate. The strain-specific databases enabled confirmation of genomic differences (i.e. large genomic regions of difference and nonsynonymous single nucleotide variants) in these two clinical M. tuberculosis isolates and allowed strain differentiation at the proteome level. Our results contribute to the growing field of clinical microbial proteogenomics and can improve our understanding of phenotypic variation in clinical M. tuberculosis isolates.

  18. Molecular characteristics of "Mycobacterium canettii" the smooth Mycobacterium tuberculosis bacilli.

    NARCIS (Netherlands)

    Fabre, M.; Hauck, Y.; Soler, C.; Koeck, J.L.; Ingen, J. van; Soolingen, D. van; Vergnaud, G.; Pourcel, C.

    2010-01-01

    Since the first discovery of the smooth tubercle (SmTB) bacilli "Mycobacterium canettii" less than 60 isolates have been reported, all but one originating from a limited geographical location, the Horn of Africa. In spite of its rarity, the SmTB lineage deserves special attention. Previous investiga

  19. Tuberculosis patients co-infected with Mycobacterium bovis and Mycobacterium tuberculosis in an urban area of Brazil

    Directory of Open Access Journals (Sweden)

    Marcio Roberto Silva

    2013-05-01

    Full Text Available In this cross-sectional study, mycobacteria specimens from 189 tuberculosis (TB patients living in an urban area in Brazil were characterised from 2008-2010 using phenotypic and molecular speciation methods (pncA gene and oxyR pseudogene analysis. Of these samples, 174 isolates simultaneously grew on Löwenstein-Jensen (LJ and Stonebrink (SB-containing media and presented phenotypic and molecular profiles of Mycobacterium tuberculosis, whereas 12 had molecular profiles of M. tuberculosis based on the DNA analysis of formalin-fixed paraffin wax-embedded tissue samples (paraffin blocks. One patient produced two sputum isolates, the first of which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, and the second of which only grew on SB media and presented phenotypic profiles of Mycobacterium bovis. One patient provided a bronchial lavage isolate, which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, but had molecular profiles of M. bovis from paraffin block DNA analysis, and one sample had molecular profiles of M. tuberculosis and M. bovis identified from two distinct paraffin blocks. Moreover, we found a low prevalence (1.6% of M. bovis among these isolates, which suggests that local health service procedures likely underestimate its real frequency and that it deserves more attention from public health officials.

  20. Comparison of Sputum-Culture Conversion for Mycobacterium bovis and M. tuberculosis.

    Science.gov (United States)

    Scott, Colleen; Cavanaugh, Joseph S; Silk, Benjamin J; Ershova, Julia; Mazurek, Gerald H; LoBue, Philip A; Moonan, Patrick K

    2017-03-01

    Current US guidelines recommend longer treatment for tuberculosis (TB) caused by pyrazinamide-resistant organisms (e.g., Mycobacterium bovis) than for M. tuberculosis TB. We compared treatment response times for patients with M. bovis TB and M. tuberculosis TB reported in the United States during 2006-2013. We included culture-positive, pulmonary TB patients with genotyping results who received standard 4-drug treatment at the time of diagnosis. Time to sputum-culture conversion was defined as time between treatment start date and date of first consistently culture-negative sputum. We analyzed 297 case-patients with M. bovis TB and 30,848 case-patients with M. tuberculosis TB. After 2 months of treatment, 71% of M. bovis and 65% of M. tuberculosis TB patients showed conversion of sputum cultures to negative. Likelihood of culture conversion was higher for M. bovis than for M. tuberculosis, even after controlling for treatment administration type, sex, and a composite indicator of bacillary burden.

  1. Draft Genome Sequence of Mycobacterium tuberculosis Clinical Strain G-12-005

    OpenAIRE

    Berland, Jean-Luc; Carvalho, Fabíola Marques; de Almeida, Luiz Gonzaga Paula; Bablishvili, Nino; Gauthier, Marie; Paranhos-Baccalà, Glaucia; de Vasconcelos, Ana Tereza Ribeiro

    2014-01-01

    Infection caused by drug-resistant Mycobacterium tuberculosis is a growing concern, especially in eastern Europe. We report an annotated draft genome sequence of M. tuberculosis strain G-12-005 obtained from a patient in Georgia.

  2. IS6110 in oriC affects the morphology and growth of Mycobacterium tuberculosis and attenuates virulence in mice.

    Science.gov (United States)

    Casart, Yveth; Turcios, Lilia; Florez, Ingrid; Jaspe, Rossana; Guerrero, Elba; de Waard, Jacobus; Aguilar, Diana; Hérnandez-Pando, Rogelio; Salazar, Leiria

    2008-11-01

    The IS6110 element is widely used in studies of molecular epidemiology of tuberculosis and it is considered the gold standard for genotyping Mycobacterium tuberculosis strains. Because of its high frequency of transposition, IS6110 is probably a major contributor to the evolution of M. tuberculosis. Nevertheless, very few studies of the effect of IS6110 insertions on the virulence of M. tuberculosis have been reported. We analysed two isogenic groups of M. tuberculosis strains isolated from the sputa of two patients. Strains belonging to the same isogenic group differed from one another by one IS6110-oriC hybridising band, but they showed identical spoligo and MIRU-VNTR profiles. Isogenic strains containing the IS6110 element in oriC exhibited a diminished growth rate and average dimensions of the bacilli were modified; moreover, they were less virulent in a mouse model.

  3. Molecular profiling of Mycobacterium tuberculosis identifies tuberculosinyl nucleoside products of the virulence-associated enzyme Rv3378c

    NARCIS (Netherlands)

    Layre, Emilie; Lee, Ho Jun; Young, David C.; Martinot, Amanda Jezek; Buter, Jeffrey; Minnaard, Adriaan J.; Annand, John W.; Fortune, Sarah M.; Snider, Barry B.; Matsunaga, Isamu; Rubin, Eric J.; Alber, Tom; Moody, D. Branch

    2014-01-01

    To identify lipids with roles in tuberculosis disease, we systematically compared the lipid content of virulent Mycobacterium tuberculosis with the attenuated vaccine strain Mycobacterium bovis bacillus Calmette-Guerin. Comparative lipidomics analysis identified more than 1,000 molecular differences

  4. [A preliminary study on the molecular characteristics of D-cycloserine resistance of Mycobacterium tuberculosis].

    Science.gov (United States)

    Li, C; Li, G L; Luo, Q; Li, S J; Wang, R B; Lou, Y L; Lyu, J X; Wan, K L

    2017-02-10

    Objective: To investigate the relationship between D-cycloserine resistance and the gene mutations of alrA, ddlA and cycA of Mycobacterium (M.) tuberculosis, as well as the association between D-cycloserine resistance and spoligotyping genotyping. Methods: A total of 145 M. tuberculosis strains were selected from the strain bank. D-cycloserine resistant phenotypes of the strains were determined by the proportion method and the minimal inhibitory concentration was determined by resazurin microtiter assay. PCR amplification and DNA direct sequencing methods were used for the analysis of gene mutations. Relationship between the resistance phenotype and genotype was analyzed by chi-square test. Results: Of the 145 clinically collected strains, 24 (16.6%) of them were D-cycloserine resistant and 121 (83.4%) were sensitive. There were only synonymous mutations noticed on alrA, ddlA and cycA in sensitive strains. Of the 24 D-cycloserine resistant strains, 3 (12.5%) isolates' cycA and 1 (4.2%) isolates' alrA happened to be non-synonymous mutations, in which the codes were 188, 318 and 508 of cycA, and 261 of alrA, respectively. Results on drug sensitivity tests confirmed the minimal inhibitory concentration of the mutant strains were all increased to some degrees. The D-cycloserine resistant rates of 88 Beijing genotype and 57 non-Beijing genotype strains were 20.5% and 10.5% , respectively, but with no statistically significant difference (χ(2) =2.47, P>0.05). Conclusions: The non-synonymous mutations of alrA and cycA might contribute to one of the mechanisms of M. tuberculosis D-cycloserine resistance. M. tuberculosis Beijing genotype or non-Beijing genotype was not considered to be associated with the D-cycloserine resistance.

  5. Association between Mycobacterium tuberculosis complex phylogenetic lineage and acquired drug resistance.

    Directory of Open Access Journals (Sweden)

    Courtney M Yuen

    Full Text Available BACKGROUND: Development of resistance to antituberculosis drugs during treatment (i.e., acquired resistance can lead to emergence of resistant strains and consequent poor clinical outcomes. However, it is unknown whether Mycobacterium tuberculosis complex species and lineage affects the likelihood of acquired resistance. METHODS: We analyzed data from the U.S. National Tuberculosis Surveillance System and National Tuberculosis Genotyping Service for tuberculosis cases during 2004-2011 with assigned species and lineage and both initial and final drug susceptibility test results. We determined univariate associations between species and lineage of Mycobacterium tuberculosis complex bacteria and acquired resistance to isoniazid, rifamycins, fluoroquinolones, and second-line injectables. We used Poisson regression with backward elimination to generate multivariable models for acquired resistance to isoniazid and rifamycins. RESULTS: M. bovis was independently associated with acquired resistance to isoniazid (adjusted prevalence ratio = 8.46, 95% CI 2.96-24.14 adjusting for HIV status, and with acquired resistance to rifamycins (adjusted prevalence ratio = 4.53, 95% CI 1.29-15.90 adjusting for homelessness, HIV status, initial resistance to isoniazid, site of disease, and administration of therapy. East Asian lineage was associated with acquired resistance to fluoroquinolones (prevalence ratio = 6.10, 95% CI 1.56-23.83. CONCLUSIONS: We found an association between mycobacterial species and lineage and acquired drug resistance using U.S. surveillance data. Prospective clinical studies are needed to determine the clinical significance of these findings, including whether rapid genotyping of isolates at the outset of treatment may benefit patient management.

  6. Mycobacterium tuberculosis complex mycobacteria as amoeba-resistant organisms.

    Directory of Open Access Journals (Sweden)

    Felix Mba Medie

    Full Text Available BACKGROUND: Most environmental non-tuberculous mycobacteria have been demonstrated to invade amoebal trophozoites and cysts, but such relationships are largely unknown for members of the Mycobacterium tuberculosis complex. An environmental source has been proposed for the animal Mycobacterium bovis and the human Mycobacterium canettii. METHODOLOGY/PRINCIPAL FINDINGS: Using optic and electron microscopy and co-culture methods, we observed that 89±0.6% of M. canettii, 12.4±0.3% of M. tuberculosis, 11.7±2% of M. bovis and 11.2±0.5% of Mycobacterium avium control organisms were phagocytized by Acanthamoeba polyphaga, a ratio significantly higher for M. canettii (P = 0.03, correlating with the significantly larger size of M. canetti organisms (P = 0.035. The percentage of intraamoebal mycobacteria surviving into cytoplasmic vacuoles was 32±2% for M. canettii, 26±1% for M. tuberculosis, 28±2% for M. bovis and 36±2% for M. avium (P = 0.57. M. tuberculosis, M. bovis and M. avium mycobacteria were further entrapped within the double wall of <1% amoebal cysts, but no M. canettii organisms were observed in amoebal cysts. The number of intracystic mycobacteria was significantly (P = 10(-6 higher for M. avium than for the M. tuberculosis complex, and sub-culturing intracystic mycobacteria yielded significantly more (P = 0.02 M. avium organisms (34×10(4 CFU/mL than M. tuberculosis (42×10(1 CFU/mL and M. bovis (35×10(1 CFU/mL in the presence of a washing fluid free of mycobacteria. Mycobacteria survived in the cysts for up to 18 days and cysts protected M. tuberculosis organisms against mycobactericidal 5 mg/mL streptomycin and 2.5% glutaraldehyde. CONCLUSIONS/SIGNIFICANCE: These data indicate that M. tuberculosis complex organisms are amoeba-resistant organisms, as previously demonstrated for non-tuberculous, environmental mycobacteria. Intercystic survival of tuberculous mycobacteria, except for M. canettii, protect them

  7. Zirconia based nucleic acid sensor for Mycobacterium tuberculosis detection

    Science.gov (United States)

    Das, Maumita; Sumana, Gajjala; Nagarajan, R.; Malhotra, B. D.

    2010-03-01

    Nanostructured zirconium oxide (ZrO2) film (particle size˜35 nm), electrochemically deposited onto gold(Au) surface, has been used to immobilize 21-mer oligonucleotide probe (ssDNA) specific to Mycobacterium tuberculosis by utilizing affinity between oxygen atom of phosphoric group and zirconium to fabricate DNA biosensor. This DNA-ZrO2/Au bioelectrode, characterized using x-ray diffraction, Fourier transform infrared spectroscopy, cyclic voltammetry, and scanning electron microscopy techniques, can be used for early and rapid diagnosis of M. tuberculosis with detection limit of 0.065 ng/μL within 60s.

  8. The complex evolution of antibiotic resistance in Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    J.D. Fonseca

    2015-03-01

    Full Text Available Multidrug-resistant and extensively drug-resistant tuberculosis (TB represent a major threat to the control of the disease worldwide. The mechanisms and pathways that result in the emergence and subsequent fixation of resistant strains of Mycobacterium tuberculosis are not fully understood and recent studies suggest that they are much more complex than initially thought. In this review, we highlight the exciting new areas of research within TB resistance that are beginning to fill these gaps in our understanding, whilst also raising new questions and providing future directions.

  9. Structures of citrate synthase and malate dehydrogenase of Mycobacterium tuberculosis.

    Science.gov (United States)

    Ferraris, Davide M; Spallek, Ralf; Oehlmann, Wulf; Singh, Mahavir; Rizzi, Menico

    2015-02-01

    The tricarboxylic acid (TCA) cycle is a central metabolic pathway of all aerobic organisms and is responsible for the synthesis of many important precursors and molecules. TCA cycle plays a key role in the metabolism of Mycobacterium tuberculosis and is involved in the adaptation process of the bacteria to the host immune response. We present here the first crystal structures of M. tuberculosis malate dehydrogenase and citrate synthase, two consecutive enzymes of the TCA, at 2.6 Å and 1.5 Å resolution, respectively. General analogies and local differences with the previously reported homologous protein structures are described. © 2014 Wiley Periodicals, Inc.

  10. Lymphatic endothelial cells are a replicative niche for Mycobacterium tuberculosis

    Science.gov (United States)

    Lerner, Thomas R.; de Souza Carvalho-Wodarz, Cristiane; Repnik, Urska; Russell, Matthew R.G.; Borel, Sophie; Diedrich, Collin R.; Rohde, Manfred; Wainwright, Helen; Collinson, Lucy M.; Wilkinson, Robert J.; Griffiths, Gareth; Gutierrez, Maximiliano G.

    2016-01-01

    In extrapulmonary tuberculosis, the most common site of infection is within the lymphatic system, and there is growing recognition that lymphatic endothelial cells (LECs) are involved in immune function. Here, we identified LECs, which line the lymphatic vessels, as a niche for Mycobacterium tuberculosis in the lymph nodes of patients with tuberculosis. In cultured primary human LECs (hLECs), we determined that M. tuberculosis replicates both in the cytosol and within autophagosomes, but the bacteria failed to replicate when the virulence locus RD1 was deleted. Activation by IFN-γ induced a cell-autonomous response in hLECs via autophagy and NO production that restricted M. tuberculosis growth. Thus, depending on the activation status of LECs, autophagy can both promote and restrict replication. Together, these findings reveal a previously unrecognized role for hLECs and autophagy in tuberculosis pathogenesis and suggest that hLECs are a potential niche for M. tuberculosis that allows establishment of persistent infection in lymph nodes. PMID:26901813

  11. Carbapenems and Rifampin Exhibit Synergy against Mycobacterium tuberculosis and Mycobacterium abscessus.

    Science.gov (United States)

    Kaushik, Amit; Makkar, Nayani; Pandey, Pooja; Parrish, Nicole; Singh, Urvashi; Lamichhane, Gyanu

    2015-10-01

    An effective regimen for treatment of tuberculosis (TB) is comprised of multiple drugs that inhibit a range of essential cellular activities in Mycobacterium tuberculosis. The effectiveness of a regimen is further enhanced if constituent drugs act with synergy. Here, we report that faropenem (a penem) or biapenem, doripenem, or meropenem (carbapenems), which belong to the β-lactam class of antibiotics, and rifampin, one of the drugs that forms the backbone of TB treatment, act with synergy when combined. One of the reasons (carba)penems are seldom used for treatment of TB is the high dosage levels required, often at the therapeutic limits. The synergistic combination of rifampin and these (carba)penems indicates that (carba)penems can be administered at dosages that are therapeutically relevant. The combination of faropenem and rifampin also limits the frequency of resistant mutants, as we were unable to obtain spontaneous mutants in the presence of these two drugs. The combinations of rifampin and (carba)penems were effective not only against drug-sensitive Mycobacterium tuberculosis but also against drug-resistant clinical isolates that are otherwise resistant to rifampin. A combination of doripenem or biapenem and rifampin also exhibited synergistic activity against Mycobacterium abscessus. Although the MICs of these three drugs alone against M. abscessus are too high to be of clinical relevance, their concentrations in combinations are therapeutically relevant; therefore, they warrant further evaluation for clinical utility to treat Mycobacterium abscessus infection, especially in cystic fibrosis patients.

  12. Epidemiological trends and clinical comparisons of Mycobacterium tuberculosis lineages in Thai TB meningitis.

    Science.gov (United States)

    Faksri, Kiatichai; Drobniewski, Francis; Nikolayevskyy, Vladyslav; Brown, Timothy; Prammananan, Therdsak; Palittapongarnpim, Prasit; Prayoonwiwat, Naraporn; Chaiprasert, Angkana

    2011-11-01

    Mycobacterium tuberculosis (MTB) strains were isolated from cerebrospinal fluids collected from individual tuberculous meningitis (TBM) patients from 1996 to 2007 (n = 184) and characterised based on IS6110-restriction fragment length polymorphism (RFLP), spoligotyping, Mycobacterium interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) and large sequence polymorphisms (LSPs). Beijing strains were found to possess the highest transmissibility and proportion in clustered isolates. Beijing strain predomination and stability, at 56% of the genotypic proportion, as well as association with drug resistance in TBM patients, was demonstrated. The proportion of Beijing sublineages revealed that the modern Beijing sublineage showed an increasing trend, whereas the ancestral Beijing sublineage showed a decreasing trend across the three periods. In contrast, there were neither clustered nor multidrug-resistance (MDR) isolates from the Euro-American (EuA) lineage, and the lineage genotypic proportion trend was also decreased. Based on LSPs, only the Beijing, Indo-Oceanic and Euro-American lineages were identified from TBM patients in Thailand. TBM mortality rates were not associated with either drug resistance or significantly different among MTB lineages. This study may support the Beijing genotype strain as most pathogenic causing TBM, with the EuA lineage genotype as the most benign of the strain genotypes tested. The analysis of drug susceptibility also revealed the trend of increasing drug resistance, especially MDR, in TBM patients in Thailand.

  13. Bloodstream Infections with Mycobacterium tuberculosis among HIV patients

    Centers for Disease Control (CDC) Podcasts

    2010-09-23

    This podcast looks at bloodstream infections with Mycobacterium tuberculosis and other pathogens among outpatients infected with HIV in Southeast Asia. CDC health scientist Kimberly McCarthy discusses the study and why bloodstream infections occur in HIV-infected populations.  Created: 9/23/2010 by National Center for Emerging and Zoonotic Infectious Diseases (NCEZID).   Date Released: 9/23/2010.

  14. Immune subdominant antigens as vaccine candidates against Mycobacterium tuberculosis.

    Science.gov (United States)

    Orr, Mark T; Ireton, Gregory C; Beebe, Elyse A; Huang, Po-Wei D; Reese, Valerie A; Argilla, David; Coler, Rhea N; Reed, Steven G

    2014-09-15

    Unlike most pathogens, many of the immunodominant epitopes from Mycobacterium tuberculosis are under purifying selection. This startling finding suggests that M. tuberculosis may gain an evolutionary advantage by focusing the human immune response against selected proteins. Although the implications of this to vaccine development are incompletely understood, it has been suggested that inducing strong Th1 responses against Ags that are only weakly recognized during natural infection may circumvent this evasion strategy and increase vaccine efficacy. To test the hypothesis that subdominant and/or weak M. tuberculosis Ags are viable vaccine candidates and to avoid complications because of differential immunodominance hierarchies in humans and experimental animals, we defined the immunodominance hierarchy of 84 recombinant M. tuberculosis proteins in experimentally infected mice. We then combined a subset of these dominant or subdominant Ags with a Th1 augmenting adjuvant, glucopyranosyl lipid adjuvant in stable emulsion, to assess their immunogenicity in M. tuberculosis-naive animals and protective efficacy as measured by a reduction in lung M. tuberculosis burden of infected animals after prophylactic vaccination. We observed little correlation between immunodominance during primary M. tuberculosis infection and vaccine efficacy, confirming the hypothesis that subdominant and weakly antigenic M. tuberculosis proteins are viable vaccine candidates. Finally, we developed two fusion proteins based on strongly protective subdominant fusion proteins. When paired with the glucopyranosyl lipid adjuvant in stable emulsion, these fusion proteins elicited robust Th1 responses and limited pulmonary M. tuberculosis for at least 6 wk postinfection with a single immunization. These findings expand the potential pool of M. tuberculosis proteins that can be considered as vaccine Ag candidates. Copyright © 2014 by The American Association of Immunologists, Inc.

  15. EmbA is an essential arabinosyltransferase in Mycobacterium tuberculosis.

    Science.gov (United States)

    Amin, Anita G; Goude, Renan; Shi, Libin; Zhang, Jian; Chatterjee, Delphi; Parish, Tanya

    2008-01-01

    The Emb proteins (EmbA, EmbB, EmbC) are mycobacterial arabinosyltransferases involved in the biogenesis of the mycobacterial cell wall. EmbA and EmbB are predicted to work in unison as a heterodimer. EmbA and EmbB are involved in the formation of the crucial terminal hexaarabinoside motif [Arabeta(1-->2)Araalpha(1-->5)] [Arabeta(1-->2)Araalpha(1-->3)]Araalpha(1-->5)Araalpha1-->(Ara(6)) in the cell wall polysaccharide arabinogalactan. Studies conducted in Mycobacterium smegmatis revealed that mutants with disruptions in embA or embB are viable, although the growth rate was affected. In contrast, we demonstrate here that embA is an essential gene in Mycobacterium tuberculosis, since a deletion of the chromosomal gene could only be achieved when a second functional copy was provided on an integrated vector. Complementation of an embA mutant of M. smegmatis by M. tuberculosis embA confirmed that it encodes a functional arabinosyltransferase. We identified a promoter for M. tuberculosis embA located immediately upstream of the gene, indicating that it is expressed independently from the upstream gene, embC. Promoter activity from P(embA)((Mtb)) was sevenfold lower when assayed in M. smegmatis compared to M. tuberculosis, indicating that the latter is not a good host for genetic analysis of M. tuberculosis embA expression. P(embA)((Mtb)) activity remained constant throughout growth phases and after stress treatment, although it was reduced during hypoxia-induced non-replicating persistence. Ethambutol exposure had no effect on P(embA)((Mtb)) activity. These data demonstrate that M. tuberculosis embA encodes a functional arabinosyltransferase which is constitutively expressed and plays a critical role in M. tuberculosis.

  16. Mycobacterium tuberculosis pili (MTP), a putative biomarker for a tuberculosis diagnostic test.

    Science.gov (United States)

    Naidoo, Natasha; Ramsugit, Saiyur; Pillay, Manormoney

    2014-05-01

    Novel biomarkers are urgently needed for point of care TB diagnostics. In this study, we investigated the potential of the pilin subunit protein encoded by the mtp gene as a diagnostic biomarker. BLAST analysis of the mtp gene on published genome databases, and amplicon sequencing were performed in Mycobacterium tuberculosis Complex (MTBC) strains and other organisms. The protein secondary structure of the amino acid sequences of non-tuberculous Mycobacteria that partially aligned with the mtp sequence was analysed with PredictProtein software. The mtp gene and corresponding amino acid sequence of MTBC were 100% homologous with H37Rv, in contrast to the partial alignment of the non-tuberculous Mycobacteria. The mtp gene was present in all 91 clinical isolates of MTBC. Except for 2 strains with point mutations, the sequence was 100% conserved among the clinical strains. The mtp gene could not be amplified in all non-tuberculous Mycobacteria and respiratory organisms. The predicted MTP protein structure of Mycobacterium avium, Mycobacterium ulcerans and Mycobacterium abscessus differed significantly from that of the M. tuberculosis, which was similar to Mycobacterium marinum. The absence of the mtp gene in non-tuberculous Mycobacteria and other respiratory bacteria suggests that its encoded product, the pilin subunit protein of M. tuberculosis may be a suitable marker for a point of care TB test.

  17. The Potential Spread of Mycobacterium tuberculosis into the Environment in the Creation of Spondylitis Tuberculosis Rabbit

    Directory of Open Access Journals (Sweden)

    Ahmad Jabir Rahyussalim

    2015-01-01

    Full Text Available Direct Mycobacterium tuberculosis inoculation on rabbit vertebral body was used in rabbit spinal infection study. The potential spread of Mycobacterium tuberculosis into the environment will be observed in order to create the conditions fulfilling biosafety aspects. Two groups of six New Zealand rabbits were treatment group (n=4 and control group (n=2. The treatment group had injection of 0.1 mL (107 cfu/mL suspension of Mycobacterium tuberculosis into the vertebral body T12. They were incubated for 2 to 14 weeks. One rabbit per period of 2, 4, 6, and 14 weeks was euthanized to collect feces, urine, saliva, and tissue lesions. The control group had only feces, urine, and saliva to detect bacteria using AFB staining, culture, and PCR. Both two groups were kept in individual cages. They were put together in a large cage for 3 hours every day to interact with each other. AFB staining, culture, and radiological examination showed negative result, but in one rabbit, histopathological examination showed positive result and PCR examination in another rabbit of the treatment group. Spreading score was 1.05% and infected score was 0 (null. The procedure did not reveal the potential spread of Mycobacterium tuberculosis into the environment.

  18. Investigation on Mycobacterium tuberculosis diversity in China and the origin of the Beijing clade.

    Directory of Open Access Journals (Sweden)

    Kanglin Wan

    Full Text Available BACKGROUND: Investigation of the genetic diversity of Mycobacterium tuberculosis in China has shown that Beijing genotype strains play a dominant role in the tuberculosis (TB epidemic. In order to examine the strain diversity in the whole country, and to study the evolutionary development of Beijing strains, we sought to genotype a large collection of isolates using different methods. METHODOLOGY/PRINCIPAL FINDINGS: We applied a 15-loci VNTR typing analysis on 1,586 isolates from the Beijing municipality and 12 Chinese provinces or autonomous regions. The data was compared to that of 900 isolates from various other worldwide geographic regions outside of China. A total of 1,162/1,586 (73.2% of the isolates, distributed into 472 VNTR types, were found to belong to the Beijing genotype family and this represented 56 to 94% of the isolates in each of the localizations. VNTR typing revealed that the majority of the non-Beijing isolates fall into two genotype families, which represented 17% of the total number of isolates, and seem largely restricted to China. A small number of East African Indian genotype strains was also observed in this collection. Ancient Beijing strains with an intact region of difference (RD 181, as well as strains presumably resembling ancestors of the whole Beijing genotype family, were mainly found in the Guangxi autonomous region. CONCLUSIONS/SIGNIFICANCE: This is the largest M. tuberculosis VNTR-based genotyping study performed in China to date. The high percentage of Beijing isolates in the whole country and the presence in the South of strains representing early branching points may be an indication that the Beijing lineage originated from China, probably in the Guangxi region. Two modern lineages are shown here to represent the majority of non-Beijing Chinese isolates. The observed geographic distribution of the different lineages within China suggests that natural frontiers are major factors in their diffusion.

  19. Investigation on Mycobacterium tuberculosis Diversity in China and the Origin of the Beijing Clade

    Science.gov (United States)

    Wan, Kanglin; Liu, Jinghua; Hauck, Yolande; Zhang, Yuanyuan; Liu, Jie; Zhao, Xiuqin; Liu, Zhiguang; Lu, Bing; Dong, Haiyan; Jiang, Yi; Kremer, Kristin; Vergnaud, Gilles; van Soolingen, Dick; Pourcel, Christine

    2011-01-01

    Background Investigation of the genetic diversity of Mycobacterium tuberculosis in China has shown that Beijing genotype strains play a dominant role in the tuberculosis (TB) epidemic. In order to examine the strain diversity in the whole country, and to study the evolutionary development of Beijing strains, we sought to genotype a large collection of isolates using different methods. Methodology/Principal Findings We applied a 15-loci VNTR typing analysis on 1,586 isolates from the Beijing municipality and 12 Chinese provinces or autonomous regions. The data was compared to that of 900 isolates from various other worldwide geographic regions outside of China. A total of 1,162/1,586 (73.2%) of the isolates, distributed into 472 VNTR types, were found to belong to the Beijing genotype family and this represented 56 to 94% of the isolates in each of the localizations. VNTR typing revealed that the majority of the non-Beijing isolates fall into two genotype families, which represented 17% of the total number of isolates, and seem largely restricted to China. A small number of East African Indian genotype strains was also observed in this collection. Ancient Beijing strains with an intact region of difference (RD) 181, as well as strains presumably resembling ancestors of the whole Beijing genotype family, were mainly found in the Guangxi autonomous region. Conclusions/Significance This is the largest M. tuberculosis VNTR-based genotyping study performed in China to date. The high percentage of Beijing isolates in the whole country and the presence in the South of strains representing early branching points may be an indication that the Beijing lineage originated from China, probably in the Guangxi region. Two modern lineages are shown here to represent the majority of non-Beijing Chinese isolates. The observed geographic distribution of the different lineages within China suggests that natural frontiers are major factors in their diffusion. PMID:22220207

  20. Genomic diversity of drug-resistant Mycobacterium tuberculosis isolates in Lisbon Portugal: Towards tuberculosis genomic epidemiology

    KAUST Repository

    Perdigão, João

    2015-03-01

    Multidrug- (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) present a challenge to disease control and elimination goals. Lisbon, Portugal, has a high TB incidence rate and unusual and successful XDR-TB strains that have been found in circulation for almost two decades. For the last 20. years, a continued circulation of two phylogenetic clades, Lisboa3 and Q1, which are highly associated with MDR and XDR, have been observed. In recent years, these strains have been well characterized regarding the molecular basis of drug resistance and have been inclusively subjected to whole genome sequencing (WGS). Researchers have been studying the genomic diversity of strains circulating in Lisbon and its genomic determinants through cutting-edge next generation sequencing. An enormous amount of whole genome sequence data are now available for the most prevalent and clinically relevant strains circulating in Lisbon.It is the persistence, prevalence and rapid evolution towards drug resistance that has prompted researchers to investigate the properties of these strains at the genomic level and in the future at a global transcriptomic level. Seventy Mycobacterium tuberculosis (MTB) isolates, mostly recovered in Lisbon, were genotyped by 24-. loci Mycobacterial Interspersed Repetitive Unit - Variable Number of Tandem Repeats (MIRU-VNTR) and the genomes sequenced using a next generation sequencing platform - Illumina HiSeq 2000.The genotyping data revealed three major clusters associated with MDR-TB (Lisboa3-A, Lisboa3-B and Q1), two of which are associated with XDR-TB (Lisboa3-B and Q1), whilst the genomic data contributed to elucidating the phylogenetic positioning of circulating MDR-TB strains, showing a high predominance of a single SNP cluster group 5. Furthermore, a genome-wide phylogeny analysis from these strains, together with 19 publicly available genomes of MTB clinical isolates, revealed two major clades responsible for MDR/XDR-TB in the region: Lisboa3 and Q

  1. Mycobacterium tuberculosis complex transmission is not associated with recent immigration (≤5 years) in Florida.

    Science.gov (United States)

    Séraphin, Marie Nancy; Lauzardo, Michael

    2015-12-01

    As tuberculosis (TB) incidence decreases in the US, foreign-born persons continue to account for a larger proportion of the burden. In these cross-sectional analyses of 1149 culture-confirmed TB cases genotyped using spoligotyping and 24-locus MIRU, we show that over a quarter of cases among the foreign-born population in Florida resulted from recent transmission of the Mycobacterium tuberculosis complex. In addition, over a third of these cases occurred among persons who had immigrated 5 years or less prior to their diagnosis. Although recent immigration was not a significant predictor of TB transmission, younger age, birthplace in the Americas, homelessness, drug use and TB lineage are risk factors for TB transmission among the foreign-born population in Florida. These data provide actionable insights into TB transmission among the foreign-born population in Florida.

  2. The relative frequency of Mycobacterium tuberculosis and Mycobacterium avium infections in HIV positive patients, Ahvaz, Iran

    Institute of Scientific and Technical Information of China (English)

    Khosravi AD; Alavi SM; Hashemzade M; Abasi E; Seghatoleslami S

    2012-01-01

    Objective:To estimate the prevalence of Mycobacterium tuberculosis (M. tuberculosis) and Mycobacterium avium (M. avium) infections in HIV-positive patients suspected to have pulmonary and extrapulmonary mycobacterial co-infection using PCR technique. Methods:Totally 50 samples comprising sputum, pleural fluid and CSF taken from HIV positive patients suspected to have mycobacterial infection, were processed. The demographic information and results of acid fast staining and culture were recorded for each patient. The PCR for detecting of M. tuberculosis comprised of specific primers targeting IS6110 gene sequence. For detecting of M. avium, PCR with primers that amplifies the mig gene were used. Results:From 50 samples processed, 45 were sputum (90%), 3 pleural fluid (6%) and 2 CSF (4%). In total, 8 (16%) were culture positive, 7 had positive acid fast staining (14%) and 13 samples (26%) were positive using PCR technique. All the positive samples were sputum and belonged to patients with pulmonary infection. Of these, 9 were positive for M. tuberculosis (69.2%) and 4 were identified as M. avium (30.8%), which 2 out of 13 positive samples showed mixed infections by both mycobacteria. Conclusions:The PCR shows the highest detection rate (26%) of mycobacteria compared with culture and acid fast staining. The majority of infections were with M. tuberculosis (18%) and this shows the importance of this mycobacterial co-infection in HIV positive patients in the region of study.

  3. 5-Arylaminouracil Derivatives: New Inhibitors of Mycobacterium tuberculosis.

    Science.gov (United States)

    Matyugina, Elena; Novikov, Mikhail; Babkov, Denis; Ozerov, Alexander; Chernousova, Larisa; Andreevskaya, Sofia; Smirnova, Tatiana; Karpenko, Inna; Chizhov, Alexander; Murthu, Pravin; Lutz, Stefan; Kochetkov, Sergei; Seley-Radtke, Katherine L; Khandazhinskaya, Anastasia L

    2015-12-01

    Three series of 5-arylaminouracil derivatives, including 5-(phenylamino)uracils, 1-(4'-hydroxy-2'-cyclopenten-1'-yl)-5-(phenylamino)uracils, and 1,3-di-(4'-hydroxy-2'-cyclopenten-1'-yl)-5-(phenylamino)uracils, were synthesized and screened for potential antimicrobial activity. Most of compounds had a negative effect on the growth of the Mycobacterium tuberculosis H37Rv strain, with 100% inhibition observed at concentrations between 5 and 40 μg/mL. Of those, 1-(4'-hydroxy-2'-cyclopenten-1'-yl)-3-(4‴-hydroxy-2‴-cyclopenten-1‴-yl)-5-(4″-butyloxyphenylamino)uracil proved to be the most active among tested compounds against the M. tuberculosis multidrug-resistant strain MS-115 (MIC90 5 μg/mL). In addition, the thymidylate kinase of M. tuberculosis was evaluated as a possible enzymatic target.

  4. Mycobacterium tuberculosis infection in a HIV-positive patient

    Directory of Open Access Journals (Sweden)

    Maria Theresa Montales

    2015-01-01

    Full Text Available Mycobacterium tuberculosis (MTB and human immunodeficiency virus (HIV coinfection remains a global public health challenge. We report a 40 year old African American male who is a known HIV-positive patient, non-compliant with his antiretrovirals and developed pulmonary tuberculosis. His chief complaints were chronic cough, fever, night sweats and undocumented weight loss. He had a prior positive T-SPOT-TB test; however, chest radiograph and sputum smear examination revealed normal results. PCR-based GeneXPERT MTB/RIF assay was ordered and confirmed MTB infection. The sputum cultures grew MTB and sensitivities showed susceptibility to all primary anti-tuberculosis medications. A delay in diagnosis and initiation of MTB therapy, in the setting of HIV or AIDS, may result in rapid disease progression and worse clinical outcome.

  5. Bisphosphonic acids as effective inhibitors of Mycobacterium tuberculosis glutamine synthetase.

    Science.gov (United States)

    Kosikowska, Paulina; Bochno, Marta; Macegoniuk, Katarzyna; Forlani, Giuseppe; Kafarski, Paweł; Berlicki, Łukasz

    2016-12-01

    Inhibition of glutamine synthetase (GS) is one of the most promising strategies for the discovery of novel drugs against tuberculosis. Forty-three bisphosphonic and bis-H-phosphinic acids of various scaffolds, bearing aromatic substituents, were screened against recombinant GS from Mycobacterium tuberculosis. Most of the studied compounds exhibited activities in micromolar range, with N-(3,5-dichlorophenyl)-2-aminoethylidenebisphoshonic acid, N-(3,5-difluorophenyl)-2-aminoethylidene-bisphoshonic acid and N-(3,4-dichlorophenyl)-1-hydroxy-1,1-ethanebisphosphonic acid showing the highest potency with kinetic parameters similar to the reference compound - L-methionine-S-sulfoximine. Moreover, these inhibitors were found to be much more effective against pathogen enzyme than against the human ortholog. Thus, with the bone-targeting properties of the bisphosphonate compounds in mind, this activity/selectivity profile makes these compounds attractive agents for the treatment of bone tuberculosis.

  6. Development of a new DNA extraction protocol for PFGE typing of Mycobacterium tuberculosis complex

    Directory of Open Access Journals (Sweden)

    A Ghodousi

    2012-03-01

    Full Text Available A modified pulsed-field gel electrophoresis (PFGE protocol was developed and applied to clinical isolates of Mycobacterium tuberculosis complex to reduce the cost of using lyticase. This protocol reduces the expense of PFGE typing of Mycobacterium tuberculosis complex as it removes the use of lyticase during the spheroplast formation from these bacteria.

  7. Mixed Infection of Mycobacterium abscessus subsp. abscessus and Mycobacterium tuberculosis in the Lung

    Science.gov (United States)

    Sohn, Sungmin; Wang, Sungho; Shi, Hyejin; Park, Sungrock; Lee, Sangki; Park, Kyoung Taek

    2017-01-01

    A mixed infection of Mycobacterium abscessus subsp. abscessus (Mab) and Mycobacterium tuberculosis (MTB) in the lung is an unusual clinical manifestation and has not yet been reported. A 61-year-old woman had been treated for Mab lung disease and concomitant pneumonia, and was diagnosed with pulmonary tuberculosis (PTB). Despite both anti-PTB and anti-Mab therapy, her entire left lung was destroyed and collapsed. She underwent left pneumonectomy and received medical therapy. We were able to successfully treat her mixed infection by pneumonectomy followed by inhaled amikacin therapy. To the best of our knowledge, thus far, this is the first description of a mixed Mab and MTB lung infection. PMID:28180105

  8. Role of Mycobacterium tuberculosis pknD in the Pathogenesis of central nervous system tuberculosis

    Directory of Open Access Journals (Sweden)

    Be Nicholas A

    2012-01-01

    Full Text Available Abstract Background Central nervous system disease is the most serious form of tuberculosis, and is associated with high mortality and severe neurological sequelae. Though recent clinical reports suggest an association of distinct Mycobacterium tuberculosis strains with central nervous system disease, the microbial virulence factors required have not been described previously. Results We screened 398 unique M. tuberculosis mutants in guinea pigs to identify genes required for central nervous system tuberculosis. We found M. tuberculosis pknD (Rv0931c to be required for central nervous system disease. These findings were central nervous system tissue-specific and were not observed in lung tissues. We demonstrated that pknD is required for invasion of brain endothelia (primary components of the blood-brain barrier protecting the central nervous system, but not macrophages, lung epithelia, or other endothelia. M. tuberculosis pknD encodes a "eukaryotic-like" serine-threonine protein kinase, with a predicted intracellular kinase and an extracellular (sensor domain. Using confocal microscopy and flow cytometry we demonstrated that the M. tuberculosis PknD sensor is sufficient to trigger invasion of brain endothelia, a process which was neutralized by specific antiserum. Conclusions Our findings demonstrate a novel in vivo role for M. tuberculosis pknD and represent an important mechanism for bacterial invasion and virulence in central nervous system tuberculosis, a devastating and understudied disease primarily affecting young children.

  9. The quiet and controversial: Ural family of Mycobacterium tuberculosis.

    Science.gov (United States)

    Mokrousov, Igor

    2012-06-01

    The absence of lateral gene exchange is a characteristic feature defining the genome evolution and clonal population structure of Mycobacterium tuberculosis. Certain of its lineages have justly attracted more attention due to their global dissemination and/or remarkable pathogenic properties. In this critical review, I discuss the population structure and genetic geography of the less 'popular' but in some aspects no less noteworthy M. tuberculosis lineage, Ural family. Its specific signature was initially defined by single copy in MIRU26, and large (>6) copy number in MIRU10 loci, and by 43-spoligotyping as absence of signals 29-31 and 33-36. Here, I suggest to subdivide Ural strains with present and absent spoligosignal 2 into primary Ural-1 and secondary Ural-2 sublineages, respectively, while 1 copy in MIRU26 is specific of Ural-1. Furthermore, three copies were recently described in MIRU10 in Ural-1 strains which highlights a high diversity of this locus in Ural genotype. The data on the two Ural sublineages were extracted from SpolDB4 database and original publications in order to trace their distribution at global and within-country levels. Importantly, the rigorous reanalysis suggested the true rate of the Ural genotype in the Ural area in Russia to be only 7%. In contrast, the frequencies of the Ural sublineages peak elsewhere: in South Ukraine and Georgia/Abkhazia (Ural-1, up to 14-19%), and in southwestern Iran (Ural-2, up to 26%). However, as this name is used since 2005, it seems most parsimonious to continue its use even if misleading. The forest graph was built on the available spoligoprofiles of Ural family strains from Eurasia. It helped to suggest routes of their primary dispersal that are discussed in the context of the known human migrations also influenced by natural barriers. The north/east Pontic area may have been an area of origin and primary dispersal of the Ural (Ural-1) genotype in Eurasia, whereas political and natural borders may have

  10. Multiplex-PCR for differentiation of Mycobacterium bovis from Mycobacterium tuberculosis complex.

    Science.gov (United States)

    Spositto, F L E; Campanerut, P A Z; Ghiraldi, L D; Leite, C Q F; Hirata, M H; Hirata, R D C; Siqueira, V L D; Cardoso, R Fressatti

    2014-01-01

    We evaluated a multiplex-PCR to differentiate Mycobacterium bovis from M. tuberculosis Complex (MTC) by one step amplification based on simultaneous detection of pncA 169 C > G change in M. bovis and the IS6110 present in MTC species. Our findings showed the proposed multiplex-PCR is a very useful tool for complementation in differentiating M. bovis from other cultured MTC species.

  11. Genetic variation of Mycobacterium tuberculosis circulating in Kharkiv Oblast, Ukraine

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    Khrapov Eugeny A

    2011-03-01

    Full Text Available Abstract Background A persistent increase of tuberculosis cases has recently been noted in the Ukraine. The reported incidence of drug-resistant isolates of M. tuberculosis is growing steadily; however, data on the genetic variation of isolates of M. tuberculosis circulating in northern Ukraine and on the spectrum and frequency of occurrence of mutations determining resistance to the principal anti-tuberculosis drugs isoniazid and rifampicin have not yet been reported. Methods Isolates of M. tuberculosis from 98 tuberculosis patients living in Kharkiv Oblast (Ukraine were analyzed using VNTR- and RFLP-IS6110-typing methods. Mutations associated with resistance to rifampicin and isoniazid were detected by RFLP-PCR methods, and also confirmed by sequencing. Results We identified 75 different genetic profiles. Thirty four (34% isolates belonged to the Beijing genotype and 23 (23% isolates belonged to the LAM family. A cluster of isolates belonging to the LAM family had significant genetic heterogeneity, indicating that this family had an ancient distribution and circulation in this geographical region. Moreover, we found a significant percentage of the isolates (36% belonged to as yet unidentified families of M. tuberculosis or had individual non-clustering genotypes. Mutations conferring rifampicin and isoniazid resistance were detected in 49% and 54% isolates, respectively. Mutations in codon 531 of the rpoB gene and codon 315 of the katG gene were predominant among drug-resistant isolates. An association was found for belonging to the LAM strain family and having multiple drug resistance (R = 0.27, p = 0.0059 and also for the presence of a mutation in codon 531 of the rpoB gene and belonging to the Beijing strain family (R = 0.2, p = 0.04. Conclusions Transmission of drug-resistant isolates seems to contribute to the spread of resistant TB in this oblast. The Beijing genotype and LAM genotype should be seen as a major cause of drug resistant TB

  12. Effect of mutation and genetic background on drug resistance in Mycobacterium tuberculosis.

    Science.gov (United States)

    Fenner, Lukas; Egger, Matthias; Bodmer, Thomas; Altpeter, Ekkehardt; Zwahlen, Marcel; Jaton, Katia; Pfyffer, Gaby E; Borrell, Sonia; Dubuis, Olivier; Bruderer, Thomas; Siegrist, Hans H; Furrer, Hansjakob; Calmy, Alexandra; Fehr, Jan; Stalder, Jesica Mazza; Ninet, Béatrice; Böttger, Erik C; Gagneux, Sebastien

    2012-06-01

    Bacterial factors may contribute to the global emergence and spread of drug-resistant tuberculosis (TB). Only a few studies have reported on the interactions between different bacterial factors. We studied drug-resistant Mycobacterium tuberculosis isolates from a nationwide study conducted from 2000 to 2008 in Switzerland. We determined quantitative drug resistance levels of first-line drugs by using Bactec MGIT-960 and drug resistance genotypes by sequencing the hot-spot regions of the relevant genes. We determined recent transmission by molecular methods and collected clinical data. Overall, we analyzed 158 isolates that were resistant to isoniazid, rifampin, or ethambutol, 48 (30.4%) of which were multidrug resistant. Among 154 isoniazid-resistant strains, katG mutations were associated with high-level and inhA promoter mutations with low-level drug resistance. Only katG(S315T) (65.6% of all isoniazid-resistant strains) and inhA promoter -15C/T (22.7%) were found in molecular clusters. M. tuberculosis lineage 2 (includes Beijing genotype) was associated with any drug resistance (adjusted odds ratio [OR], 3.0; 95% confidence interval [CI], 1.7 to 5.6; P mutations (OR, 6.4; 95% CI, 2.0 to 20.7; P = 0.002). We found that the genetic strain background influences the level of isoniazid resistance conveyed by particular mutations (interaction tests of drug resistance mutations across all lineages; P tuberculosis drug resistance mutations were associated with various levels of drug resistance and transmission, and M. tuberculosis lineages were associated with particular drug resistance-conferring mutations and phenotypic drug resistance. Our study also supports a role for epistatic interactions between different drug resistance mutations and strain genetic backgrounds in M. tuberculosis drug resistance.

  13. 儿童结核病101例临床分离结核分枝杆菌多位点串联重复序列分型%Genotyping of 101 Mycobacterium tuberculosis isolates from pediatrics tuberculosis in Chongqing by multiple locus VNTR analysis

    Institute of Scientific and Technical Information of China (English)

    王均; 黄延风; 张爱华; 许红梅

    2011-01-01

    Objective To study the distribution of the variable number tandem repeats (VNTR) in clinically isolated strains Mycobacterium tuberculosis (M. tuberculosis ) from pediatrics tuberculosis in Chongqing and explore the effective loci combination of them by multiple locus VNTR analysis (MLVA). Methods Twenty-four standardized MLVA loci was used to type the 101 clinically isolated M. tuberculosis strains, and the results were analyzed by BioNumerics 6.1 software, which including dendrogram and Hunter-Gaston index (HGI) of 24 loci. The HGI of different loci set (12 loci, 15 loci, and 24 loci) was evaluated. Results Twenty-four loci showed different polymorphism. All the 101 clinical isolates were typed into one group that displaying 83 genotypes, in which 69 strains (68.32%, 69/101 ) were typed 69 genotypes respectively, and 32 strains (31.68%) were typed 14 genotypes which showed cluster rate of 17.82%. The HGI of 24 loci was from 0. 168 to 0. 829, the number of VNTR loci with HGI higher than 0.5 was 16. Three loci set displayed different polymorphism. HGI of 12 loci, 15 loci, and 24 loci set were 0.995, 0.996, and 0.996 respectively. Conclusion The clinical strains of M. tuberculosis isolated from pediatrics tuberculosis in Chongqing show definite polymorphism and the standardization 15 loci VNTR set of MLVA can be used in the molecular epidemiological study of these strains in Chongqing.%目的 了解重庆地区儿童结核病中结核分枝杆菌临床分离株的数目可变串联重复序列(variable number tandem repeats,VNTR)的分布特征,寻找适合的VNTR位点组合.方法 采用多位点串联重复序列(multiple locus VNTR analysis,MLVA)分型方法,选择标化的24个VNTR位点,对101例结核分枝杆菌临床分离株DNA进行检测,结果采用BioNumerics 6.1数据库软件进行聚类分析和单位点Hunter-Gaston分辨率指数(Hunter-Gaston index,HGI)分析,并比较分析国际推荐的分组(12、15、24位点)的

  14. Culture and molecular method for detection of Mycobacterium tuberculosis complex and Mycobacterium avium subsp. paratuberculosis in milk and dairy products.

    Science.gov (United States)

    Messelhäusser, U; Kämpf, P; Hörmansdorfer, S; Wagner, B; Schalch, B; Busch, U; Höller, C; Wallner, P; Barth, G; Rampp, A

    2012-01-01

    A combined molecular and cultural method for the detection of the Mycobacterium tuberculosis complex (MTBC) and Mycobacterium avium subsp. paratuberculosis was developed and tested with artificially contaminated milk and dairy products. Results indicate that the method can be used for a reliable detection as a basis for first risk assessments.

  15. Inactivation of Mycobacterium paratuberculosis and Mycobacterium tuberculosis in fresh soft cheese by gamma radiation

    Energy Technology Data Exchange (ETDEWEB)

    Badr, Hesham M., E-mail: heshambadr_aea@yahoo.co.uk [Atomic Energy Authority, Nuclear Research Center, Abou Zaabal, P.O. Box 13759 Cairo (Egypt)

    2011-11-15

    The effectiveness of gamma irradiation on the inactivation of Mycobacterium paratuberculosis, Mycobacterium bovis and Mycobacterium tuberculosis in fresh soft cheese that prepared from artificially inoculated milk samples was studied. Irradiation at dose of 2 kGy was sufficient for the complete inactivation of these mycobacteria as they were not detected in the treated samples during storage at 4{+-}1 {sup o}C for 15 days. Moreover, irradiation of cheese samples, that were prepared from un-inoculated milk, at this effective dose had no significant effects on their gross composition and contents from riboflavin, niacin and pantothenic acid, while significant decreases in vitamin A and thiamin were observed. In addition, irradiation of cheese samples had no significant effects on their pH and nitrogen fractions contents, except for the contents of ammonia, which showed a slight, but significant, increases due to irradiation. The analysis of cheese fats indicated that irradiation treatment induced significant increase in their oxidation parameters and contents from free fatty acids; however, the observed increases were relatively low. On the other hand, irradiation of cheese samples induced no significant alterations on their sensory properties. Thus, irradiation dose of 2 kGy can be effectively applied to ensure the safety of soft cheese with regards to these harmful mycobacteria. - Highlights: > We examined the effectiveness of gamma irradiation on inactivation of Mycobacterium paratuberculosis, Mycobacterium bovis and Mycobacterium tuberculosis in fresh soft cheese. > Irradiation at dose of 2 kGy was sufficient for complete inactivation of these mycobacteria. > Irradiation of cheese samples induced no significant alterations on their sensory properties.

  16. The human antibody response to the surface of Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Casey C Perley

    Full Text Available Vaccine-induced human antibodies to surface components of Haemophilus influenzae and Streptococcus pneumonia are correlated with protection. Monoclonal antibodies to surface components of Mycobacterium tuberculosis are also protective in animal models. We have characterized human antibodies that bind to the surface of live M. tuberculosis.Plasma from humans with latent tuberculosis (TB infection (n = 23, active TB disease (n = 40, and uninfected controls (n = 9 were assayed by ELISA for reactivity to the live M. tuberculosis surface and to inactivated M. tuberculosis fractions (whole cell lysate, lipoarabinomannan, cell wall, and secreted proteins.When compared to uninfected controls, patients with active TB disease had higher antibody titers to the surface of live M. tuberculosis (Δ = 0.72 log10, whole cell lysate (Δ = 0.82 log10, and secreted proteins (Δ = 0.62 log10, though there was substantial overlap between the two groups. Individuals with active disease had higher relative IgG avidity (Δ = 1.4 to 2.6 to all inactivated fractions. Surprisingly, the relative IgG avidity to the live M. tuberculosis surface was lower in the active disease group than in uninfected controls (Δ =  -1.53, p = 0.004. Patients with active disease had higher IgG than IgM titers for all inactivated fractions (ratios, 2.8 to 10.1, but equal IgG and IgM titers to the live M. tuberculosis surface (ratio, 1.1. Higher antibody titers to the M. tuberculosis surface were observed in active disease patients who were BCG-vaccinated (Δ = 0.55 log10, p = 0.008, foreign-born (Δ = 0.61 log10, p = 0.004, or HIV-seronegative (Δ = 0.60 log10, p = 0.04. Higher relative IgG avidity scores to the M. tuberculosis surface were also observed in active disease patients who were BCG-vaccinated (Δ = 1.12, p < 0.001 and foreign-born (Δ = 0.87, p = 0.01.Humans with active TB disease produce antibodies to the surface of M. tuberculosis with low avidity and with a low IgG/IgM ratio

  17. The Human Antibody Response to the Surface of Mycobacterium tuberculosis

    Science.gov (United States)

    Perley, Casey C.; Frahm, Marc; Click, Eva M.; Dobos, Karen M.; Ferrari, Guido; Stout, Jason E.; Frothingham, Richard

    2014-01-01

    Background Vaccine-induced human antibodies to surface components of Haemophilus influenzae and Streptococcus pneumonia are correlated with protection. Monoclonal antibodies to surface components of Mycobacterium tuberculosis are also protective in animal models. We have characterized human antibodies that bind to the surface of live M. tuberculosis. Methods Plasma from humans with latent tuberculosis (TB) infection (n = 23), active TB disease (n = 40), and uninfected controls (n = 9) were assayed by ELISA for reactivity to the live M. tuberculosis surface and to inactivated M. tuberculosis fractions (whole cell lysate, lipoarabinomannan, cell wall, and secreted proteins). Results When compared to uninfected controls, patients with active TB disease had higher antibody titers to the surface of live M. tuberculosis (Δ = 0.72 log10), whole cell lysate (Δ = 0.82 log10), and secreted proteins (Δ = 0.62 log10), though there was substantial overlap between the two groups. Individuals with active disease had higher relative IgG avidity (Δ = 1.4 to 2.6) to all inactivated fractions. Surprisingly, the relative IgG avidity to the live M. tuberculosis surface was lower in the active disease group than in uninfected controls (Δ = –1.53, p = 0.004). Patients with active disease had higher IgG than IgM titers for all inactivated fractions (ratios, 2.8 to 10.1), but equal IgG and IgM titers to the live M. tuberculosis surface (ratio, 1.1). Higher antibody titers to the M. tuberculosis surface were observed in active disease patients who were BCG-vaccinated (Δ = 0.55 log10, p = 0.008), foreign-born (Δ = 0.61 log10, p = 0.004), or HIV-seronegative (Δ = 0.60 log10, p = 0.04). Higher relative IgG avidity scores to the M. tuberculosis surface were also observed in active disease patients who were BCG-vaccinated (Δ = 1.12, p<0.001) and foreign-born (Δ = 0.87, p = 0.01). Conclusions/Significance Humans

  18. High resolution, on-line identification of strains from the Mycobacterium tuberculosis complex based on tandem repeat typing

    Directory of Open Access Journals (Sweden)

    Denoeud France

    2002-11-01

    Full Text Available Abstract Background Currently available reference methods for the molecular epidemiology of the Mycobacterium tuberculosis complex either lack sensitivity or are still too tedious and slow for routine application. Recently, tandem repeat typing has emerged as a potential alternative. This report contributes to the development of tandem repeat typing for M. tuberculosis by summarising the existing data, developing additional markers, and setting up a freely accessible, fast, and easy to use, internet-based service for strain identification. Results A collection of 21 VNTRs incorporating 13 previously described loci and 8 newly evaluated markers was used to genotype 90 strains from the M. tuberculosis complex (M. tuberculosis (64 strains, M. bovis (9 strains including 4 BCG representatives, M. africanum (17 strains. Eighty-four different genotypes are defined. Clustering analysis shows that the M. africanum strains fall into three main groups, one of which is closer to the M. tuberculosis strains, and an other one is closer to the M. bovis strains. The resulting data has been made freely accessible over the internet http://bacterial-genotyping.igmors.u-psud.fr/bnserver to allow direct strain identification queries. Conclusions Tandem-repeat typing is a PCR-based assay which may prove to be a powerful complement to the existing epidemiological tools for the M. tuberculosis complex. The number of markers to type depends on the identification precision which is required, so that identification can be achieved quickly at low cost in terms of consumables, technical expertise and equipment.

  19. Synthetic Long Peptide Derived from Mycobacterium tuberculosis Latency Antigen Rv1733c Protects against Tuberculosis.

    Science.gov (United States)

    Coppola, Mariateresa; van den Eeden, Susan J F; Wilson, Louis; Franken, Kees L M C; Ottenhoff, Tom H M; Geluk, Annemieke

    2015-09-01

    Responsible for 9 million new cases of active disease and nearly 2 million deaths each year, tuberculosis (TB) remains a global health threat of overwhelming dimensions. Mycobacterium bovis BCG, the only licensed vaccine available, fails to confer lifelong protection and to prevent reactivation of latent infection. Although 15 new vaccine candidates are now in clinical trials, an effective vaccine against TB remains elusive, and new strategies for vaccination are vital. BCG vaccination fails to induce immunity against Mycobacterium tuberculosis latency antigens. Synthetic long peptides (SLPs) combined with adjuvants have been studied mostly for therapeutic cancer vaccines, yet not for TB, and proved to induce efficient antitumor immunity. This study investigated an SLP derived from Rv1733c, a major M. tuberculosis latency antigen which is highly expressed by "dormant" M. tuberculosis and well recognized by T cells from latently M. tuberculosis-infected individuals. In order to assess its in vivo immunogenicity and protective capacity, Rv1733c SLP in CpG was administered to HLA-DR3 transgenic mice. Immunization with Rv1733c SLP elicited gamma interferon-positive/tumor necrosis factor-positive (IFN-γ(+)/TNF(+)) and IFN-γ(+) CD4(+) T cells and Rv1733c-specific antibodies and led to a significant reduction in the bacterial load in the lungs of M. tuberculosis-challenged mice. This was observed both in a pre- and in a post-M. tuberculosis challenge setting. Moreover, Rv1733c SLP immunization significantly boosted the protective efficacy of BCG, demonstrating the potential of M. tuberculosis latency antigens to improve BCG efficacy. These data suggest a promising role for M. tuberculosis latency antigen Rv1733c-derived SLPs as a novel TB vaccine approach, both in a prophylactic and in a postinfection setting.

  20. Enhanced Amplified Mycobacterium Tuberculosis Direct Test for Detection of Mycobacterium tuberculosis Complex in Positive BACTEC 12B Broth Cultures of Respiratory Specimens

    OpenAIRE

    1999-01-01

    The reliability of the Gen-Probe enhanced Amplified Mycobacterium Tuberculosis Direct Test (MTD) for identification of Mycobacterium tuberculosis complex (MTBC) in BACTEC 12B broth cultures of respiratory specimens was evaluated by testing aliquots from 268 bottles with a growth index of ≥50. MTD results were compared to those obtained by usual laboratory protocol, whereby MTBC was identified by DNA probe (Gen-Probe, Inc.) testing sediment from broth samples or colonies on a solid medium. For...

  1. Inhibition of Mycobacterium tuberculosis Glutamine Synthetase as a Novel Antibiotic Strategy against Tuberculosis: Demonstration of Efficacy In Vivo

    OpenAIRE

    2003-01-01

    Tuberculosis remains one of humankind's greatest killers, and new therapeutic strategies are needed to combat the causative agent, Mycobacterium tuberculosis, which is rapidly developing resistance to conventional antibiotics. Using the highly demanding guinea pig model of pulmonary tuberculosis, we have investigated the feasibility of inhibiting M. tuberculosis glutamine synthetase (GS), an enzyme that plays a key role in both nitrogen metabolism and cell wall biosynthesis, as a novel antibi...

  2. Interplay between Mutations and Efflux in Drug Resistant Clinical Isolates of Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Miguel Viveiros

    2017-04-01

    Full Text Available Numerous studies show efflux as a universal bacterial mechanism contributing to antibiotic resistance and also that the activity of the antibiotics subject to efflux can be enhanced by the combined use of efflux inhibitors. Nevertheless, the contribution of efflux to the overall drug resistance levels of clinical isolates of Mycobacterium tuberculosis is poorly understood and still is ignored by many. Here, we evaluated the contribution of drug efflux plus target-gene mutations to the drug resistance levels in clinical isolates of M. tuberculosis. A panel of 17 M. tuberculosis clinical strains were characterized for drug resistance associated mutations and antibiotic profiles in the presence and absence of efflux inhibitors. The correlation between the effect of the efflux inhibitors and the resistance levels was assessed by quantitative drug susceptibility testing. The bacterial growth/survival vs. growth inhibition was analyzed through the comparison between the time of growth in the presence and absence of an inhibitor. For the same mutation conferring antibiotic resistance, different MICs were observed and the different resistance levels found could be reduced by efflux inhibitors. Although susceptibility was not restored, the results demonstrate the existence of a broad-spectrum synergistic interaction between antibiotics and efflux inhibitors. The existence of efflux activity was confirmed by real-time fluorometry. Moreover, the efflux pump genes mmr, mmpL7, Rv1258c, p55, and efpA were shown to be overexpressed in the presence of antibiotics, demonstrating the contribution of these efflux pumps to the overall resistance phenotype of the M. tuberculosis clinical isolates studied, independently of the genotype of the strains. These results showed that the drug resistance levels of multi- and extensively-drug resistant M. tuberculosis clinical strains are a combination between drug efflux and the presence of target-gene mutations, a reality

  3. Molecular diversity of Mycobacterium tuberculosis strains indifferent provinces of Iran.

    Directory of Open Access Journals (Sweden)

    Mohadese Mozafari

    2013-12-01

    Full Text Available Molecular epidemiology tools are widely used in determining epidemiology of tuberculosis. Spoligotyping is a molecular epidemiology method that is used for characterization and typing of Mycobacterium tuberculosis complex strains. The method is based on polymorphism of the chromosomal DR locus consisting of identical 36-bp DRs alternating with 35-41 unique spacers. The objective of this study was to investigate the prevalence of M. tuberculosis spoligotypes in different provinces of Iran.M. tuberculosis strains were isolated from TB patients of Mycobacteriology Research center (MRC. DNA was extracted from patient's clinical samples. PCR was performed by using of specific primers for DR region. The amplified DNA was hybridized to the spoligotyping Membrane. Hybridized DNA was detected with ECL detection kit and by exposing ECL Hyperfilm to the membrane. The obtained result was entered to a binary format and was analyzed using SpolDB4 database.Spoligotyping resulted in 136 different patterns. Out of 1242 M. tuberculosis strains, 1165 strains (93.8% were classified into 59 clusters and the remaining strains (6.2 % were singleton.The results of present study showed that strains of CAS family were more prevalent than other strains in Iran. Other prevalent families were Haarlem, T and Beijing, respectively.

  4. Gene Transfer in Mycobacterium tuberculosis: Shuttle Phasmids to Enlightenment.

    Science.gov (United States)

    Jacobs, William R

    2014-04-01

    Infectious diseases have plagued humankind throughout history and have posed serious public health problems. Yet vaccines have eradicated smallpox and antibiotics have drastically decreased the mortality rate of many infectious agents. These remarkable successes in the control of infections came from knowing the causative agents of the diseases, followed by serendipitous discoveries of attenuated viruses and antibiotics. The discovery of DNA as genetic material and the understanding of how this information translates into specific phenotypes have changed the paradigm for developing new vaccines, drugs, and diagnostic tests. Knowledge of the mechanisms of immunity and mechanisms of action of drugs has led to new vaccines and new antimicrobial agents. The key to the acquisition of the knowledge of these mechanisms has been identifying the elemental causes (i.e., genes and their products) that mediate immunity and drug resistance. The identification of these genes is made possible by being able to transfer the genes or mutated forms of the genes into causative agents or surrogate hosts. Such an approach was limited in Mycobacterium tuberculosis by the difficulty of transferring genes or alleles into M. tuberculosis or a suitable surrogate mycobacterial host. The construction of shuttle phasmids-chimeric molecules that replicate in Escherichia coli as plasmids and in mycobacteria as mycobacteriophages-was instrumental in developing gene transfer systems for M. tuberculosis. This review will discuss M. tuberculosis genetic systems and their impact on tuberculosis research.

  5. Killing of Mycobacterium tuberculosis by neutrophils: a nonoxidative process.

    Science.gov (United States)

    Jones, G S; Amirault, H J; Andersen, B R

    1990-09-01

    To determine the role of oxygen radicals in the killing of Mycobacterium tuberculosis by neutrophils, the effects of free-radical inhibitors and enzymes, catalase, superoxide dismutase, taurine, deferoxamine, and histidine were evaluated. Changes in the viability of M. tuberculosis were determined by agar plate colony counts and a radiometric assay. No impairment in killing was seen with any of the inhibitors or enzymes. Patients with chronic granulomatous disease (CGD) have a defect in the NADPH oxidase pathway, causing their neutrophils to be unable to generate oxygen radicals. If these radicals are involved in killing, then CGD neutrophils should be less effective killers of M. tuberculosis than normal neutrophils. There was no evidence by either measure of M. tuberculosis viability that CGD neutrophils were less bactericidal than normal neutrophils. Killing by normal neutrophils was also effective in the absence of serum. These results lead to the conclusion that the mechanism by which M. tuberculosis is killed by neutrophils is independent of the oxygen metabolic burst.

  6. How B cells shape the immune response against Mycobacterium tuberculosis.

    Science.gov (United States)

    Maglione, Paul J; Chan, John

    2009-03-01

    Extensive work illustrating the importance of cellular immune mechanisms for protection against Mycobacterium tuberculosis has largely relegated B-cell biology to an afterthought within the tuberculosis (TB) field. However, recent studies have illustrated that B lymphocytes, through a variety of interactions with the cellular immune response, play previously underappreciated roles in shaping host defense against non-viral intracellular pathogens, including M. tuberculosis. Work in our laboratory has recently shown that, by considering these lymphocytes more broadly within their variety of interactions with cellular immunity, B cells have a significant impact on the outcome of airborne challenge with M. tuberculosis as well as the resultant inflammatory response. In this review, we advocate for a revised view of TB immunology in which roles of cellular and humoral immunity are not mutually exclusive. In the context of our current understanding of host defense against non-viral intracellular infections, we review recent data supporting a more significant role of B cells during M. tuberculosis infection than previously thought.

  7. Masquerading Mycobacterium: Rectal Growth or Tuberculosis?

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    Nabajit Choudhury

    2013-01-01

    Full Text Available A 37-year old male presented to us with history of lower abdominal pain for 6 months. His physical examination revealed a rectal mass of approximately 1centimeter. He was investigated for possible rectal growth with sigmoidoscopy and biopsy. The histopathological examination (HPE showed a non-specific chronic inflammation in the tissue from the mass. Another tissue from the mass was sent for polymerase chain reaction (PCR for tuberculosis, which turned out to be positive. The patient was started on standard anti tubercular (ATT regimen and responded completely to the treatment. We discuss the patient and review some of the available literature on the topic and discuss the issue of considering a diagnosis of tuberculosis in cases with rectal mass specially when it has become a major public health issue with increasing number of HIV (Human Immunodeficiency Virus infected patients.

  8. Genomic Epidemiology of a Major Mycobacterium tuberculosis Outbreak

    DEFF Research Database (Denmark)

    Folkvardsen, Dorte Bek; Norman, Anders; Andersen, Åse Bengård

    2017-01-01

    Since 1992, Denmark has documented the largest outbreak of tuberculosis in Scandinavia ascribed to a single genotype, termed C2/1112-15. As of spring 2017, the International Reference Laboratory of Mycobacteriology in Copenhagen has collected and identified isolates from more than a thousand cases...

  9. Dielectrophoretic characterization of antibiotic-treated Mycobacterium tuberculosis complex cells.

    Science.gov (United States)

    Inoue, Shinnosuke; Lee, Hyun-Boo; Becker, Annie L; Weigel, Kris M; Kim, Jong-Hoon; Lee, Kyong-Hoon; Cangelosi, Gerard A; Chung, Jae-Hyun

    2015-10-01

    Multi-drug resistant tuberculosis (MDR-TB) has become a serious concern for proper treatment of patients. As a phenotypic method, dielectrophoresis can be useful but is yet to be attempted to evaluate Mycobacterium tuberculosis complex cells. This paper investigates the dielectrophoretic behavior of Mycobacterium bovis (Bacillus Calmette-Guérin, BCG) cells that are treated with heat or antibiotics rifampin (RIF) or isoniazid (INH). The experimental parameters are designed on the basis of our sensitivity analysis. The medium conductivity (σ(m)) and the frequency (f) for a crossover frequency (f(xo1)) test are decided to detect the change of σ(m)-f(xo1) in conjunction with the drug mechanism. Statistical modeling is conducted to estimate the distributions of viable and nonviable cells from the discrete measurement of f (xo1). Finally, the parameters of the electrophysiology of BCG cells, C(envelope) and σ(cyto), are extracted through a sampling algorithm. This is the first evaluation of the dielectrophoresis (DEP) approach as a means to assess the effects of antimicrobial drugs on M. tuberculosis complex cells.

  10. Targeting Mycobacterium tuberculosis topoisomerase I by small-molecule inhibitors.

    Science.gov (United States)

    Godbole, Adwait Anand; Ahmed, Wareed; Bhat, Rajeshwari Subray; Bradley, Erin K; Ekins, Sean; Nagaraja, Valakunja

    2015-03-01

    We describe inhibition of Mycobacterium tuberculosis topoisomerase I (MttopoI), an essential mycobacterial enzyme, by two related compounds, imipramine and norclomipramine, of which imipramine is clinically used as an antidepressant. These molecules showed growth inhibition of both Mycobacterium smegmatis and M. tuberculosis cells. The mechanism of action of these two molecules was investigated by analyzing the individual steps of the topoisomerase I (topoI) reaction cycle. The compounds stimulated cleavage, thereby perturbing the cleavage-religation equilibrium. Consequently, these molecules inhibited the growth of the cells overexpressing topoI at a low MIC. Docking of the molecules on the MttopoI model suggested that they bind near the metal binding site of the enzyme. The DNA relaxation activity of the metal binding mutants harboring mutations in the DxDxE motif was differentially affected by the molecules, suggesting that the metal coordinating residues contribute to the interaction of the enzyme with the drug. Taken together, the results highlight the potential of these small molecules, which poison the M. tuberculosis and M. smegmatis topoisomerase I, as leads for the development of improved molecules to combat mycobacterial infections. Moreover, targeting metal coordination in topoisomerases might be a general strategy to develop new lead molecules.

  11. Mycobacterium tuberculosis: ecology and evolution of a human bacterium.

    Science.gov (United States)

    Bañuls, Anne-Laure; Sanou, Adama; Anh, Nguyen Thi Van; Godreuil, Sylvain

    2015-11-01

    Some species of the Mycobacterium tuberculosis complex (MTBC), particularly Mycobacterium tuberculosis, which causes human tuberculosis (TB), are the first cause of death linked to a single pathogen worldwide. In the last decades, evolutionary studies have much improved our knowledge on MTBC history and have highlighted its long co-evolution with humans. Its ability to remain latent in humans, the extraordinary proportion of asymptomatic carriers (one-third of the entire human population), the deadly epidemics and the observed increasing level of resistance to antibiotics are proof of its evolutionary success. Many MTBC molecular signatures show not only that these bacteria are a model of adaptation to humans but also that they have influenced human evolution. Owing to the unbalance between the number of asymptomatic carriers and the number of patients with active TB, some authors suggest that infection by MTBC could have a protective role against active TB disease and also against other pathologies. However, it would be inappropriate to consider these infectious pathogens as commensals or symbionts, given the level of morbidity and mortality caused by TB.

  12. Genetic Lineages of Mycobacterium tuberculosis Isolates in Isfahan, Iran.

    Science.gov (United States)

    Riyahi Zaniani, Fatemeh; Moghim, Sharareh; Mirhendi, Hossein; Ghasemian Safaei, Hajieh; Fazeli, Hossein; Salehi, Mahshid; Nasr Esfahani, Bahram

    2017-01-01

    In this study, we aimed to identify the genetic lineages of Mycobacterium tuberculosis isolates in Isfahan via the mycobacterial interspersed repetitive-unit-variable number tandem repeat typing method based on 15 loci. Forty-nine M. tuberculosis isolates were collected between 2013 and 2015 from Tuberculosis patients in Mollahadi Sabzevari Tuberculosis Center in Isfahan. All isolates were typed by 15-locus MIRU-VNTR typing. The highest percentage of isolates, 44.89 % (22/49), belonged to the Euro-American lineage, while the frequencies of the East-African-Indian, East-Asian, and Indo-Oceanic lineages were 28.57 % (14/49), 24.4 % (12/49), and 2.04 % (1/49), respectively. Among the 22 isolates of the Euro-American lineage, those belonging to the NEW-1 sub-lineage were most prevalent (24.4 %). Approximately, the same proportion of isolates belonging to the Delhi/CAS, Beijing, and NEW-1 sub-lineages were identified in Iranian and Afghan immigrant patients. The Delhi/CAS and Beijing sub-lineage isolates were prevalent among patients who had been previously treated for TB. Results showed that all of the 49 MIRU-VNTR patterns were unique and the clustering rate of the 15-locus MIRU-VNTR was 0.0 (minimum recent transmission). The results of this study show that the lineages of M. tuberculosis isolates in Isfahan are similar to those reported in the Eastern Mediterranean region (indicative of the epidemiological relationship between the countries in the region). The low clustering rate in our results reveals that transmission of tuberculosis in Isfahan is, in most cases, a reactivation of previous tuberculosis infection and the role of recently transmitted disease is minor.

  13. Combating highly resistant emerging pathogen Mycobacterium abscessus and Mycobacterium tuberculosis with novel salicylanilide esters and carbamates.

    Science.gov (United States)

    Baranyai, Zsuzsa; Krátký, Martin; Vinšová, Jarmila; Szabó, Nóra; Senoner, Zsuzsanna; Horváti, Kata; Stolaříková, Jiřina; Dávid, Sándor; Bősze, Szilvia

    2015-08-28

    In the Mycobacterium genus over one hundred species are already described and new ones are periodically reported. Species that form colonies in a week are classified as rapid growers, those requiring longer periods (up to three months) are the mostly pathogenic slow growers. More recently, new emerging species have been identified to lengthen the list, all rapid growers. Of these, Mycobacterium abscessus is also an intracellular pathogen and it is the most chemotherapy-resistant rapid-growing mycobacterium. In addition, the cases of multidrug-resistant Mycobacterium tuberculosis infection are also increasing. Therefore there is an urgent need to find new active molecules against these threatening strains. Based on previous results, a series of salicylanilides, salicylanilide 5-chloropyrazinoates and carbamates was designed, synthesized and characterised. The compounds were evaluated for their in vitro activity on M. abscessus, susceptible M. tuberculosis H37Rv, multidrug-resistant (MDR) M. tuberculosis MDR A8, M. tuberculosis MDR 9449/2006 and on the extremely-resistant Praha 131 (XDR) strains. All derivatives exhibited a significant activity with minimum inhibitory concentrations (MICs) in the low micromolar range. Eight salicylanilide carbamates and two salicylanilide esters exhibited an excellent in vitro activity on M. abscessus with MICs from 0.2 to 2.1 μM, thus being more effective than ciprofloxacin and gentamicin. This finding is potentially promising, particularly, as M. abscessus is a threateningly chemotherapy-resistant species. M. tuberculosis H37Rv was inhibited with MICs from 0.2 μM, and eleven compounds have lower MICs than isoniazid. Salicylanilide esters and carbamates were found that they were effective also on MDR and XDR M. tuberculosis strains with MICs ≥1.0 μM. The in vitro cytotoxicity (IC50) was also determined on human MonoMac-6 cells, and selectivity index (SI) of the compounds was established. In general, salicylanilide

  14. Diffuse Type Primary Mycobacterium Tuberculosis of the Breast: A Case Report

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hyun A; Kang, Bong Joo; Kim, Sung Hun; Kim, Hnana; Lee, Ah Won [Seoul St. Mary' s Hospital, The Catholic University of Korea, Seoul (Korea, Republic of)

    2011-12-15

    Tuberculous mastitis is a rare manifestation of mycobacterium tuberculosis infection. It mimics inflammatory breast cancer or other pyogenic inflammations. In most of the tuberculous mastitis reports, coexisting or prior tuberculosis infection and secondary infection of the breast by direct spread via axillary or cervical lymphadenopathy, or hematogenous spread have been noted. We describe the mammographic and ultrasonographic findings of a case of diffuse type mycobacterium tuberculosis of the breast showing diffuse edema which was confirmed as tuberculosis through biopsy and had no evidence of old or concurrent pulmonary tuberculosis on chest computed tomography

  15. Tuberculosis Caused by Mycobacterium bovis in a Capybara (Hydrochoerus hydrochaeris).

    Science.gov (United States)

    Mol, J P S; Carvalho, T F; Fonseca, A A; Sales, E B; Issa, M A; Rezende, L C; Hodon, M A; Tinoco, H P; Malta, M C C; Pessanha, A T; Pierezan, F; Mota, P M P C; Paixão, T A; Santos, R L

    2016-01-01

    Tuberculosis, associated with Mycobacterium bovis, was diagnosed post mortem in an adult female capybara (Hydrochoerus hydrochaeris), kept at the Pampulha Ecological Park, Belo Horizonte, Brazil, in a large metropolitan area. On post-mortem examination, there were numerous firm white nodules scattered throughout all lobes of both lungs. Tissue samples were collected for histological and microbiological examination. Microscopically, the pulmonary nodules were multifocal to coalescing granulomas and intralesional acid-fast bacilli were evident in Ziehl-Neelsen-stained sections of the lung and spleen. Colonies with morphological features of Mycobacterium spp. were isolated from lung samples and conventional polymerase chain reaction (PCR) with genomic DNA from the isolates was positive for M. bovis; sequencing indicated 100% identity with the region of difference 4 (RD4) of M. bovis. In addition, M. bovis DNA was detected in the lung by quantitative PCR. The finding of M. bovis in a capybara indicates a potential public health risk in a zoological collection.

  16. High prevalence of shared international type 53 among Mycobacterium tuberculosis complex strains in retreated patients from Cote d'Ivoire.

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    Timothée Ouassa

    Full Text Available BACKGROUND: Genotyping methods are useful tools to provide information on tuberculosis epidemic. They can allow a better response from health authorities and the implementation of measures for tuberculosis control. This study aimed to identify the main lineages and clades of Mycobacterium tuberculosis complex strains circulating in Côte d'Ivoire. METHODS/MAIN FINDINGS: Strains isolated from sputum samples of patients ongoing retreatment from all the country were characterized by spoligotyping and by MIRU-VNTR. Profiles obtained by spoligotyping were first compared to the SITVIT/SpolDB4 database for family assignment. Of 194 strains analysed, 146 (75.3% belonged to the T lineage. The most predominant spoligotype was the shared international type 53 with 135 strains (69.6%. In contrast with neighbouring countries, LAM (11 strains, 5.7% and H (9 strains 4.6% lineages were slightly represented. Only 3 Beijing strains (1.5% and 4 strains of Mycobacterium africanum (2% were found. Analysis of the results obtained with MIRU-VNTR revealed also a high level of clustering. CONCLUSION/SIGNIFICANCE: The population of Mycobacterium tuberculosis complex strains among retreatment cases in Côte d'Ivoire exhibits a low diversity, allowing to assume recent transmission and locally based infection.

  17. Characterisation of Mycobacterium tuberculosis isolates lacking IS6110 in Viet Nam.

    NARCIS (Netherlands)

    Huyen, M.N.; Tiemersma, E.W.; Kremer, K.; Haas, P. de; Lan, N.T.; Buu, T.N.; Sola, C.; Cobelens, F.G.; Soolingen, D. van

    2013-01-01

    SETTING: The molecular diagnosis of tuberculosis (TB) in Viet Nam is often based on the detection of insertion sequence (IS) 6110 in Mycobacterium tuberculosis. However, 8-11% of M. tuberculosis strains in South-East Asia do not contain this target and this undermines the validity of these molecular

  18. The zoonotic importance of Mycobacterium tuberculosis : transmission from human to monkey : case report

    Directory of Open Access Journals (Sweden)

    A.L. Michel

    1998-07-01

    Full Text Available A case of zoonotic Mycobacterium tuberculosis infection in a marmoset (Callithrix jacchus is reported. Genomic typing of the relevant M. tuberculosis isolates strongly suggests that the marmoset, which was kept as companion animal, acquired the disease from an infected member in the household who had been treated for pulmonary tuberculosis 8 years prior to this case.

  19. Characterisation of pks15/1 in clinical isolates of Mycobacterium tuberculosis from Mexico

    Directory of Open Access Journals (Sweden)

    Roberto Zenteno-Cuevas

    2013-09-01

    Full Text Available Tuberculosis (TB is an infectocontagious respiratory disease caused by members of the Mycobacterium tuberculosis complex. A 7 base pair (bp deletion in the locus polyketide synthase (pks15/1 is described as polymorphic among members of the M. tuberculosis complex, enabling the identification of Euro-American, Indo-Oceanic and Asian lineages. The aim of this study was to characterise this locus in TB isolates from Mexico. One hundred twenty clinical isolates were recovered from the states of Veracruz and Estado de Mexico. We determined the nucleotide sequence of a ± 400 bp fragment of the locus pks15/1, while genotypic characterisation was performed by spoligotyping. One hundred and fifty isolates contained the 7 bp deletion, while five had the wild type locus. Lineages X (22%, LAM (18% and T (17% were the most frequent; only three (2% of the isolates were identified as Beijing and two (1% EAI-Manila. The wild type pks15/1 locus was observed in all Asian lineage isolates tested. Our results confirm the utility of locus pks15/1 as a molecular marker for identifying Asian lineages of the M. tuberculosis complex. This marker could be of great value in the epidemiological surveillance of TB, especially in countries like Mexico, where the prevalence of such lineages is unknown.

  20. Genomic epidemiology of multidrug-resistant Mycobacterium tuberculosis during transcontinental spread.

    Science.gov (United States)

    Coscolla, Mireia; Barry, Pennan M; Oeltmann, John E; Koshinsky, Heather; Shaw, Tambi; Cilnis, Martin; Posey, Jamie; Rose, Jordan; Weber, Terry; Fofanov, Viacheslav Y; Gagneux, Sebastien; Kato-Maeda, Midori; Metcalfe, John Z

    2015-07-15

    The transcontinental spread of multidrug-resistant (MDR) tuberculosis is poorly characterized in molecular epidemiologic studies. We used genomic sequencing to understand the establishment and dispersion of MDR Mycobacterium tuberculosis within a group of immigrants to the United States. We used a genomic epidemiology approach to study a genotypically matched (by spoligotype, IS6110 restriction fragment length polymorphism, and mycobacterial interspersed repetitive units-variable number of tandem repeat signature) lineage 2/Beijing MDR strain implicated in an outbreak of tuberculosis among refugees in Thailand and consecutive cases within California. All 46 MDR M. tuberculosis genomes from both Thailand and California were highly related, with a median difference of 10 single-nucleotide polymorphisms (SNPs). The Wat Tham Krabok (WTK) strain is a new sequence type distinguished from all known Beijing strains by 55 SNPs and a genomic deletion (Rv1267c) associated with increased fitness. Sequence data revealed a highly prevalent MDR strain that included several closely related but distinct allelic variants within Thailand, rather than the occurrence of a single outbreak. In California, sequencing data supported multiple independent introductions of WTK with subsequent transmission and reactivation within the state, as well as a potential super spreader with a prolonged infectious period. Twenty-seven drug resistance-conferring mutations and 4 putative compensatory mutations were found within WTK strains. Genomic sequencing has substantial epidemiologic value in both low- and high-burden settings in understanding transmission chains of highly prevalent MDR strains.

  1. Spoligotyping of Mycobacterium tuberculosis isolates at a tertiary care hospital in India.

    Science.gov (United States)

    Suzana, Shirly; Shanmugam, Sivakumar; Uma Devi, K R; Swarna Latha, P N; Michael, Joy S

    2017-06-01

    Spoligotyping is a valuable genotyping tool to study the genetic diversity and molecular epidemiology of Mycobacterium tuberculosis (M. tb). The aim of this study was to analyse different spoligotype patterns of M. tb strains isolated from patients with tuberculosis from different parts of India. A total of 163 M. tb isolates were spoligotyped between January 2014 and January 2015. About 47% (n = 77) were from patients with extrapulmonary tuberculosis; of these, 10 were MDR, and seven were Pre-XDR. Of the 86 M. tb isolates from patients with pulmonary tuberculosis, 25 were MDR, and 25 were Pre-XDR. We found 61 spoligo patterns, 128 clusters in the spoligotype data base (spoldb4 data base) with spoligo international type (SIT) number and 35 true unique isolates. The most pre-dominant spoligotype was EAI lineage (56), followed by Beijing (28), CAS (20), T(9), U(7), X(3), H(3), BOVIS_1 BCG(1) and LAM(1). Although our study identified EAI, CAS and Beijing strain lineages as pre-dominant, we also found a large number of orphan strains (20%) in our study. Beijing strains were more significantly associated with MDR TB than CAS and EAI lineages. Further studies on large sample sizes would help to clearly describe the epidemiology of M. tb in India. © 2017 John Wiley & Sons Ltd.

  2. Prevalence of Multidrug Resistant Mycobacterium tuberculosis by Mycobacteria growth

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    Livani S

    2012-01-01

    Full Text Available Background and objectives: Identification and monitoring ofmultidrugresistant Mycobacterium tuberculosis strains (MDR ishighlighted by the high risk of their spreading in different areas.Prevalence of these strains was evaluated in Golestan province innortheast of Iran.Material and Methods: Drug susceptibility testing to Isoniazid andrifampin was carried out for 148 clinical samples that had grown inMycobacteria growth indicator tube (MGIT system, according to themanufacturer's instructions (Becton-Dickinson, USA. The associationof drug resistance frequency with demographic characteristics andgrowth time were investigated. The appropriate statistical tests, X2 andstudent Ttest were performed for comparison of these variants. A pvalue>0.05 was considered significant in all cases.Results: The turnaround time required for growth of Mycobacteriumtuberculosis in MGIT system was between 2 to 55 days (mean16.3±10.4 days. Of all samples studied, 17.6% and 3.4% wereresistant to Isoniazid and rifampin, respectively, and 3.4% (5 sampleswere MDR (CI 95%; 1- 6%. The turnaround time required fordetermining MDR cases was 9.6 days. No statistically significantassociation was found between the resistance to the drugs and none ofthe factors including sex, age, type of clinical sample, and positivity ofthe smear.Conclusion: The prevalence of MDR in the studied region wasdetermined to be 3.4% which is similar to the country-wideevaluations. The turnaround time for Mycobacterium growth and antidrug susceptibility result can be shortened by MGIT method.Key words: Mycobacterium tuberculosis, Mycobacterium GrowthIndicator Tube, Multidrug Resistant

  3. Using approximate Bayesian computation to estimate tuberculosis transmission parameters from genotype data.

    Science.gov (United States)

    Tanaka, Mark M; Francis, Andrew R; Luciani, Fabio; Sisson, S A

    2006-07-01

    Tuberculosis can be studied at the population level by genotyping strains of Mycobacterium tuberculosis isolated from patients. We use an approximate Bayesian computational method in combination with a stochastic model of tuberculosis transmission and mutation of a molecular marker to estimate the net transmission rate, the doubling time, and the reproductive value of the pathogen. This method is applied to a published data set from San Francisco of tuberculosis genotypes based on the marker IS6110. The mutation rate of this marker has previously been studied, and we use those estimates to form a prior distribution of mutation rates in the inference procedure. The posterior point estimates of the key parameters of interest for these data are as follows: net transmission rate, 0.69/year [95% credibility interval (C.I.) 0.38, 1.08]; doubling time, 1.08 years (95% C.I. 0.64, 1.82); and reproductive value 3.4 (95% C.I. 1.4, 79.7). These figures suggest a rapidly spreading epidemic, consistent with observations of the resurgence of tuberculosis in the United States in the 1980s and 1990s.

  4. Anti-Mycobacterium tuberculosis activity of fungus Phomopsis stipata

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    Karina Andrade de Prince

    2012-03-01

    Full Text Available Our purpose was to determine the anti-Mycobacterium tuberculosis activity of the metabolites produced by the endophitic fungus Phomopsis stipata (Lib. B. Sutton, (Diaporthaceae, cultivated in different media. The antimycobacterial activity was assessed through the Resazurin Microtiter Assay (REMA and the cytotoxicity test performed on macrophage cell line. The extracts derived from fungi grown on Corn Medium and Potato Dextrose Broth presented the smallest values of Minimum Inhibitory Concentration (MIC and low cytotoxicity, which implies a high selectivity index. This is the first report on the chemical composition and antitubercular activity of metabolites of P. stipata, as well as the influence of culture medium on these properties.

  5. Detection of Mycobacterium tuberculosis and Mycobacterium bovis from human sputum samples through multiplex PCR.

    Science.gov (United States)

    Jabbar, Abdul; Khan, Jafar; Ullah, Aman; Rehman, Hazir; Ali, Ijaz

    2015-07-01

    Tuberculosis (TB) has a long history and being present even before the start of recording history. It has left detrimental effects on all aspect of the life and geared the developments in the science of health. TB is caused by Mycobacterium tuberculosis complex (MTBC) including five species M. tuberculosis, M. bovis, M. africanum, M. canetti, and M. microti. M. tuberculosis and M. bovis infect both animals and humans. Therefore, differentiation of these two closely related species is very important for epidemiological and management purpose. We undertook the present study to characterize mycobacteria isolated from sputum of known TB patients by conventional methods and further, by multiplex PCR (mPCR) to detect the prevalence of Zoonotic TB (TB caused by M. bovis). Sputum samples from TB patient were collected from two tertiary care hospitals in Peshawar i.e. Lady Reading Hospital and Hayatabad Medical Complex. All the samples were subjected to Ziehl Neelsen (ZN) stain, culture on Lowenstein Jensen (LJ) and Stone Brink medium, Nitrate reduction test and multiplex PCR. A total of hundred mycobacterial strains were isolated from these samples on the basis of ZN staining, cultural and biochemical methods. Later on, these isolates were subjected to multiplex PCR by using pncATB-1.2 and pncAMT-2 primers specific to M. tuberculosis and JB21, JB22 primers specific to M. bovis. By means of conventional method, these hundred cultures isolates were differentiated into M. tuberculosis (ninety six) and M. bovis (four). Furthermore, by mPCR, it was determined that out of hundred isolates, ninety-eight were identified as M. tuberculosis and two isolates as M. bovis. This molecular method enables to differentiate M. bovis from M. tuberculosis in human sputum.

  6. Predominance of Mycobacterium tuberculosis EAI and Beijing lineages in Yangon, Myanmar.

    Science.gov (United States)

    Phyu, Sabai; Stavrum, Ruth; Lwin, Thandar; Svendsen, Øyvind S; Ti, Ti; Grewal, Harleen M S

    2009-02-01

    Isolates of the Mycobacterium tuberculosis Beijing lineage are associated with high rates of transmission, hypervirulence and drug resistance. The Beijing lineage has been shown to dominate the tuberculosis (TB) epidemic in East Asia; however, the diversity and frequency of M. tuberculosis genotypes from Myanmar are unknown. We present the first comprehensive study describing the M. tuberculosis isolates circulating in Yangon, Myanmar. Thus, 310 isolates from pulmonary TB patients from Yangon, Myanmar, were genotyped by spoligotyping and IS6110-based restriction fragment length polymorphism analysis (IS6110 RFLP). The most frequent lineages observed were the East African-Indian (EAI; 48.4%; n = 150) and Beijing (31.9%; n = 99) lineages. Isolates belonging to the most frequent shared types (STs), ST1 (n = 98; Beijing), ST292 (n = 28; EAI), and ST89 (n = 11; EAI), had >or=75% similarity in their IS6110 patterns. Five of 11 Beijing isolates comprising five clusters with identical IS6110 RFLP patterns could be discriminated by mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) analysis. Of the 150 EAI isolates, 40 isolates (26.7%) had only one IS6110 copy, and 17 of these isolates could be discriminated by MIRU-VNTR analysis. The findings from this study suggest that although there is a predominance of the ancient EAI lineage in Yangon, the TB epidemic in Yangon is driven by clonal expansion of the ST1 genotype. The Beijing lineage isolates (21.4%) were more likely (P = 0.009) than EAI lineage isolates to be multidrug resistant (MDR) (1.3%; odds ratio, 3.2, adjusted for the patients' history of exposure to anti-TB drugs), suggesting that the spread of MDR Beijing isolates is a major problem in Yangon.

  7. Immunoinformatics study on highly expressed Mycobacterium tuberculosis genes during infection.

    Science.gov (United States)

    Nguyen Thi, Le Thuy; Sarmiento, Maria Elena; Calero, Romel; Camacho, Frank; Reyes, Fatima; Hossain, Md Murad; Gonzalez, Gustavo Sierra; Norazmi, Mohd Nor; Acosta, Armando

    2014-09-01

    The most important targets for vaccine development are the proteins that are highly expressed by the microorganisms during infection in-vivo. A number of Mycobacterium tuberculosis (Mtb) proteins are also reported to be expressed in-vivo at different phases of infection. In the present study, we analyzed multiple published databases of gene expression profiles of Mtb in-vivo at different phases of infection in animals and humans and selected 38 proteins that are highly expressed in the active, latent and reactivation phases. We predicted T- and B-cell epitopes from the selected proteins using HLAPred for T-cell epitope prediction and BCEPred combined with ABCPred for B-cell epitope prediction. For each selected proteins, regions containing both T- and B-cell epitopes were identified which might be considered as important candidates for vaccine design against tuberculosis.

  8. EVOLUTION OF MYCOBACTERIUM TUBERCULOSIS AND IMPLICATIONS FOR VACCINE DEVELOPMENT.

    Science.gov (United States)

    Gagneux, Sebastien

    2016-04-01

    Tuberculosis (TB) is a growing public health threat, particularly in the face of the global epidemics of multidrug resistance. Given the limited efficacy of the current TB vaccine and the recent clinical failure of the most advanced new TB vaccine candidate, novel concepts for vaccine design should be explored. Most T cell antigens in the human-adapted Mycobacterium tuberculosis complex (MTBC) are evolutionarily conserved and under strong purifying selection, indicating that host immune responses targeting these antigens might not be protective. By contrast, a few highly variable T cell epitopes have recently been discovered, which could serve as alternative vaccine antigens. Moreover, there is increasing evidence that the human-adapted MTBC has been co-evolving with the human host for a long time. Hence, studying the interaction between bacterial and human genetic diversity might help identify additional targets that could be exploited for TB vaccine development.

  9. Antimycobacterial Metabolism: Illuminating Mycobacterium tuberculosis Biology and Drug Discovery.

    Science.gov (United States)

    Awasthi, Divya; Freundlich, Joel S

    2017-09-01

    Bacteria are capable of performing a number of biotransformations that may activate or deactivate xenobiotics. Recent efforts have utilized metabolomics techniques to study the fate of small-molecule antibacterials within the targeted organism. Examples involving Mycobacterium tuberculosis are reviewed and analyzed with regard to the insights they provide as to both activation and deactivation of the antibacterial. The studies, in particular, shed light on biosynthetic transformations performed by M. tuberculosis while suggesting avenues for the evolution of chemical tools, highlighting potential areas for drug discovery, and mechanisms of approved drugs. A two-pronged approach investigating the metabolism of antibacterials within both the host and bacterium is outlined and will be of value to both the chemical biology and drug discovery fields. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Streptomyces as host for recombinant production of Mycobacterium tuberculosis proteins.

    Science.gov (United States)

    Vallin, Carlos; Ramos, Astrid; Pimienta, Elsa; Rodríguez, Caridad; Hernández, Tairí; Hernández, Ivones; Del Sol, Ricardo; Rosabal, Grisel; Van Mellaert, Lieve; Anné, Jozef

    2006-01-01

    The 45/47 kDa APA protein (Rv1860) of Mycobacterium tuberculosis was produced by Streptomyces lividans. The recombinant protein could be recovered from the culture medium of an S. lividans clone containing the apa gene under control of the promoter and signal sequence of the Streptomyces coelicolor agarase gene. The recombinant protein production was further scaled-up using fermentation conditions. The APA protein was subsequently purified from the culture supernatant by means of immunochromatography. About 80 mg of recombinant protein were obtained per liter of culture media. In vivo tests with the APA protein purified from S. lividans TK24/pRGAPA1 revealed that the recombinant protein was antigenic and could induce high titers of specific antibodies in the mouse biological model. Results obtained concerning heterologous production of APA, its immunogenic and antigenic capacity, demonstrated the potential of S. lividans as a valuable host for the production of recombinant proteins from M. tuberculosis.

  11. Structural enzymology of sulphur metabolism in Mycobacterium tuberculosis.

    Science.gov (United States)

    Schnell, Robert; Schneider, Gunter

    2010-05-21

    The emergence of multidrug-resistant strains of Mycobacterium tuberculosis poses a serious threat to human health and has led to world-wide efforts focusing on the development of novel vaccines and antibiotics against this pathogen. Sulphur metabolism in this organism has been linked to essential processes such as virulence and redox defence. The cysteine biosynthetic pathway is up-regulated in models of persistent M. tuberculosis infections and provides potential targets for novel anti-mycobacterial agents, directed specifically toward the pathogen in its persistent phase. Functional and structural characterization of enzymes from sulfur metabolism establishes a necessary framework for the design of strong binding inhibitors that might be developed into new drugs. This review summarizes recent progress in the elucidation of the structural enzymology of the sulphate reduction and cysteine biosynthesis pathways.

  12. Benzothiazinones kill Mycobacterium tuberculosis by blocking arabinan synthesis.

    Science.gov (United States)

    Makarov, Vadim; Manina, Giulia; Mikusova, Katarina; Möllmann, Ute; Ryabova, Olga; Saint-Joanis, Brigitte; Dhar, Neeraj; Pasca, Maria Rosalia; Buroni, Silvia; Lucarelli, Anna Paola; Milano, Anna; De Rossi, Edda; Belanova, Martina; Bobovska, Adela; Dianiskova, Petronela; Kordulakova, Jana; Sala, Claudia; Fullam, Elizabeth; Schneider, Patricia; McKinney, John D; Brodin, Priscille; Christophe, Thierry; Waddell, Simon; Butcher, Philip; Albrethsen, Jakob; Rosenkrands, Ida; Brosch, Roland; Nandi, Vrinda; Bharath, Sowmya; Gaonkar, Sheshagiri; Shandil, Radha K; Balasubramanian, Venkataraman; Balganesh, Tanjore; Tyagi, Sandeep; Grosset, Jacques; Riccardi, Giovanna; Cole, Stewart T

    2009-05-08

    New drugs are required to counter the tuberculosis (TB) pandemic. Here, we describe the synthesis and characterization of 1,3-benzothiazin-4-ones (BTZs), a new class of antimycobacterial agents that kill Mycobacterium tuberculosis in vitro, ex vivo, and in mouse models of TB. Using genetics and biochemistry, we identified the enzyme decaprenylphosphoryl-beta-d-ribose 2'-epimerase as a major BTZ target. Inhibition of this enzymatic activity abolishes the formation of decaprenylphosphoryl arabinose, a key precursor that is required for the synthesis of the cell-wall arabinans, thus provoking cell lysis and bacterial death. The most advanced compound, BTZ043, is a candidate for inclusion in combination therapies for both drug-sensitive and extensively drug-resistant TB.

  13. Unraveling Mycobacterium tuberculosis genomic diversity and evolution in Lisbon, Portugal, a highly drug resistant setting

    KAUST Repository

    Perdigão, João

    2014-11-18

    Background Multidrug- (MDR) and extensively drug resistant (XDR) tuberculosis (TB) presents a challenge to disease control and elimination goals. In Lisbon, Portugal, specific and successful XDR-TB strains have been found in circulation for almost two decades. Results In the present study we have genotyped and sequenced the genomes of 56 Mycobacterium tuberculosis isolates recovered mostly from Lisbon. The genotyping data revealed three major clusters associated with MDR-TB, two of which are associated with XDR-TB. Whilst the genomic data contributed to elucidate the phylogenetic positioning of circulating MDR-TB strains, showing a high predominance of a single SNP cluster group 5. Furthermore, a genome-wide phylogeny analysis from these strains, together with 19 publicly available genomes of Mycobacterium tuberculosis clinical isolates, revealed two major clades responsible for M/XDR-TB in the region: Lisboa3 and Q1 (LAM). The data presented by this study yielded insights on microevolution and identification of novel compensatory mutations associated with rifampicin resistance in rpoB and rpoC. The screening for other structural variations revealed putative clade-defining variants. One deletion in PPE41, found among Lisboa3 isolates, is proposed to contribute to immune evasion and as a selective advantage. Insertion sequence (IS) mapping has also demonstrated the role of IS6110 as a major driver in mycobacterial evolution by affecting gene integrity and regulation. Conclusions Globally, this study contributes with novel genome-wide phylogenetic data and has led to the identification of new genomic variants that support the notion of a growing genomic diversity facing both setting and host adaptation.

  14. Genomics and Machine Learning for Taxonomy Consensus: The Mycobacterium tuberculosis Complex Paradigm.

    Directory of Open Access Journals (Sweden)

    Jérôme Azé

    Full Text Available Infra-species taxonomy is a prerequisite to compare features such as virulence in different pathogen lineages. Mycobacterium tuberculosis complex taxonomy has rapidly evolved in the last 20 years through intensive clinical isolation, advances in sequencing and in the description of fast-evolving loci (CRISPR and MIRU-VNTR. On-line tools to describe new isolates have been set up based on known diversity either on CRISPRs (also known as spoligotypes or on MIRU-VNTR profiles. The underlying taxonomies are largely concordant but use different names and offer different depths. The objectives of this study were 1 to explicit the consensus that exists between the alternative taxonomies, and 2 to provide an on-line tool to ease classification of new isolates. Genotyping (24-VNTR, 43-spacers spoligotypes, IS6110-RFLP was undertaken for 3,454 clinical isolates from the Netherlands (2004-2008. The resulting database was enlarged with African isolates to include most human tuberculosis diversity. Assignations were obtained using TB-Lineage, MIRU-VNTRPlus, SITVITWEB and an algorithm from Borile et al. By identifying the recurrent concordances between the alternative taxonomies, we proposed a consensus including 22 sublineages. Original and consensus assignations of the all isolates from the database were subsequently implemented into an ensemble learning approach based on Machine Learning tool Weka to derive a classification scheme. All assignations were reproduced with very good sensibilities and specificities. When applied to independent datasets, it was able to suggest new sublineages such as pseudo-Beijing. This Lineage Prediction tool, efficient on 15-MIRU, 24-VNTR and spoligotype data is available on the web interface "TBminer." Another section of this website helps summarizing key molecular epidemiological data, easing tuberculosis surveillance. Altogether, we successfully used Machine Learning on a large dataset to set up and make available the first

  15. Characterization of rifampin-resistant isolates of Mycobacterium tuberculosis from Sichuan in China.

    Science.gov (United States)

    Tang, Ke; Sun, Honghu; Zhao, Yuding; Guo, Jianhua; Zhang, Congcong; Feng, Qin; He, Yongquan; Luo, Mei; Li, Yuqing; Sun, Qun

    2013-01-01

    Rifampin is a key component of standard short-course first-line therapy against Mycobacterium tuberculosis, and rifampin resistance of this pathogen has been reported to be related to rpoB gene mutations. The objective of this study was to characterize the rpoB gene mutations in rifampin-resistant M. tuberculosis isolates circulated in Sichuan. Sequencing of rpoB gene and spoligotyping were performed on 268 randomly selected isolates from January 2008 to May 2010. The results indicated that 207 (97.2%) rifampin-resistant isolates had mutations at 26 codons in the amplified region with 50 different genotypes, while 1 (1.8%) of 55 susceptible isolates had a nonsense mutation. The most common mutations were in codon 531 (55.9%), 526 (16.4%), 516 (10.3%) and 511 (8.9%). Among the 213 resistant isolates, 150 (70.4%) belonged to the Beijing family and mutation at codon 531 (TCG→TTG) was associated with Beijing genotype (χ(2), 9.8305; p, 0.0017). It is demonstrated that the frequency of 511 (CTG→CCG) mutations in Sichuan was higher than in other provinces of China, as well as other geographic regions worldwide. It is suggested that only a small portion (2.7%) of rifampin-resistant Beijing genotype isolates in Sichuan be spread by clonal expansion during the study period.

  16. IS6110 restriction fragment length polymorphism of Mycobacterium tuberculosis isolated from patients with pulmonary tuberculosis in Campinas, Brazil: evidence of intercontinental distribution of strains

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    Ana Lucia Roscani Calusni

    2003-07-01

    Full Text Available Tuberculosis (TB is a major concern in developing countries. In Brazil, few genotyping studies have been conducted to verify the number of IS6110 copies present in local prevalent strains of Mycobacterium tuberculosis, the distribution and clustering of strains. IS6110 DNA fingerprinting was performed on a sample of M. tuberculosis isolates from patients with AFB smear-positive pulmonary TB, at a hospital in Brazil. The IS6110 profiles were analyzed and compared to a M. tuberculosis database of the Houston Tuberculosis Initiative, Houston, US. Seventy-six fingerprints were obtained from 98 patients. All M. tuberculosis strains had an IS6110 copy number between 5-21 allowing for differentiation of the isolates. Human immunodeficiency virus infection was confirmed in nearly half the patients of whom data was available. Fifty-eight strains had unique patterns, while 17 strains were grouped in 7 clusters (2 to 6 strains. When compared to the HTI database, 6 strains matched isolates from El Paso, Ciudad de Juarez, Houston, and New York. Recently acquired infections were documented in 19% of cases. The community transmission of infection is intense, since some clustered strains were recovered during the four-year study period. The intercontinental dissemination of M. tuberculosis strains is suspected by demonstration of identical fingerprints in a distant country.

  17. Native New Zealand plants with inhibitory activity towards Mycobacterium tuberculosis

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    Swift Simon

    2010-06-01

    Full Text Available Abstract Background Plants have long been investigated as a source of antibiotics and other bioactives for the treatment of human disease. New Zealand contains a diverse and unique flora, however, few of its endemic plants have been used to treat tuberculosis. One plant, Laurelia novae-zelandiae, was reportedly used by indigenous Maori for the treatment of tubercular lesions. Methods Laurelia novae-zelandiae and 44 other native plants were tested for direct anti-bacterial activity. Plants were extracted with different solvents and extracts screened for inhibition of the surrogate species, Mycobacterium smegmatis. Active plant samples were then tested for bacteriostatic activity towards M. tuberculosis and other clinically-important species. Results Extracts of six native plants were active against M. smegmatis. Many of these were also inhibitory towards M. tuberculosis including Laurelia novae-zelandiae (Pukatea. M. excelsa (Pohutukawa was the only plant extract tested that was active against Staphylococcus aureus. Conclusions Our data provide support for the traditional use of Pukatea in treating tuberculosis. In addition, our analyses indicate that other native plant species possess antibiotic activity.

  18. Inhibition studies of Mycobacterium tuberculosis salicylate synthase (MbtI).

    Science.gov (United States)

    Manos-Turvey, Alexandra; Bulloch, Esther M M; Rutledge, Peter J; Baker, Edward N; Lott, J Shaun; Payne, Richard J

    2010-07-05

    Mycobacterium tuberculosis salicylate synthase (MbtI), a member of the chorismate-utilizing enzyme family, catalyses the first committed step in the biosynthesis of the siderophore mycobactin T. This complex secondary metabolite is essential for both virulence and survival of M. tuberculosis, the etiological agent of tuberculosis (TB). It is therefore anticipated that inhibitors of this enzyme may serve as TB therapies with a novel mode of action. Herein we describe the first inhibition study of M. tuberculosis MbtI using a library of functionalized benzoate-based inhibitors designed to mimic the substrate (chorismate) and intermediate (isochorismate) of the MbtI-catalyzed reaction. The most potent inhibitors prepared were those designed to mimic the enzyme intermediate, isochorismate. These compounds, based on a 2,3-dihydroxybenzoate scaffold, proved to be low-micromolar inhibitors of MbtI. The most potent inhibitors in this series possessed hydrophobic enol ether side chains at C3 in place of the enol-pyruvyl side chain found in chorismate and isochorismate.

  19. High throughput phenotypic analysis of Mycobacterium tuberculosis and Mycobacterium bovis strains' metabolism using biolog phenotype microarrays.

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    Bhagwati Khatri

    Full Text Available Tuberculosis is a major human and animal disease of major importance worldwide. Genetically, the closely related strains within the Mycobacterium tuberculosis complex which cause disease are well-characterized but there is an urgent need better to understand their phenotypes. To search rapidly for metabolic differences, a working method using Biolog Phenotype MicroArray analysis was developed. Of 380 substrates surveyed, 71 permitted tetrazolium dye reduction, the readout over 7 days in the method. By looking for ≥5-fold differences in dye reduction, 12 substrates differentiated M. tuberculosis H37Rv and Mycobacterium bovis AF2122/97. H37Rv and a Beijing strain of M. tuberculosis could also be distinguished in this way, as could field strains of M. bovis; even pairs of strains within one spoligotype could be distinguished by 2 to 3 substrates. Cluster analysis gave three clear groups: H37Rv, Beijing, and all the M. bovis strains. The substrates used agreed well with prior knowledge, though an unexpected finding that AF2122/97 gave greater dye reduction than H37Rv with hexoses was investigated further, in culture flasks, revealing that hexoses and Tween 80 were synergistic for growth and used simultaneously rather than in a diauxic fashion. Potential new substrates for growth media were revealed, too, most promisingly N-acetyl glucosamine. Osmotic and pH arrays divided the mycobacteria into two groups with different salt tolerance, though in contrast to the substrate arrays the groups did not entirely correlate with taxonomic differences. More interestingly, these arrays suggested differences between the amines used by the M. tuberculosis complex and enteric bacteria in acid tolerance, with some hydrophobic amino acids being highly effective. In contrast, γ-aminobutyrate, used in the enteric bacteria, had no effect in the mycobacteria. This study proved principle that Phenotype MicroArrays can be used with slow-growing pathogenic mycobacteria

  20. High throughput phenotypic analysis of Mycobacterium tuberculosis and Mycobacterium bovis strains' metabolism using biolog phenotype microarrays.

    Science.gov (United States)

    Khatri, Bhagwati; Fielder, Mark; Jones, Gareth; Newell, William; Abu-Oun, Manal; Wheeler, Paul R

    2013-01-01

    Tuberculosis is a major human and animal disease of major importance worldwide. Genetically, the closely related strains within the Mycobacterium tuberculosis complex which cause disease are well-characterized but there is an urgent need better to understand their phenotypes. To search rapidly for metabolic differences, a working method using Biolog Phenotype MicroArray analysis was developed. Of 380 substrates surveyed, 71 permitted tetrazolium dye reduction, the readout over 7 days in the method. By looking for ≥5-fold differences in dye reduction, 12 substrates differentiated M. tuberculosis H37Rv and Mycobacterium bovis AF2122/97. H37Rv and a Beijing strain of M. tuberculosis could also be distinguished in this way, as could field strains of M. bovis; even pairs of strains within one spoligotype could be distinguished by 2 to 3 substrates. Cluster analysis gave three clear groups: H37Rv, Beijing, and all the M. bovis strains. The substrates used agreed well with prior knowledge, though an unexpected finding that AF2122/97 gave greater dye reduction than H37Rv with hexoses was investigated further, in culture flasks, revealing that hexoses and Tween 80 were synergistic for growth and used simultaneously rather than in a diauxic fashion. Potential new substrates for growth media were revealed, too, most promisingly N-acetyl glucosamine. Osmotic and pH arrays divided the mycobacteria into two groups with different salt tolerance, though in contrast to the substrate arrays the groups did not entirely correlate with taxonomic differences. More interestingly, these arrays suggested differences between the amines used by the M. tuberculosis complex and enteric bacteria in acid tolerance, with some hydrophobic amino acids being highly effective. In contrast, γ-aminobutyrate, used in the enteric bacteria, had no effect in the mycobacteria. This study proved principle that Phenotype MicroArrays can be used with slow-growing pathogenic mycobacteria and already has

  1. Drug-resistant tuberculosis in Central Mozambique: the role of a rapid genotypic susceptibility testing.

    Science.gov (United States)

    Namburete, Evangelina Inácio; Tivane, Inês; Lisboa, Miguelhete; Passeri, Margarida; Pocente, Renata; Ferro, Josefo Joao; Harrison, Lee H; Bollela, Valdes Roberto

    2016-08-17

    Genotypic molecular testing may be very helpful for tuberculosis (TB) drug-resistance surveillance and for treatment guidance in low resource settings. Descriptive analysis of M. tuberculosis isolates from Beira Central Hospital, Mozambique, during 2014-2015. Genotype MTBDRplus and MTBDRsl were used and patient medical records reviewed. To explore genotypic susceptibility profile of Mycobacterium tuberculosis, to first and second line drugs (SLD) in Beira Mozambique. Of 155 isolates, 16.1 % (25) were multidrug resistant (MDR), 8.4 % (13) isoniazid-monoresistant and 1.3 % (2) rifampicin-monoresistant. Among MDR-TB, 22.2 % showed primary and 77.8 % represented acquired resistance. The majority of patients with drug resistance had a history of previous TB treatment. Among 125 isolates tested for ethambutol and SLD, 7.2 % (9) were resistant to ethambutol, 4.8 % (6) to fluoroquinolones and 0.8 % (1) to ethambutol and fluoroquinolones. Resistance to injectable SLD was not detected. As far as we know this is the first report of a genotypic testing used to provide information about SLD resistance in Mozambique, where phenotypic susceptibility testing is usually unavailable. Extensively drug resistant TB was not detected in this isolates from Beira Mozambique.

  2. Genotypic characterization by spoligotyping and VNTR typing of Mycobacterium bovis and Mycobacterium caprae isolates from cattle of Tunisia.

    Science.gov (United States)

    Lamine-Khemiri, Hela; Martínez, Remigio; García-Jiménez, Waldo Luis; Benítez-Medina, Jose Manuel; Cortés, Maria; Hurtado, Inés; Abassi, Mohammed Salah; Khazri, Imed; Benzarti, Mohammed; Hermoso-de-Mendoza, Javier

    2014-02-01

    This work is an approach to the molecular epidemiology of Mycobacterium tuberculosis complex (MTBC) bovine infections in Tunisia. A total of 35 MTBC isolates from both lateral retropharyngeal lymph node samples of cattle slaughtered in different Tunisian regions were genotyped by spoligotyping and variable number tandem repeat typing (VNTR)-typing. Spoligotyping allowed to identify two profiles not previously registered, namely SB2024, a Mycobacterium caprae isolate from Nabeul Region (North East Tunisia), the first description of this species in the country, and SB2025 (Mycobacterium bovis) from Sfax Region (Southern Tunisia). A second M. caprae isolate with a spoligotyping profile previously described in Europe mainland, SB0418, was also isolated from a bovine of Sfax region. Both isolates suggest the possibility of a widespread distribution of this species in the country. The predominant spoligotype was SB0120, present in all Tunisian regions selected for the study but Nabeul. Molecular typing also allowed to describe a mixed infection caused by two different M. bovis isolates (SB0120 and SB0848) in the same animal. VNTR typing was highly discriminant by testing a panel of six loci. Loci QUB3232 and QUB11b were the most discriminant, whereas ETR-D and QUB11a had the lower diversity index. The value of allelic diversity can significantly vary among countries; thus, it is important to standardize a panel of loci for future inter-laboratory comparisons. Although VNTR typing proved to be useful for an efficient discrimination among MTBC isolates, especially in combination with spoligotyping, further studies are needed in order to assess the genetic diversity of the MTBC in Tunisia.

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

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    Azar Dokht Khosravi

    2014-10-01

    Full Text Available 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.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.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.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.

  4. Impaired Cytokine but Enhanced Cytotoxic Marker Expression in Mycobacterium tuberculosis-Induced CD8+ T Cells in Individuals With Type 2 Diabetes and Latent Mycobacterium tuberculosis Infection.

    Science.gov (United States)

    Kumar, Nathella Pavan; Moideen, Kadar; George, Parakkal Jovvian; Dolla, Chandrakumar; Kumaran, Paul; Babu, Subash

    2016-03-01

    Type 2 diabetes mellitus (DM) is a risk factor for tuberculosis among individuals with latent Mycobacterium tuberculosis infection. To explore the influence of DM on CD8(+) T-cell responses during latent M. tuberculosis infection, we estimated the cytokine and cytotoxic marker expression pattern in individuals with latent M. tuberculosis infection with DM and those with latent M. tuberculosis infection without DM. Among individuals with latent M. tuberculosis infection, those with DM had diminished frequencies of CD8(+) T-helper type 1 (Th1), Th2, and Th17 cells following stimulation by M. tuberculosis antigen and enhanced frequencies of CD8(+) T cells expressing cytotoxic markers, compared with those without DM. Thus, our results suggest that coincident DM modulates CD8(+) T-cell function during latent M. tuberculosis infection.

  5. [Advances in the research of an animal model of wound due to Mycobacterium tuberculosis infection].

    Science.gov (United States)

    Chen, Ling; Jia, Chiyu

    2015-12-01

    Tuberculosis ranks as the second deadly infectious disease worldwide. The incidence of tuberculosis is high in China. Refractory wound caused by Mycobacterium tuberculosis infection ranks high in misdiagnosis, and it is accompanied by a protracted course, and its pathogenic mechanism is still not so clear. In order to study its pathogenic mechanism, it is necessary to reproduce an appropriate animal model. Up to now the study of the refractory wound caused by Mycobacterium tuberculosis infection is just beginning, and there is still no unimpeachable model for study. This review describes two models which may reproduce a wound similar to the wound caused by Mycobacterium tuberculosis infection, so that they could be used to study the pathogenesis and characteristics of a tuberculosis wound in an animal.

  6. Molecular characteristics of rifampin and isoniazid resistant Mycobacterium tuberculosis strains from Beijing, China

    Institute of Scientific and Technical Information of China (English)

    JIAO Wei-wei; Mokrousov Igor; SUN Gui-zhi; LI Mo; LIU Jia-wen; Narvskaya Olga; SHEN A-dong

    2007-01-01

    Background China is one of the high burden countries of Mycobacterium tuberculosis (TB) infection globally, with high incidence and mortality. We studied the molecular characteristics of rifampin (RIF) and isoniazid (INH) resistant Mycobacterium tuberculosis strains from Beijing, China, in order to find out the genetic marker for rapid detection of specific drug resistance.Methods Forty pansusceptible and 81 resistant strains of Mycobacterium tuberculosis isolated from Beijing, China during 2002-2005 were analyzed. The modified rifampin oligonucleotide (RIFO) assay based on reverse line blot hybridization was used to detect mutations in the 81 bp hot-spot region of rpoB gene, which is associated with RIF resistance. The INH resistance associated genes, regulatory region mab-inhA (-15C/T) and structural gene katG S315T were detected by reverse line blot hybridization and PCR-restriction fragment length polymorphism (RFLP) method respectively. All the strains were typed by spoligotying and the Beijing genotype was further subdivided by NTF locus analysis. The distribution of drug resistance associated mutations in the above genes was compared in these groups.Results Sixty-five (91.5%) of 71 RIF resistant and 52 (92.9%) of 56 multidrug-resistant (MDR, Ⅰ.e. Resistant to at least RIF and INH) strains were found to harbor mutations in the rpoB hot-spot region. No mutation was detected in RIF sensitive strains. The specificity and sensitivity of the modified RIFO assay were 100% and 91.5%, respectively. katG315 AGC>ACC and inhA-15C>T mutations were found in 40 (60.6%) and 10 (15.2%) of 66 Ⅰ NH resistant strains, respectively;7.6% of INH-resistant strains had mutations in both of these genes. Therefore, a combined use of both katG315 and inhA-15 identified 68.2% of INH-resistant strains. The Beijing genotype accounted for 91.7% of total strains and was further subdivided into "modern" (76.6%) and "ancestral" (23.4%) group. There is no significant difference between

  7. Conspicuous multidrug-resistant Mycobacterium tuberculosis cluster strains do not trespass country borders in Latin America and Spain.

    Science.gov (United States)

    Ritacco, Viviana; Iglesias, María-José; Ferrazoli, Lucilaine; Monteserin, Johana; Dalla Costa, Elis R; Cebollada, Alberto; Morcillo, Nora; Robledo, Jaime; de Waard, Jacobus H; Araya, Pamela; Aristimuño, Liselotte; Díaz, Raúl; Gavin, Patricia; Imperiale, Belen; Simonsen, Vera; Zapata, Elsa M; Jiménez, María S; Rossetti, Maria L; Martin, Carlos; Barrera, Lucía; Samper, Sofia

    2012-06-01

    Multidrug-resistant Mycobacterium tuberculosis strain diversity in Ibero-America was examined by comparing extant genotype collections in national or state tuberculosis networks. To this end, genotypes from over 1000 patients with multidrug-resistant tuberculosis diagnosed from 2004 through 2008 in Argentina, Brazil, Chile, Colombia, Venezuela and Spain were compared in a database constructed ad hoc. Most of the 116 clusters identified by IS6110 restriction fragment length polymorphism were small and restricted to individual countries. The three largest clusters, of 116, 49 and 25 patients, were found in Argentina and corresponded to previously documented locally-epidemic strains. Only 13 small clusters involved more than one country, altogether accounting for 41 patients, of whom 13 were, in turn, immigrants from Latin American countries different from those participating in the study (Peru, Ecuador and Bolivia). Most of these international clusters belonged either to the emerging RD(Rio) LAM lineage or to the Haarlem family of M. tuberculosis and four were further split by country when analyzed with spoligotyping and rifampin resistance-conferring mutations, suggesting that they did not represent ongoing transnational transmission events. The Beijing genotype accounted for 1.3% and 10.2% of patients with multidrug-resistant tuberculosis in Latin America and Spain, respectively, including one international cluster of two cases. In brief, Euro-American genotypes were widely predominant among multidrug-resistant M. tuberculosis strains in Ibero-America, reflecting closely their predominance in the general M. tuberculosis population in the region, and no evidence was found of acknowledged outbreak strains trespassing country borders. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. A high-throughput cidality screen for Mycobacterium tuberculosis.

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    Parvinder Kaur

    Full Text Available Exposure to Mycobacterium tuberculosis (Mtb aerosols is a major threat to tuberculosis (TB researchers, even in bio-safety level-3 (BSL-3 facilities. Automation and high-throughput screens (HTS in BSL3 facilities are essential for minimizing manual aerosol-generating interventions and facilitating TB research. In the present study, we report the development and validation of a high-throughput, 24-well 'spot-assay' for selecting bactericidal compounds against Mtb. The bactericidal screen concept was first validated in the fast-growing surrogate Mycobacterium smegmatis (Msm and subsequently confirmed in Mtb using the following reference anti-tubercular drugs: rifampicin, isoniazid, ofloxacin and ethambutol (RIOE, acting on different targets. The potential use of the spot-assay to select bactericidal compounds from a large library was confirmed by screening on Mtb, with parallel plating by the conventional gold standard method (correlation, r2 = 0.808. An automated spot-assay further enabled an MBC90 determination on resistant and sensitive Mtb clinical isolates. The implementation of the spot-assay in kinetic screens to enumerate residual Mtb after either genetic silencing (anti-sense RNA, AS-RNA or chemical inhibition corroborated its ability to detect cidality. This relatively simple, economical and quantitative HTS considerably minimized the bio-hazard risk and enabled the selection of novel vulnerable Mtb targets and mycobactericidal compounds. Thus, spot-assays have great potential to impact the TB drug discovery process.

  9. Modeling Phenotypic Metabolic Adaptations of Mycobacterium tuberculosis H37Rv under Hypoxia

    Science.gov (United States)

    2012-09-13

    Tuberculosis in humans and its epidemiology , diagnosis and treatment in the United States. Int J Tuberc Lung Dis 14: 1226–1232. 4. Horsburgh CR, Jr., Rubin...Modeling Phenotypic Metabolic Adaptations of Mycobacterium tuberculosis H37Rv under Hypoxia Xin Fang, Anders Wallqvist, Jaques Reifman* DoD... tuberculosis , the causative agent of tuberculosis (TB), to successfully infect human hosts. Adaptations allow the organism to evade the host immune

  10. Defining multidrug-resistant tuberculosis: correlating GenoType MTBDRplus assay results with minimum inhibitory concentrations.

    Science.gov (United States)

    Kambli, Priti; Ajbani, Kanchan; Sadani, Meeta; Nikam, Chaitali; Shetty, Anjali; Udwadia, Zarir; Georghiou, Sophia B; Rodwell, Timothy C; Catanzaro, Antonino; Rodrigues, Camilla

    2015-05-01

    This study correlates MICs of rifampicin (RIF) and isoniazid (INH) with GenoType MTBDRplus assay results for drug-resistant Mycobacterium tuberculosis (MTB) clinical isolates. MICs of RIF and INH were established for 84 and 90 isolates, respectively, testing 7 concentrations of each drug. Genotypic resistance to each drug was determined by GenoType MTBDRplus assay with 50 representative mutations confirmed by pyrosequencing, with mutations in the rpoB gene associated with RIF resistance and mutations in the katG and/or inhA genes associated with INH resistance. Based upon the correlation of MICs with specific genetic profiles, relative resistance levels were established for each isolate. Results indicate that MTB phenotypic resistance, currently based upon the testing of isolate susceptibility to a single drug concentration, may be more accurately profiled via quantitative MICs, and therefore, the correlation of molecular diagnostic results with specific MICs may allow for more optimal treatment of infections.

  11. Novel multiplex real-time PCR diagnostic assay for identification and differentiation of Mycobacterium tuberculosis, Mycobacterium canettii, and Mycobacterium tuberculosis complex strains.

    Science.gov (United States)

    Reddington, Kate; O'Grady, Justin; Dorai-Raj, Siobhan; Maher, Majella; van Soolingen, Dick; Barry, Thomas

    2011-02-01

    Tuberculosis (TB) in humans is caused by members of the Mycobacterium tuberculosis complex (MTC). Rapid detection of the MTC is necessary for the timely initiation of antibiotic treatment, while differentiation between members of the complex may be important to guide the appropriate antibiotic treatment and provide epidemiological information. In this study, a multiplex real-time PCR diagnostics assay using novel molecular targets was designed to identify the MTC while simultaneously differentiating between M. tuberculosis and M. canettii. The lepA gene was targeted for the detection of members of the MTC, the wbbl1 gene was used for the differentiation of M. tuberculosis and M. canettii from the remainder of the complex, and a unique region of the M. canettii genome, a possible novel region of difference (RD), was targeted for the specific identification of M. canettii. The multiplex real-time PCR assay was tested using 125 bacterial strains (64 MTC isolates, 44 nontuberculosis mycobacteria [NTM], and 17 other bacteria). The assay was determined to be 100% specific for the mycobacteria tested. Limits of detection of 2.2, 2.17, and 0.73 cell equivalents were determined for M. tuberculosis/M. canettii, the MTC, and M. canettii, respectively, using probit regression analysis. Further validation of this diagnostics assay, using clinical samples, should demonstrate its potential for the rapid, accurate, and sensitive diagnosis of TB caused by M. tuberculosis, M. canettii, and the other members of the MTC.

  12. Effect of common and experimental anti-tuberculosis treatments on Mycobacterium tuberculosis growing as biofilms

    Directory of Open Access Journals (Sweden)

    James P. Dalton

    2016-11-01

    Full Text Available Much is known regarding the antibiotic susceptibility of planktonic cultures of Mycobacterium tuberculosis, the bacterium responsible for the lung disease tuberculosis (TB. As planktonically-grown M. tuberculosis are unlikely to be entirely representative of the bacterium during infection, we set out to determine how effective a range of anti-mycobacterial treatments were against M. tuberculosis growing as a biofilm, a bacterial phenotype known to be more resistant to antibiotic treatment. Light levels from bioluminescently-labelled M. tuberculosis H37Rv (strain BSG001 were used as a surrogate for bacterial viability, and were monitored before and after one week of treatment. After treatment, biofilms were disrupted, washed and inoculated into fresh broth and plated onto solid media to rescue any surviving bacteria. We found that in this phenotypic state M. tuberculosis was resistant to the majority of the compounds tested. Minimum inhibitory concentrations (MICs increased by 20-fold to greater than 1,000-fold, underlying the potential of this phenotype to cause significant problems during treatment.

  13. A case report of vascular catheter-associated bacteremia caused by Mycobacterium tuberculosis in a non-immunosuppressed patient

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    PETRILLO Victor Flávio

    1999-01-01

    Full Text Available Mycobacterium tuberculosis was isolated from a central venous catheter in a non-immunosuppressed patient with systemic tuberculosis. This case report represents a very uncommon form of isolation of Mycobacterium tuberculosis. A total improvement was obtained after treatment.

  14. Comparing Galactan Biosynthesis in Mycobacterium tuberculosis and Corynebacterium diphtheriae.

    Science.gov (United States)

    Wesener, Darryl A; Levengood, Matthew R; Kiessling, Laura L

    2017-02-17

    The suborder Corynebacterineae encompasses species like Corynebacterium glutamicum, which has been harnessed for industrial production of amino acids, as well as Corynebacterium diphtheriae and Mycobacterium tuberculosis, which cause devastating human diseases. A distinctive component of the Corynebacterineae cell envelope is the mycolyl-arabinogalactan (mAG) complex. The mAG is composed of lipid mycolic acids, and arabinofuranose (Araf) and galactofuranose (Galf) carbohydrate residues. Elucidating microbe-specific differences in mAG composition could advance biotechnological applications and lead to new antimicrobial targets. To this end, we compare and contrast galactan biosynthesis in C. diphtheriae and M. tuberculosis In each species, the galactan is constructed from uridine 5'-diphosphate-α-d-galactofuranose (UDP-Galf), which is generated by the enzyme UDP-galactopyranose mutase (UGM or Glf). UGM and the galactan are essential in M. tuberculosis, but their importance in Corynebacterium species was not known. We show that small molecule inhibitors of UGM impede C. glutamicum growth, suggesting that the galactan is critical in corynebacteria. Previous cell wall analysis data suggest the galactan polymer is longer in mycobacterial species than corynebacterial species. To explore the source of galactan length variation, a C. diphtheriae ortholog of the M. tuberculosis carbohydrate polymerase responsible for the bulk of galactan polymerization, GlfT2, was produced, and its catalytic activity was evaluated. The C. diphtheriae GlfT2 gave rise to shorter polysaccharides than those obtained with the M. tuberculosis GlfT2. These data suggest that GlfT2 alone can influence galactan length. Our results provide tools, both small molecule and genetic, for probing and perturbing the assembly of the Corynebacterineae cell envelope.

  15. Rapid susceptibility testing of Mycobacterium avium complex and Mycobacterium tuberculosis isolated from AIDS patients

    Science.gov (United States)

    Dhople, Arvind M.

    1994-01-01

    In ominous projections issued by both U.S. Public Health Service and the World Health Organization, the epidemic of HIV infection will continue to rise more rapidly worldwide than predicted earlier. The AIDS patients are susceptible to diseases called opportunistic infections of which tuberculosis and Mycobacterium avium complex (MAC) infection are most common. This has created an urgent need to uncover new drugs for the treatment of these infections. In the seventies, NASA scientists at Goddard Space Flight Center, Greenbelt, MD, had adopted a biochemical indicator, adenosine triphosphate (ATP), to detect presence of life in extraterrestrial space. We proposed to develop ATP assay technique to determine sensitivity of antibacterial compounds against MAC and M. tuberculosis.

  16. Inferring patient to patient transmission of Mycobacterium tuberculosis from whole genome sequencing data

    NARCIS (Netherlands)

    J.M. Bryant (Josephine); A. Schürch (Anita); H. van Deutekom (Henk); S.R. Harris (Simon); J.L. de Beer (Jessica); V. de Jager (Victor); K. Kremer (Kristin); S.A.F.T. van Hijum (Sacha); R.J. Siezen (Roland); M.W. Borgdorff (Martien ); S.D. Bentley (Stephen); J. Parkhill (Julian); D. van Soolingen (Dick)

    2013-01-01

    textabstractBackground: Mycobacterium tuberculosis is characterised by limited genomic diversity, which makes the application of whole genome sequencing particularly attractive for clinical and epidemiological investigation. However, in order to confidently infer transmission events, an accurate kno

  17. Inferring patient to patient transmission of Mycobacterium tuberculosis from whole genome sequencing data

    NARCIS (Netherlands)

    Bryant, J.M.; Schürch, A.C.; Deutekom, van H.; Harris, S.R.; Beer, de J.L.; Jager, de V.C.L.; Kremer, K.; Hijum, van S.A.F.T.; Siezen, R.J.; Borgdorff, M.; Bentley, S.D.; Parkhill, J.; Soolingen, van D.

    2013-01-01

    BACKGROUND: Mycobacterium tuberculosis is characterised by limited genomic diversity, which makes the application of whole genome sequencing particularly attractive for clinical and epidemiological investigation. However, in order to confidently infer transmission events, an accurate knowledge of th

  18. Inferring patient to patient transmission of Mycobacterium tuberculosis from whole genome sequencing data.

    NARCIS (Netherlands)

    Bryant, J.M.; Schurch, A.C.; Deutekom, H. van; Harris, S.R.; Beer, J.L. de; Jager, V. de; Kremer, K.; Hijum, S.A.F.T. van; Siezen, R.J.; Borgdorff, M.; Bentley, S.D.; Parkhill, J.; Soolingen, D. van

    2013-01-01

    BACKGROUND: Mycobacterium tuberculosis is characterised by limited genomic diversity, which makes the application of whole genome sequencing particularly attractive for clinical and epidemiological investigation. However, in order to confidently infer transmission events, an accurate knowledge of th

  19. An Acute Disseminated Encephalomyelitis Case due to Mycobacterium tuberculosis

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    Hale Turan Özden

    2016-03-01

    Full Text Available Acute disseminated encephalomyelitis (ADEM is an inflammatory and demyelinating disorder of the central nervous system that is characterized by multifocal involvement of the white matter. Our patient was a 27-year-old female patient who had given birth to a baby with caesarean in another hospital. After four days upon the parturition, she was admitted to our hospital’s general intensive care unit with a poor general status, confusion and a fever. She was diagnosed with ADEM according to the clinical, laboratory and radiological findings. In addition to her antibiotic treatment (meropenem that had been given in the previous health care facility, corticosteroid therapy was also started. The patient passed away due to the ventilator-associated pneumonia infection on the 10th day of her admission. Mycobacterium tuberculosis proliferation was observed in the cerebrospinal fluid after her death. As it is reported in literature, tuberculosis is a rare cause of ADEM. In conclusion, it should be noted that M. tuberculosis can be a rare cause of ADEM in regions where the disease is endemic. J Microbiol Infect Dis 2016;6(1: 28-31

  20. PolyTB: A genomic variation map for Mycobacterium tuberculosis

    KAUST Repository

    Coll, Francesc

    2014-02-15

    Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is the second major cause of death from an infectious disease worldwide. Recent advances in DNA sequencing are leading to the ability to generate whole genome information in clinical isolates of M. tuberculosis complex (MTBC). The identification of informative genetic variants such as phylogenetic markers and those associated with drug resistance or virulence will help barcode Mtb in the context of epidemiological, diagnostic and clinical studies. Mtb genomic datasets are increasingly available as raw sequences, which are potentially difficult and computer intensive to process, and compare across studies. Here we have processed the raw sequence data (>1500 isolates, eight studies) to compile a catalogue of SNPs (n = 74,039, 63% non-synonymous, 51.1% in more than one isolate, i.e. non-private), small indels (n = 4810) and larger structural variants (n = 800). We have developed the PolyTB web-based tool (http://pathogenseq.lshtm.ac.uk/polytb) to visualise the resulting variation and important meta-data (e.g. in silico inferred strain-types, location) within geographical map and phylogenetic views. This resource will allow researchers to identify polymorphisms within candidate genes of interest, as well as examine the genomic diversity and distribution of strains. PolyTB source code is freely available to researchers wishing to develop similar tools for their pathogen of interest. 2014 Elsevier Ltd. All rights reserved.

  1. Mycobacterium tuberculosis Transcription Machinery: Ready To Respond to Host Attacks.

    Science.gov (United States)

    Flentie, Kelly; Garner, Ashley L; Stallings, Christina L

    2016-05-01

    Regulating responses to stress is critical for all bacteria, whether they are environmental, commensal, or pathogenic species. For pathogenic bacteria, successful colonization and survival in the host are dependent on adaptation to diverse conditions imposed by the host tissue architecture and the immune response. Once the bacterium senses a hostile environment, it must enact a change in physiology that contributes to the organism's survival strategy. Inappropriate responses have consequences; hence, the execution of the appropriate response is essential for survival of the bacterium in its niche. Stress responses are most often regulated at the level of gene expression and, more specifically, transcription. This minireview focuses on mechanisms of regulating transcription initiation that are required by Mycobacterium tuberculosis to respond to the arsenal of defenses imposed by the host during infection. In particular, we highlight how certain features of M. tuberculosis physiology allow this pathogen to respond swiftly and effectively to host defenses. By enacting highly integrated and coordinated gene expression changes in response to stress,M. tuberculosis is prepared for battle against the host defense and able to persist within the human population.

  2. Standartization of broth microdilution method for Mycobacterium tuberculosis

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    Leite Clarice Queico Fujimura

    2000-01-01

    Full Text Available Indirect drug susceptibility tests of Mycobacterium tuberculosis was done to investigate the accuracy and feasibility of a broth microdilution method (BMM for determining minimal inhibitory concentrations of conventional drugs against M. tuberculosis. Test drugs included isoniazid (H, rifampicin (R, ethambutol (E, streptomycin (S and pyrazinamide (Z. Fifty isolates of M. tuberculosis from patients who had never received drug therapy, and H37Rv strain for control, were evaluated in the system. When comparing this method with the gold standard proportional method in Lowenstein-Jensen medium, sensitivity of 100% for all drugs and specifities of 91, 100, 96, 98 and 85% were observed respectively for H, R, E, S and Z. The BMM was read faster (14-20 days than the proportional method (20-28 days. The microdilution method evaluated allows the testing of multiple drugs in multiple concentrations. It is easy to perform and does not require special equipment or expensive supplies. In contrast to radiometric method it does not use radioactive material.

  3. Detection of Mycobacterium tuberculosis resistance mutations to rifampin and isoniazid by real-time PCR

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    Hristea A

    2010-01-01

    Full Text Available Objective: The objective of our study was to evaluate the use of a real-time polymerase chain reaction (PCR-based technique for the prediction of phenotypic resistance of Mycobacterium tuberculosis. Materials and Methods: We tested 67 M tuberculosis strains (26 drug resistant and 41 drug susceptible using a method recommended for the LightCycler platform. The susceptibility testing was performed by the absolute concentration method. For rifampin resistance, two regions of the rpoB gene were targeted, while for identification of isoniazid resistance, we searched for mutations in katG and inhA genes. Results: The sensitivity and specificity of this method for rapid detection of mutations for isoniazid resistance were 96% (95% CI: 88% to 100% and 95% (95% CI: 89% to 100%, respectively. For detection of rifampin resistance, the sensitivity and specificity were 92% (95% CI: 81% to 100% and 74% (95% CI: 61% to 87%, respectively. The main isoniazid resistance mechanism identified in our isolates is related to changes in the katG gene that encodes catalase. We found that for rifampin resistance the concordance between the predicted and observed phenotype was less than satisfactory. Conclusions: Using this method, the best accuracy for genotyping compared with phenotypic resistance testing was obtained for detecting isoniazid resistance mutations. Although real-time PCR assay may be a valuable diagnostic tool, it is not yet completely satisfactory for detection of drug resistance mutations in M tuberculosis.

  4. Molecular analysis of Mycobacterium tuberculosis from tuberculous meningitis patients in Thailand.

    Science.gov (United States)

    Yorsangsukkamol, Jutaporn; Chaiprasert, Angkana; Prammananan, Therdsak; Palittapongarnpim, Prasit; Limsoontarakul, Sunsern; Prayoonwiwat, Naraporn

    2009-07-01

    The first retrospective molecular characterization of Mycobacterium tuberculosis from cerebrospinal fluid of 158 tuberculous meningitis (TBM) in Thailand, collected between 1995 and 2005, was performed by Southern-blot hybridization with an IS6110 probe and spoligotyping on 152 and 147 isolates, respectively. Antituberculous drug susceptibility testing was performed in 149 patients, 118 of whom (79.2%) contained pan-sensitive strains and eight (5.4%) harbored multidrug-resistant M. tuberculosis. IS6110 RFLP typing revealed 110 RFLP patterns with 57.9% of patients infected with the Beijing genotype. This percentage was significantly higher than that in a previous report from pulmonary tuberculosis patients. Fifteen of 18 TBM patients (83%) aged <15 years were infected with Beijing isolates (OR=4.47, p=0.018). There were 40 spoligotypes, with 118 patients (80.3%) being clustered. The biggest cluster, which consisted of 84 patients, was the Beijing spoligotype (57.1%). There were 16 novel spoligotypes from 16 patients compared to the Fourth International Spoligotyping Database, SpolDB4. Sixty-four percent of the patients were male, and the mean age of patients was 33.8 years. Beijing isolates from 2001 to 2005 were found in higher percentages than those from 1995 to 2000, but this difference was not significant (p=0.28).

  5. Molecular epidemiology of Mycobacterium tuberculosis in aboriginal peoples of Taiwan, 2006-2011.

    Science.gov (United States)

    Chen, Yih-Yuan; Chang, Jia-Ru; Huang, Wei-Feng; Kuo, Shu-Chen; Yeh, Jun-Jun; Lee, Jen-Jyh; Jang, Chang-Sheng; Sun, Jun-Ren; Chiueh, Tzong-Shi; Su, Ih-Jen; Dou, Horng-Yunn

    2014-04-01

    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.

  6. "Pseudo-Beijing": evidence for convergent evolution in the direct repeat region of Mycobacterium tuberculosis.

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    Lukas Fenner

    Full Text Available BACKGROUND: Mycobacterium tuberculosis has a global population structure consisting of six main phylogenetic lineages associated with specific geographic regions and human populations. One particular M. tuberculosis genotype known as "Beijing" has repeatedly been associated with drug resistance and has been emerging in some parts of the world. "Beijing" strains are traditionally defined based on a characteristic spoligotyping pattern. We used three alternative genotyping techniques to revisit the phylogenetic classification of M. tuberculosis complex (MTBC strains exhibiting the typical "Beijing" spoligotyping pattern. METHODS AND FINDINGS: MTBC strains were obtained from an ongoing molecular epidemiological study in Switzerland and Nepal. MTBC genotyping was performed based on SNPs, genomic deletions, and 24-loci MIRU-VNTR. We identified three MTBC strains from patients originating from Tibet, Portugal and Nepal which exhibited a spoligotyping patterns identical to the classical Beijing signature. However, based on three alternative molecular markers, these strains were assigned to Lineage 3 (also known as Delhi/CAS rather than to Lineage 2 (also known as East-Asian lineage. Sequencing of the RD207 in one of these strains showed that the deletion responsible for this "Pseudo-Beijing" spoligotype was about 1,000 base pairs smaller than the usual deletion of RD207 in classical "Beijing" strains, which is consistent with an evolutionarily independent deletion event in the direct repeat (DR region of MTBC. CONCLUSIONS: We provide an example of convergent evolution in the DR locus of MTBC, and highlight the limitation of using spoligotypes for strain classification. Our results indicate that a proportion of "Beijing" strains may have been misclassified in the past. Markers that are more phylogenetically robust should be used when exploring strain-specific differences in experimental or clinical phenotypes.

  7. Systems-based approaches to probing metabolic variation within the Mycobacterium tuberculosis complex.

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    Emma K Lofthouse

    Full Text Available The Mycobacterium tuberculosis complex includes bovine and human strains of the tuberculosis bacillus, including Mycobacterium tuberculosis, Mycobacterium bovis and the Mycobacterium bovis BCG vaccine strain. M. bovis has evolved from a M. tuberculosis-like ancestor and is the ancestor of the BCG vaccine. The pathogens demonstrate distinct differences in virulence, host range and metabolism, but the role of metabolic differences in pathogenicity is poorly understood. Systems biology approaches have been used to investigate the metabolism of M. tuberculosis, but not to probe differences between tuberculosis strains. In this study genome scale metabolic networks of M. bovis and M. bovis BCG were constructed and interrogated, along with a M. tuberculosis network, to predict substrate utilisation, gene essentiality and growth rates. The models correctly predicted 87-88% of high-throughput phenotype data, 75-76% of gene essentiality data and in silico-predicted growth rates matched measured rates. However, analysis of the metabolic networks identified discrepancies between in silico predictions and in vitro data, highlighting areas of incomplete metabolic knowledge. Additional experimental studies carried out to probe these inconsistencies revealed novel insights into the metabolism of these strains. For instance, that the reduction in metabolic capability observed in bovine tuberculosis strains, as compared to M. tuberculosis, is not reflected by current genetic or enzymatic knowledge. Hence, the in silico networks not only successfully simulate many aspects of the growth and physiology of these mycobacteria, but also provide an invaluable tool for future metabolic studies.

  8. Differences in T-cell responses between Mycobacterium tuberculosis and Mycobacterium africanum-infected patients.

    Science.gov (United States)

    Tientcheu, Leopold D; Sutherland, Jayne S; de Jong, Bouke C; Kampmann, Beate; Jafali, James; Adetifa, Ifedayo M; Antonio, Martin; Dockrell, Hazel M; Ota, Martin O

    2014-05-01

    In The Gambia, Mycobacterium tuberculosis (Mtb) and Mycobacterium africanum (Maf) are major causes of tuberculosis (TB). Maf is more likely to cause TB in immune suppressed individuals, implying differences in virulence. Despite this, few studies have assessed the underlying immunity to the two pathogens in human. In this study, we analyzed T-cell responses from 19 Maf- and 29 Mtb-infected HIV-negative patients before and after TB chemotherapy following overnight stimulation of whole blood with TB-specific antigens. Before treatment, percentages of early secreted antigenic target-6(ESAT-6)/culture filtrate protein-10(CFP-10) and purified protein derivative-specific single-TNF-α-producing CD4(+) and CD8(+) T cells were significantly higher while single-IL-2-producing T cells were significantly lower in Maf- compared with Mtb-infected patients. Purified protein derivative-specific polyfunctional CD4(+) T cells frequencies were significantly higher before than after treatment, but there was no difference between the groups at both time points. Furthermore, the proportion of CD3(+) CD11b(+) T cells was similar in both groups pretreatment, but was significantly lower with higher TNF-α, IL-2, and IFN-γ production in Mtb- compared with that of Maf-infected patients posttreatment. Our data provide evidence of differences in T-cell responses to two mycobacterial strains with differing virulence, providing some insight into TB pathogenesis with different Mtb strains that could be prospectively explored as biomarkers for TB protection or susceptibility.

  9. A pilot study to determine genetic polymorphism in Mycobacterium tuberculosis isolates in Central India

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    P Desikan

    2012-01-01

    Full Text Available This study was carried out to identify predominant spoligotypes responsible for transmission and prevalence of tuberculosis in central India since there is no data available about the genetic biodiversity of Mycobacterium tuberculosis isolates from patients with tuberculosis in this region. 35 strains of Mycobacterium tuberculosis were subjected to spoligotyping according to the standard protocol. A total of 25 strains out of the 35 (71.42% could be grouped in to 6 clusters. The largest cluster comprised 8 isolates. Unique (Non-clustered spoligotypes were seen in 10 isolates, Nine strains did not match the data base (Spol DB-4 data base. The results indicate that there may be a number of orphan strains unique to this geographical area. Further studies on a larger sample size derived from this area would help us delineate the epidemiology of Mycobacterium tuberculosis infection in this area.

  10. Impact of β-lactamase inhibition on the activity of ceftaroline against Mycobacterium tuberculosis and Mycobacterium abscessus.

    Science.gov (United States)

    Dubée, Vincent; Soroka, Daria; Cortes, Mélanie; Lefebvre, Anne-Laure; Gutmann, Laurent; Hugonnet, Jean-Emmanuel; Arthur, Michel; Mainardi, Jean-Luc

    2015-05-01

    The production of β-lactamases Bla(Mab) and BlaC contributes to β-lactam resistance in Mycobacterium abscessus and Mycobacterium tuberculosis, respectively. Ceftaroline was efficiently hydrolyzed by these enzymes. Inhibition of M. tuberculosis BlaC by clavulanate decreased the ceftaroline MIC from ≥ 256 to 16 to 64 μg/ml, but these values are clinically irrelevant. In contrast, the ceftaroline-avibactam combination should be evaluated against M. abscessus since it inhibited growth at lower and potentially achievable drug concentrations.

  11. Prevalence of latent Mycobacterium tuberculosis infection in prisoners.

    Science.gov (United States)

    Navarro, Pedro Daibert de; Almeida, Isabela Neves de; Kritski, Afrânio Lineu; Ceccato, Maria das Graças; Maciel, Mônica Maria Delgado; Carvalho, Wânia da Silva; Miranda, Silvana Spindola de

    2016-01-01

    To determine the prevalence of and the factors associated with latent Mycobacterium tuberculosis infection (LTBI) in prisoners in the state of Minas Gerais, Brazil. This was a cross-sectional cohort study conducted in two prisons in Minas Gerais. Tuberculin skin tests were performed in the individuals who agreed to participate in the study. A total of 1,120 individuals were selected for inclusion in this study. The prevalence of LTBI was 25.2%. In the multivariate analysis, LTBI was associated with self-reported contact with active tuberculosis patients within prisons (adjusted OR = 1.51; 95% CI: 1.05-2.18) and use of inhaled drugs (adjusted OR = 1.48; 95% CI: 1.03-2.13). Respiratory symptoms were identified in 131 (11.7%) of the participants. Serological testing for HIV was performed in 940 (83.9%) of the participants, and the result was positive in 5 (0.5%). Two cases of active tuberculosis were identified during the study period. Within the prisons under study, the prevalence of LTBI was high. In addition, LTBI was associated with self-reported contact with active tuberculosis patients and with the use of inhaled drugs. Our findings demonstrate that it is necessary to improve the conditions in prisons, as well as to introduce strategies, such as chest X-ray screening, in order to detect tuberculosis cases and, consequently, reduce M. tuberculosis infection within the prison system. Determinar a prevalência e os fatores associados à infecção latente por Mycobacterium tuberculosis (ILTB) em pessoas privadas de liberdade no Estado de Minas Gerais. Estudo de coorte transversal realizado em duas penitenciárias em Minas Gerais. Foi realizada a prova tuberculínica nos indivíduos que aceitaram participar do estudo. Foram selecionados 1.120 indivíduos para a pesquisa. A prevalência da ILTB foi de 25,2%. Na análise multivariada, a ILTB esteve associada com relato de contato com caso de tuberculose ativa dentro da penitenciária (OR ajustada = 1,51; IC95%: 1

  12. DETECTION OF MYCOBACTERIUM TUBERCULOSIS IN BLOOD FOR DIAGNOSIS OF GENERALISED TUBERCULOSIS IN HIV-POSITIVE PATIENTS

    Directory of Open Access Journals (Sweden)

    V. N. Zimina

    2017-01-01

    Full Text Available Objective: To study the informative value of the detection of mycobacteria in blood with the cultural method in patients with suspected tuberculous sepsis and to determine the most significant clinical and laboratory criteria for testing. Materials and methods: The investigation to detect M.tuberculosis was fulfilled in 159 HIV-positive patients with suspected tuberculosis sepsis. Blood culture was completed with culture medium Myco/F Lytic Culture Vials and analyzer BACTEC 9050. Results: Mycobacteria were detected in blood of 19 patients (11,9% of all patients: in 18 patients the growth of М. tuberculosis complex was detected (25,3% of all patients with diagnosed tuberculosis and in 1 patient it was Mycobacterium avium complex (0,6% of all patients. It was shown, that the probability of M.tuberculosis detection was especially associated with the severity of the disease, immunosupression (less than 100 cells/mkl, hemoglobin quantity less than 90 g/l (levels were determined through the seeking for the most significant cutoffs. It was not proofed, that meningoencephalitis develops more often in patients with proven bacteremia. There were no evident differences in detection frequency of mycobacteria in sputum between patients with tuberculous sepsis and without it.

  13. Genotyping of Mycobacterium leprae in Myanmar and possible transmission modes.

    Science.gov (United States)

    Aye, Khin Saw; Oo, Yin Thet Nu; Kyaw, Kyaw; Win, Aye Aye; Matsuoka, Masanori

    2012-09-01

    The polymorphism of TTC repeats in Mycobacterium leprae was examined using bacilli from slit skin samples of leprosy patients attending at Central Special Skin Clinic, Yangon General Hospital and nasal swabs of their contacts to elucidate the possible mode of leprosy transmission. It was found that bacilli with different TTC genotypes were distributed among same household contacts and also harbored bacilli in patients were different TTC genotype from that harbored on the nasal mucus of the healthy contacts. Genotypes of TTC repeats were found to differ between husband under treatment and his wife and also mother under treatment and her sons living in same house. This study revealed that TTC genotype of bacilli harbored by household contacts was different with the TTC genotype by index cases. These results indicate that the family members get transmission from outside the dwellings rather than from commonly supposed their MB index cases. There might have been some infectious sources to which the populace had been commonly exposed outside the dwellings.

  14. Two new rapid SNP-typing methods for classifying Mycobacterium tuberculosis complex into the main phylogenetic lineages.

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    David Stucki

    Full Text Available There is increasing evidence that strain variation in Mycobacterium tuberculosis complex (MTBC might influence the outcome of tuberculosis infection and disease. To assess genotype-phenotype associations, phylogenetically robust molecular markers and appropriate genotyping tools are required. Most current genotyping methods for MTBC are based on mobile or repetitive DNA elements. Because these elements are prone to convergent evolution, the corresponding genotyping techniques are suboptimal for phylogenetic studies and strain classification. By contrast, single nucleotide polymorphisms (SNP are ideal markers for classifying MTBC into phylogenetic lineages, as they exhibit very low degrees of homoplasy. In this study, we developed two complementary SNP-based genotyping methods to classify strains into the six main human-associated lineages of MTBC, the "Beijing" sublineage, and the clade comprising Mycobacterium bovis and Mycobacterium caprae. Phylogenetically informative SNPs were obtained from 22 MTBC whole-genome sequences. The first assay, referred to as MOL-PCR, is a ligation-dependent PCR with signal detection by fluorescent microspheres and a Luminex flow cytometer, which simultaneously interrogates eight SNPs. The second assay is based on six individual TaqMan real-time PCR assays for singleplex SNP-typing. We compared MOL-PCR and TaqMan results in two panels of clinical MTBC isolates. Both methods agreed fully when assigning 36 well-characterized strains into the main phylogenetic lineages. The sensitivity in allele-calling was 98.6% and 98.8% for MOL-PCR and TaqMan, respectively. Typing of an additional panel of 78 unknown clinical isolates revealed 99.2% and 100% sensitivity in allele-calling, respectively, and 100% agreement in lineage assignment between both methods. While MOL-PCR and TaqMan are both highly sensitive and specific, MOL-PCR is ideal for classification of isolates with no previous information, whereas TaqMan is faster

  15. Characterisation of methionine adenosyltransferase from Mycobacterium smegmatis and M. tuberculosis

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    Knodel Marvin H

    2003-06-01

    Full Text Available Abstract Background Tuberculosis remains a serious world-wide health threat which requires the characterisation of novel drug targets for the development of future antimycobacterials. One of the key obstacles in the definition of new targets is the large variety of metabolic alterations that occur between cells in the active growth and chronic/dormant phases of tuberculosis. The ideal biochemical target should be active in both growth phases. Methionine adenosyltransferase, which catalyses the formation of S-adenosylmethionine from methionine and ATP, is involved in polyamine biosynthesis during active growth and is also required for the methylation and cyclopropylation of mycolipids necessary for survival in the chronic phase. Results The gene encoding methionine adenosyltransferase has been cloned from Mycobacterium tuberculosis and the model organism M. smegmatis. Both enzymes retained all amino acids known to be involved in catalysing the reaction. While the M. smegmatis enzyme could be functionally expressed, the M. tuberculosis homologue was insoluble and inactive under a large variety of expression conditions. For the M. smegmatis enzyme, the Vmax for S-adenosylmethionine formation was 1.30 μmol/min/mg protein and the Km for methionine and ATP was 288 μM and 76 μM respectively. In addition, the enzyme was competitively inhibited by 8-azaguanine and azathioprine with a Ki of 4.7 mM and 3.7 mM respectively. Azathioprine inhibited the in vitro growth of M. smegmatis with a minimal inhibitory concentration (MIC of 500 μM, while the MIC for 8-azaguanine was >1.0 mM. Conclusion The methionine adenosyltransferase from both organisms had a primary structure very similar those previously characterised in other prokaryotic and eukaryotic organisms. The kinetic properties of the M. smegmatis enzyme were also similar to known prokaryotic methionine adenosyltransferases. Inhibition of the enzyme by 8-azaguanine and azathioprine provides a starting

  16. Inactivation of Mycobacterium paratuberculosis and Mycobacterium tuberculosis in fresh soft cheese by gamma radiation

    Science.gov (United States)

    Badr, Hesham M.

    2011-11-01

    The effectiveness of gamma irradiation on the inactivation of Mycobacterium paratuberculosis, Mycobacterium bovis and Mycobacterium tuberculosis in fresh soft cheese that prepared from artificially inoculated milk samples was studied. Irradiation at dose of 2 kGy was sufficient for the complete inactivation of these mycobacteria as they were not detected in the treated samples during storage at 4±1 °C for 15 days. Moreover, irradiation of cheese samples, that were prepared from un-inoculated milk, at this effective dose had no significant effects on their gross composition and contents from riboflavin, niacin and pantothenic acid, while significant decreases in vitamin A and thiamin were observed. In addition, irradiation of cheese samples had no significant effects on their pH and nitrogen fractions contents, except for the contents of ammonia, which showed a slight, but significant, increases due to irradiation. The analysis of cheese fats indicated that irradiation treatment induced significant increase in their oxidation parameters and contents from free fatty acids; however, the observed increases were relatively low. On the other hand, irradiation of cheese samples induced no significant alterations on their sensory properties. Thus, irradiation dose of 2 kGy can be effectively applied to ensure the safety of soft cheese with regards to these harmful mycobacteria.

  17. Influence of antituberculosis drug resistance and Mycobacterium tuberculosis lineage on outcome in HIV-associated tuberculous meningitis.

    Science.gov (United States)

    Tho, Dau Quang; Török, M Estée; Yen, Nguyen Thi Bich; Bang, Nguyen Duc; Lan, Nguyen Thi Ngoc; Kiet, Vo Sy; van Vinh Chau, Nguyen; Dung, Nguyen Huy; Day, Jeremy; Farrar, Jeremy; Wolbers, Marcel; Caws, Maxine

    2012-06-01

    HIV-associated tuberculous meningitis (TBM) has high mortality. Aside from the devastating impact of multidrug resistance (MDR) on survival, little is understood about the influence of other bacterial factors on outcome. This study examined the influence of Mycobacterium tuberculosis drug resistance, bacterial lineage, and host vaccination status on outcome in patients with HIV-associated TBM. Mycobacterium tuberculosis isolates from the cerebrospinal fluid of 186 patients enrolled in two studies of HIV-associated TBM in Ho Chi Minh City, Vietnam, were tested for resistance to first-line antituberculosis drugs. Lineage genotyping was available for 122 patients. The influence of antituberculosis drug resistance and M. tuberculosis lineage on 9-month mortality was analyzed using Kaplan-Meier survival analysis and Cox multiple regression models. Isoniazid (INH) resistance without rifampin resistance was associated with increased mortality (adjusted hazard ratio [HR], 1.78, 95% confidence interval [CI], 1.18 to 2.66; P = 0.005), and multidrug resistance was uniformly fatal (n = 8/8; adjusted HR, 5.21, 95% CI, 2.38 to 11.42; P tuberculosis lineage are important determinants of mortality in patients with HIV-associated TBM. Interventions which target these factors may help reduce the unacceptably high mortality in patients with TBM.

  18. Consequences of Mycobacterium tuberculosis genetic diversity in the context of HIV co-infection for laboratory diagnosis of tuberculosis in Africa

    NARCIS (Netherlands)

    Ssengooba, W.

    2017-01-01

    Willy Ssengooba’s thesis evaluates the consequences of the genetic diversity of Mycobacterium tuberculosis, the causative agent of tuberculosis, for diagnosis of this disease tuberculosis in Africa, often in HIV co-infected patients. It addresses three main sub-themes around M. tuberculosis genetic

  19. [Susceptibilities of Mycobacterium tuberculosis strains collected from regional tuberculosis laboratories to major antituberculous drugs].

    Science.gov (United States)

    Sayğan, M Bakir; Ocak, Fatih; Cesur, Salih; Tarhan, Gülnur; Ceyhan, Ismail; Gümüişlü, Feyzullah; Beker, Gülşan; Güner, Uğur; Coşkun, Erol

    2007-07-01

    The aim of this study was to investigate the susceptibility rates of Mycobacterium tuberculosis strains sent to Refik Saydam Hygiene Center, Tuberculosis Reference and Research Laboratory, Ankara, from seven different regional tuberculosis laboratories between the 1999-2002 period against major antituberculous drugs. The sensitivities of a total 505 M. tuberculosis strains to isoniazid (INAH), rifampicin (RIF), streptomycin (SM) and ethambutol (EMB) were determined by using proportion method in Lowenstein-Jensen medium. Of the strains, 385 (76.2%) were found sensitive to all of the tested drugs, while 120 strains were resistant to at least one of the antituberculous drugs. The resistant strains showed 14 different resistance patterns. The resistance rates were detected as 13.3% for INAH and RIF (67 strains of each), 9.1% for SM (46 strains), and 3.4% (17 strains) for EMB. Multidrug resistant (INAH+RIF) M. tuberculosis was 7.9% (40 strains). The highest resistance rate to INAH, RIF and EMB (21.2%, 21.2% and 10.6%, respectively) was detected in the isolates which were sent from Bursa province (located in northwestern Turkey); the highest SM (18.8%) and multidrug resistance (INAH+RIF) rates (18.8% and 10.6%, respectively) were detected in the strains sent from Elazig and Van provinces (both located in eastern Turkey). Since the inappropriate use of the first and second line antituberculous drugs leads to the development and spread of the resistant strains, "Directly Observed Therapy Shortcourse (DOTS)" is a very important practice. Therefore regional tuberculosis laboratories should be worth considering as the chains of a well-organized national laboratory network, in order to detect the antituberculous drug resistance patterns of the M. tuberculosis strains over the country.

  20. Innate and Adaptive Cellular Immune Responses to Mycobacterium tuberculosis Infection.

    Science.gov (United States)

    Mayer-Barber, Katrin D; Barber, Daniel L

    2015-07-17

    Host resistance to Mycobacterium tuberculosis (Mtb) infection requires the coordinated efforts of innate and adaptive immune cells. Diverse pulmonary myeloid cell populations respond to Mtb with unique contributions to both host-protective and potentially detrimental inflammation. Although multiple cell types of the adaptive immune system respond to Mtb infection, CD4 T cells are the principal antigen-specific cells responsible for containment of Mtb infection, but they can also be major contributors to disease during Mtb infection in several different settings. Here, we will discuss the role of different myeloid populations as well as the dual nature of CD4 T cells in Mtb infection with a primary focus on data generated using in vivo cellular immunological studies in experimental animal models and in humans when available. Copyright © 2015 Cold Spring Harbor Laboratory Press; all rights reserved.

  1. Mycobacterium tuberculosis: an emerging disease of free-ranging wildlife.

    Science.gov (United States)

    Alexander, Kathleen A; Pleydell, Eve; Williams, Mark C; Lane, Emily P; Nyange, John F C; Michel, Anita L

    2002-06-01

    Expansion of ecotourism-based industries, changes in land-use practices, and escalating competition for resources have increased contact between free-ranging wildlife and humans. Although human presence in wildlife areas may provide an important economic benefit through ecotourism, exposure to human pathogens may represent a health risk for wildlife. This report is the first to document introduction of a primary human pathogen into free-ranging wildlife. We describe outbreaks of Mycobacterium tuberculosis, a human pathogen, in free-ranging banded mongooses (Mungos mungo) in Botswana and suricates (Suricata suricatta) in South Africa. Wildlife managers and scientists must address the potential threat that humans pose to the health of free-ranging wildlife.

  2. An elucidation of neutrophil functions against Mycobacterium tuberculosis infection.

    Science.gov (United States)

    Morris, Devin; Nguyen, Thien; Kim, John; Kassissa, Christine; Khurasany, Melissa; Luong, Jennifer; Kasko, Sarah; Pandya, Shalin; Chu, Michael; Chi, Po-Ting; Ly, Judy; Lagman, Minette; Venketaraman, Vishwanath

    2013-01-01

    We characterized the functions of neutrophils in response to Mycobacterium tuberculosis (M. tb) infection, with particular reference to glutathione (GSH). We examined the effects of GSH in improving the ability of neutrophils to control intracellular M. tb infection. Our findings indicate that increasing the intracellular levels of GSH with a liposomal formulation of GSH (L-GSH) resulted in reduction in the levels of free radicals and increased acidification of M. tb containing phagosomes leading to the inhibition in the growth of M. tb. This inhibitory mechanism is dependent on the presence of TNF-α and IL-6. Our studies demonstrate a novel regulatory mechanism adapted by the neutrophils to control M. tb infection.

  3. An Elucidation of Neutrophil Functions against Mycobacterium tuberculosis Infection

    Directory of Open Access Journals (Sweden)

    Devin Morris

    2013-01-01

    Full Text Available We characterized the functions of neutrophils in response to Mycobacterium tuberculosis (M. tb infection, with particular reference to glutathione (GSH. We examined the effects of GSH in improving the ability of neutrophils to control intracellular M. tb infection. Our findings indicate that increasing the intracellular levels of GSH with a liposomal formulation of GSH (L-GSH resulted in reduction in the levels of free radicals and increased acidification of M. tb containing phagosomes leading to the inhibition in the growth of M. tb. This inhibitory mechanism is dependent on the presence of TNF-α and IL-6. Our studies demonstrate a novel regulatory mechanism adapted by the neutrophils to control M. tb infection.

  4. Mycobacterium tuberculosis Whole Genome Sequences From Southern India Suggest Novel Resistance Mechanisms and the Need for Region-Specific Diagnostics.

    Science.gov (United States)

    Manson, Abigail L; Abeel, Thomas; Galagan, James E; Sundaramurthi, Jagadish Chandrabose; Salazar, Alex; Gehrmann, Thies; Shanmugam, Siva Kumar; Palaniyandi, Kannan; Narayanan, Sujatha; Swaminathan, Soumya; Earl, Ashlee M

    2017-06-01

    India is home to 25% of all tuberculosis cases and the second highest number of multidrug resistant cases worldwide. However, little is known about the genetic diversity and resistance determinants of Indian Mycobacterium tuberculosis, particularly for the primary lineages found in India, lineages 1 and 3. We whole genome sequenced 223 randomly selected M. tuberculosis strains from 196 patients within the Tiruvallur and Madurai districts of Tamil Nadu in Southern India. Using comparative genomics, we examined genetic diversity, transmission patterns, and evolution of resistance. Genomic analyses revealed (11) prevalence of strains from lineages 1 and 3, (11) recent transmission of strains among patients from the same treatment centers, (11) emergence of drug resistance within patients over time, (11) resistance gained in an order typical of strains from different lineages and geographies, (11) underperformance of known resistance-conferring mutations to explain phenotypic resistance in Indian strains relative to studies focused on other geographies, and (11) the possibility that resistance arose through mutations not previously implicated in resistance, or through infections with multiple strains that confound genotype-based prediction of resistance. In addition to substantially expanding the genomic perspectives of lineages 1 and 3, sequencing and analysis of M. tuberculosis whole genomes from Southern India highlight challenges of infection control and rapid diagnosis of resistant tuberculosis using current technologies. Further studies are needed to fully explore the complement of diversity and resistance determinants within endemic M. tuberculosis populations.

  5. Added value of IP-10 as a read-out of Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Jenum, Synne; Dhanasekaran, Sivmakumaran; Ritz, Christian;

    2016-01-01

    We have explored the added value of IFNγ-inducible protein 10 as a read-out of Mycobacterium tuberculosis specific immunity in young Indian children where the sensitivity of the IGRA for tuberculosis (TB) is poor. Reduced frequency of indeterminate results and an increased sensitivity for TB...

  6. Increased risk of Mycobacterium tuberculosis infection in household child contacts exposed to passive tobacco smoke.

    Science.gov (United States)

    Sridhar, Saranya; Karnani, Nisha; Connell, David W; Millington, Kerry A; Dosanjh, Davinder; Bakir, Mustafa; Soysal, Ahmet; Deeks, Jonathan; Lalvani, Ajit

    2014-12-01

    Risk factors associated with Mycobacterium tuberculosis infection were investigated in a prospective cohort of household child tuberculosis contacts. A significantly increased risk of acquiring infection was associated with exposure to passive cigarette smoke, higher number of index cases, younger age and reduced household monthly income.

  7. Sterilization of Mycobacterium tuberculosis Erdman Samples by Antimicrobial Fixation in a Biosafety Level 3 Laboratory

    OpenAIRE

    Schwebach, J. Reid; Jacobs, William R.; Casadevall, Arturo

    2001-01-01

    Incomplete sterilization of Mycobacterium tuberculosis Erdman cultures followed 1 h of incubation in low concentrations of glutaraldehyde (0.5 and 1.0%) or azide. In contrast, 2.5% glutaraldehyde, paraformaldehyde (2 or 4%), Vesphine IIse or 5% formalin sterilized these samples after 1 h. These results suggest caution in removing fixed M. tuberculosis samples from biosafety level 3.

  8. Infection of great apes and a zoo keeper with the same Mycobacterium tuberculosis spoligotype

    NARCIS (Netherlands)

    Akkerman, O.W.; Werf, van de T.S.; Rietkerk, F.; Eger, A.; Soolingen, D.; Loo, v.d. K.; Zanden, v.d. A.G.M.

    2014-01-01

    An animal keeper was diagnosed with pulmonary tuberculosis (TB) after bi-annual screening for latent TB infection in zoo employees. In the same period, several bonobos of the zoo were suffering from TB as well. The Mycobacterium tuberculosis strains from both the animal keeper and the bonobos appear

  9. Draft Genome Sequence of Mycobacterium tuberculosis Clinical Strain G-12-005

    Science.gov (United States)

    de Carvalho, Fabíola Marques; de Almeida, Luiz Gonzaga Paula; Bablishvili, Nino; Gauthier, Marie; Paranhos-Baccalà, Glaucia; de Vasconcelos, Ana Tereza Ribeiro

    2014-01-01

    Infection caused by drug-resistant Mycobacterium tuberculosis is a growing concern, especially in eastern Europe. We report an annotated draft genome sequence of M. tuberculosis strain G-12-005 obtained from a patient in Georgia. PMID:24812221

  10. Mycobacterium tuberculosis Rv1987 induces Th2 immune responses and enhances Mycobacterium smegmatis survival in mice.

    Science.gov (United States)

    Sha, Shanshan; Shi, Xiaoxia; Deng, Guoying; Chen, Lina; Xin, Yi; Ma, Yufang

    2017-04-01

    Mycobacterium tuberculosis can interfere with host immune response and escape clearance through its specific antigens. M. tuberculosis Rv1987 encoded by region of difference (RD)-2 gene is a secretory protein with immunogenic potency. Here, we investigated the impact of Rv1987 on host cytokine responses and T cell polarization in mouse aerosol model. A recombinant M. smegmatis mc(2)155 strain that overexpressed Rv1987 protein (named MS1987) was constructed and used to infect C57BL/6 mice. The mc(2)155 harbored the empty vector (named MSVec) was as a control. The results showed that MS1987 challenged mice promoted Th2-biased cytokine responses with lower secretion of IFN-γ but higher production of IL-4 and Rv1987-specific IgG antibody compared to MSVec infected mice. Neutrophilic inflammation and high bacterial burden were observed in the lung tissues of MS1987 infected mice probably own to the failed Th1 cell immunity. Besides, subcutaneous injection of Rv1987 protein could mediate the Th1 cytokine responses caused by M. bovis BCG in mice. These results indicated that M. tuberculosis Rv1987 protein could modulate host immune response towards Th2 profile, which probably contributed to the immune evasion of bacteria from host elimination. Copyright © 2017 Elsevier GmbH. All rights reserved.

  11. CCL2 responses to Mycobacterium tuberculosis are associated with disease severity in tuberculosis.

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    Zahra Hasan

    Full Text Available BACKGROUND: Leucocyte activating chemokines such as CCL2, CCL3, and CXCL8 together with proinflammatory IFNgamma, TNFalpha and downmodulatory IL10 play a central role in the restriction of M. tuberculosis infections, but is unclear whether these markers are indicative of tuberculosis disease severity. METHODOLOGY: We investigated live M. tuberculosis- and M. bovis BCG-induced peripheral blood mononuclear cell responses in patients with tuberculosis (TB and healthy endemic controls (ECs, n = 36. TB patients comprised pulmonary (PTB, n = 34 and extrapulmonary groups, subdivided into those with less severe localized extrapulmonary TB (L-ETB, n = 16 or severe disseminated ETB (D-ETB, n = 16. Secretion of CCL2, IFNgamma, IL10 and CCL3, and mRNA expression of CCL2, TNFalpha, CCL3 and CXCL8 were determined. RESULTS: M. tuberculosis- and BCG-induced CCL2 secretion was significantly increased in both PTB and D-ETB (p<0.05, p<0.01 as compared with L-ETB patients. CCL2 secretion in response to M. tuberculosis was significantly greater than to BCG in the PTB and D-ETB groups. M. tuberculosis-induced CCL2 mRNA transcription was greater in PTB than L-ETB (p = 0.023, while CCL2 was reduced in L-ETB as compared with D-ETB (p = 0.005 patients. M. tuberculosis-induced IFNgamma was greater in L-ETB than PTB (p = 0.04, while BCG-induced IFNgamma was greater in L-ETB as compared with D-ETB patients (p = 0.036. TNFalpha mRNA expression was raised in PTB as compared with L-ETB group in response to M. tuberculosis (p = 0.02 and BCG (p = 0.03. Mycobacterium-induced CCL3 and CXCL8 was comparable between TB groups. CONCLUSIONS: The increased CCL2 and TNFalpha in PTB patients may support effective leucocyte recruitment and M. tuberculosis localization. CCL2 alone is associated with severity of TB, possibly due to increased systemic inflammation found in severe disseminated TB or due to increased monocyte infiltration to lung parenchyma in pulmonary disease.

  12. Global Efforts in the Development of Vaccines for Tuberculosis: Requirements for Improved Vaccines Against Mycobacterium tuberculosis.

    Science.gov (United States)

    Méndez-Samperio, P

    2016-10-01

    Currently, more than 9.0 million people develop acute pulmonary tuberculosis (TB) each year and about 1.5 million people worldwide die from this infection. Thus, developing vaccines to prevent active TB disease remains a priority. This article discusses recent progress in the development of new vaccines against TB and focusses on the main requirements for development of improved vaccines against Mycobacterium tuberculosis (M. tb). Over the last two decades, significant progress has been made in TB vaccine development, and some TB vaccine candidates have currently completed a phase III clinical trial. The potential public health benefits of these vaccines are possible, but it will need much more effort, including new global governance investment on this research. This investment would certainly be less than the annual global financial toll of TB treatment.

  13. Validation of a real-time PCR assay for the molecular identification of Mycobacterium tuberculosis.

    Science.gov (United States)

    Sales, Mariana L; Fonseca Júnior, Antônio Augusto; Orzil, Lívia; Alencar, Andrea Padilha; Silva, Marcio Roberto; Issa, Marina Azevedo; Soares Filho, Paulo Martins; Lage, Andrey Pereira; Heinemann, Marcos Bryan

    2014-01-01

    Mycobacterium tuberculosis is the major cause of tuberculosis in humans. This bacillus gained prominence with the occurrence of HIV, presenting itself as an important opportunistic infection associated with acquired immunodeficiency syndrome (AIDS). The current study aimed to develop a real-time PCR using Eva Green technology for molecular identification of M. tuberculosis isolates. The primers were designed to Rv1510 gene. Ninety nine samples of M. tuberculosis and sixty samples of M. bovis were tested and no sample of the bovine bacillus was detected by the qPCR. Statistical tests showed no difference between the qPCR and biochemical tests used to identify the Mycobacterium tuberculosis. The correlation between tests was perfect with Kappa index of 1.0 (p tuberculosis in samples of bacterial suspension. TB reference laboratories (health and agriculture sectors), public health programs and epidemiological studies probably may benefit from such method.

  14. Zoonotic tuberculosis in human beings caused by Mycobacterium bovis-a call for action.

    Science.gov (United States)

    Olea-Popelka, Francisco; Muwonge, Adrian; Perera, Alejandro; Dean, Anna S; Mumford, Elizabeth; Erlacher-Vindel, Elisabeth; Forcella, Simona; Silk, Benjamin J; Ditiu, Lucica; El Idrissi, Ahmed; Raviglione, Mario; Cosivi, Ottorino; LoBue, Philip; Fujiwara, Paula I

    2017-01-01

    Mycobacterium tuberculosis is recognised as the primary cause of human tuberculosis worldwide. However, substantial evidence suggests that the burden of Mycobacterium bovis, the cause of bovine tuberculosis, might be underestimated in human beings as the cause of zoonotic tuberculosis. In 2013, results from a systematic review and meta-analysis of global zoonotic tuberculosis showed that the same challenges and concerns expressed 15 years ago remain valid. These challenges faced by people with zoonotic tuberculosis might not be proportional to the scientific attention and resources allocated in recent years to other diseases. The burden of zoonotic tuberculosis in people needs important reassessment, especially in areas where bovine tuberculosis is endemic and where people live in conditions that favour direct contact with infected animals or animal products. As countries move towards detecting the 3 million tuberculosis cases estimated to be missed annually, and in view of WHO's end TB strategy endorsed by the health authorities of WHO Member States in 2014 to achieve a world free of tuberculosis by 2035, we call on all tuberculosis stakeholders to act to accurately diagnose and treat tuberculosis caused by M bovis in human beings. Copyright © 2017 World Health Organization. Published by Elsevier Ltd/Inc/BV. All rights reserved. Published by Elsevier Ltd.. All rights reserved.

  15. How dormant is Mycobacterium tuberculosis during latency? A study integrating genomics and molecular epidemiology

    DEFF Research Database (Denmark)

    Yang, Zhenhua; Rosenthal, Mariana; Rosenberg, Noah A;

    2011-01-01

    Mycobacterium tuberculosis may survive for decades in the human body in a state termed latent tuberculosis infection (LTBI). We investigated the occurrence during LTBI of insertion/deletion events in a selected set of mononucleotide simple sequence repeats, DNA sequence changes in four M....... tuberculosis genes, and large sequence variations in 4750 M. tuberculosis open reading frames. We studied 13 paired M. tuberculosis clinical isolates, with each pair representing a reactivation of LTBI more than three decades after primary infection. Absence of sequence variations between paired isolates...

  16. How dormant is Mycobacterium tuberculosis during latency? A study integrating genomics and molecular epidemiology.

    Science.gov (United States)

    Yang, Zhenhua; Rosenthal, Mariana; Rosenberg, Noah A; Talarico, Sarah; Zhang, Lixin; Marrs, Carl; Thomsen, Vibeke Østergaard; Lillebaek, Troels; Andersen, Aase B

    2011-07-01

    Mycobacterium tuberculosis may survive for decades in the human body in a state termed latent tuberculosis infection (LTBI). We investigated the occurrence during LTBI of insertion/deletion events in a selected set of mononucleotide simple sequence repeats, DNA sequence changes in four M. tuberculosis genes, and large sequence variations in 4750 M. tuberculosis open reading frames. We studied 13 paired M. tuberculosis clinical isolates, with each pair representing a reactivation of LTBI more than three decades after primary infection. Absence of sequence variations between paired isolates in nearly all investigated loci suggests a low likelihood of bacterial replication during LTBI.

  17. Biochemical Characterization of Uracil Phosphoribosyltransferase from Mycobacterium tuberculosis

    Science.gov (United States)

    Villela, Anne Drumond; Ducati, Rodrigo Gay; Rosado, Leonardo Astolfi; Bloch, Carlos Junior; Prates, Maura Vianna; Gonçalves, Danieli Cristina; Ramos, Carlos Henrique Inacio; Basso, Luiz Augusto; Santos, Diogenes Santiago

    2013-01-01

    Uracil phosphoribosyltransferase (UPRT) catalyzes the conversion of uracil and 5-phosphoribosyl-α-1-pyrophosphate (PRPP) to uridine 5′-monophosphate (UMP) and pyrophosphate (PPi). UPRT plays an important role in the pyrimidine salvage pathway since UMP is a common precursor of all pyrimidine nucleotides. Here we describe cloning, expression and purification to homogeneity of upp-encoded UPRT from Mycobacterium tuberculosis (MtUPRT). Mass spectrometry and N-terminal amino acid sequencing unambiguously identified the homogeneous protein as MtUPRT. Analytical ultracentrifugation showed that native MtUPRT follows a monomer-tetramer association model. MtUPRT is specific for uracil. GTP is not a modulator of MtUPRT ativity. MtUPRT was not significantly activated or inhibited by ATP, UTP, and CTP. Initial velocity and isothermal titration calorimetry studies suggest that catalysis follows a sequential ordered mechanism, in which PRPP binding is followed by uracil, and PPi product is released first followed by UMP. The pH-rate profiles indicated that groups with pK values of 5.7 and 8.1 are important for catalysis, and a group with a pK value of 9.5 is involved in PRPP binding. The results here described provide a solid foundation on which to base upp gene knockout aiming at the development of strategies to prevent tuberculosis. PMID:23424660

  18. Amperometric immunosensor for rapid detection of Mycobacterium tuberculosis

    Science.gov (United States)

    Hiraiwa, Morgan; Kim, Jong-Hoon; Lee, Hyun-Boo; Inoue, Shinnosuke; Becker, Annie L.; Weigel, Kris M.; Cangelosi, Gerard A.; Lee, Kyong-Hoon; Chung, Jae-Hyun

    2015-05-01

    Tuberculosis (TB) has been a major public health problem, which can be better controlled by using accurate and rapid diagnosis in low-resource settings. A simple, portable, and sensitive detection method is required for point-of-care (POC) settings. This paper studies an amperometric biosensor using a microtip immunoassay for a rapid and low-cost detection of Mycobacterium tuberculosis (MTB) in sputum. MTB in sputum is specifically captured on the functionalized microtip surface and detected by electric current. According to the numerical study, the current signal on the microtip surface is linearly changed with increasing immersion depth. Using a reference microtip, the immersion depth is compensated for a sensing microtip. On the microtip surface, target bacteria are concentrated and organized by a coffee-ring effect, which amplifies the electric current. To enhance the signal-to-noise ratio, both the sample processing and rinsing steps are presented with the use of deionized water as a medium for the amperometric measurement. When applied to cultured MTB cells spiked into human sputum, the detection limit was 100 CFU mL-1, comparable to a more labor-intensive fluorescence detection method reported previously.

  19. Succinate dehydrogenase is the regulator of respiration in Mycobacterium tuberculosis.

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    Travis Hartman

    2014-11-01

    Full Text Available In chronic infection, Mycobacterium tuberculosis bacilli are thought to enter a metabolic program that provides sufficient energy for maintenance of the protonmotive force, but is insufficient to meet the demands of cellular growth. We sought to understand this metabolic downshift genetically by targeting succinate dehydrogenase, the enzyme which couples the growth processes controlled by the TCA cycle with the energy production resulting from the electron transport chain. M. tuberculosis contains two operons which are predicted to encode succinate dehydrogenase enzymes (sdh-1 and sdh-2; we found that deletion of Sdh1 contributes to an inability to survive long term stationary phase. Stable isotope labeling and mass spectrometry revealed that Sdh1 functions as a succinate dehydrogenase during aerobic growth, and that Sdh2 is dispensable for this catalysis, but partially overlapping activities ensure that the loss of one enzyme can incompletely compensate for loss of the other. Deletion of Sdh1 disturbs the rate of respiration via the mycobacterial electron transport chain, resulting in an increased proportion of reduced electron carrier (menaquinol which leads to increased oxygen consumption. The loss of respiratory control leads to an inability to recover from stationary phase. We propose a model in which succinate dehydrogenase is a governor of cellular respiration in the adaptation to low oxygen environments.

  20. Structural Basis of Mycobacterium tuberculosis Transcription and Transcription Inhibition.

    Science.gov (United States)

    Lin, Wei; Mandal, Soma; Degen, David; Liu, Yu; Ebright, Yon W; Li, Shengjian; Feng, Yu; Zhang, Yu; Mandal, Sukhendu; Jiang, Yi; Liu, Shuang; Gigliotti, Matthew; Talaue, Meliza; Connell, Nancy; Das, Kalyan; Arnold, Eddy; Ebright, Richard H

    2017-04-20

    Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, which kills 1.8 million annually. Mtb RNA polymerase (RNAP) is the target of the first-line antituberculosis drug rifampin (Rif). We report crystal structures of Mtb RNAP, alone and in complex with Rif, at 3.8-4.4 Å resolution. The results identify an Mtb-specific structural module of Mtb RNAP and establish that Rif functions by a steric-occlusion mechanism that prevents extension of RNA. We also report non-Rif-related compounds-Nα-aroyl-N-aryl-phenylalaninamides (AAPs)-that potently and selectively inhibit Mtb RNAP and Mtb growth, and we report crystal structures of Mtb RNAP in complex with AAPs. AAPs bind to a different site on Mtb RNAP than Rif, exhibit no cross-resistance with Rif, function additively when co-administered with Rif, and suppress resistance emergence when co-administered with Rif. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Roles of Mucosal Immunity against Mycobacterium tuberculosis Infection.

    Science.gov (United States)

    Li, Wu; Deng, Guangcun; Li, Min; Liu, Xiaoming; Wang, Yujiong

    2012-01-01

    Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is one of the world's leading infectious causes of morbidity and mortality. As a mucosal-transmitted pathogen, Mtb infects humans and animals mainly through the mucosal tissue of the respiratory tract. Apart from providing a physical barrier against the invasion of pathogen, the major function of the respiratory mucosa may be to serve as the inductive sites to initiate mucosal immune responses and sequentially provide the first line of defense for the host to defend against this pathogen. A large body of studies in the animals and humans have demonstrated that the mucosal immune system, rather than the systemic immune system, plays fundamental roles in the host's defense against Mtb infection. Therefore, the development of new vaccines and novel delivery routes capable of directly inducing respiratory mucosal immunity is emphasized for achieving enhanced protection from Mtb infection. In this paper, we outline the current state of knowledge regarding the mucosal immunity against Mtb infection, including the development of TB vaccines, and respiratory delivery routes to enhance mucosal immunity are discussed.

  2. Genetics-directed drug discovery for combating Mycobacterium tuberculosis infection.

    Science.gov (United States)

    Quan, Yuan; Xiong, Le; Chen, Jing; Zhang, Hong-Yu

    2017-02-01

    Mycobacterium tuberculosis (Mtb), the pathogen of tuberculosis (TB), is one of the most infectious bacteria in the world. The traditional strategy to combat TB involves targeting the pathogen directly; however, the rapid evolution of drug resistance lessens the efficiency of this anti-TB method. Therefore, in recent years, some researchers have turned to an alternative anti-TB strategy, which hinders Mtb infection through targeting host genes. In this work, using a theoretical genetic analysis, we identified 170 Mtb infection-associated genes from human genetic variations related to Mtb infection. Then, the agents targeting these genes were identified to have high potential as anti-TB drugs. In particular, the agents that can target multiple Mtb infection-associated genes are more druggable than the single-target counterparts. These potential anti-TB agents were further screened by gene expression data derived from connectivity map. As a result, some agents were revealed to have high interest for experimental evaluation. This study not only has important implications for anti-TB drug discovery, but also provides inspirations for streamlining the pipeline of modern drug discovery.

  3. Roles of Mucosal Immunity against Mycobacterium tuberculosis Infection

    Directory of Open Access Journals (Sweden)

    Wu Li

    2012-01-01

    Full Text Available Mycobacterium tuberculosis (Mtb, the causative agent of tuberculosis (TB, is one of the world's leading infectious causes of morbidity and mortality. As a mucosal-transmitted pathogen, Mtb infects humans and animals mainly through the mucosal tissue of the respiratory tract. Apart from providing a physical barrier against the invasion of pathogen, the major function of the respiratory mucosa may be to serve as the inductive sites to initiate mucosal immune responses and sequentially provide the first line of defense for the host to defend against this pathogen. A large body of studies in the animals and humans have demonstrated that the mucosal immune system, rather than the systemic immune system, plays fundamental roles in the host’s defense against Mtb infection. Therefore, the development of new vaccines and novel delivery routes capable of directly inducing respiratory mucosal immunity is emphasized for achieving enhanced protection from Mtb infection. In this paper, we outline the current state of knowledge regarding the mucosal immunity against Mtb infection, including the development of TB vaccines, and respiratory delivery routes to enhance mucosal immunity are discussed.

  4. Biochemical characterization of uracil phosphoribosyltransferase from Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Anne Drumond Villela

    Full Text Available Uracil phosphoribosyltransferase (UPRT catalyzes the conversion of uracil and 5-phosphoribosyl-α-1-pyrophosphate (PRPP to uridine 5'-monophosphate (UMP and pyrophosphate (PP(i. UPRT plays an important role in the pyrimidine salvage pathway since UMP is a common precursor of all pyrimidine nucleotides. Here we describe cloning, expression and purification to homogeneity of upp-encoded UPRT from Mycobacterium tuberculosis (MtUPRT. Mass spectrometry and N-terminal amino acid sequencing unambiguously identified the homogeneous protein as MtUPRT. Analytical ultracentrifugation showed that native MtUPRT follows a monomer-tetramer association model. MtUPRT is specific for uracil. GTP is not a modulator of MtUPRT ativity. MtUPRT was not significantly activated or inhibited by ATP, UTP, and CTP. Initial velocity and isothermal titration calorimetry studies suggest that catalysis follows a sequential ordered mechanism, in which PRPP binding is followed by uracil, and PP(i product is released first followed by UMP. The pH-rate profiles indicated that groups with pK values of 5.7 and 8.1 are important for catalysis, and a group with a pK value of 9.5 is involved in PRPP binding. The results here described provide a solid foundation on which to base upp gene knockout aiming at the development of strategies to prevent tuberculosis.

  5. [Risk of Mycobacterium tuberculosis infection in Jalisco, Mexico].

    Science.gov (United States)

    Gloyd, S; López, J L; Mercado, F J; Durning, J

    1991-11-01

    Using a modified cluster sample design, skin tests with two TU PPD were performed on 4,083 first-grade children (mean age 6,7 years) in the state of Jalisco, Mexico, to estimate the annual risk of infection from Mycobacterium tuberculosis. The proportion of PPD reactions (a diameter of 10 mm or more of induration) was 7.6% in the Guadalajara metropolitan area and 5.5% in the rest of Jalisco. In the total sample, the proportion was 7.4% among children with scars attributed to BCG vaccine and 4.5% in children without BCG scars. The weighted proportion of children with PPD reactions of 10 mm or more was 6.8% statewide. The average annual risk of infection estimated from the group that had not received BCG vaccine was 0.82%. The results suggest that the incidence of smear-positive pulmonary tuberculosis is almost 10 times greater than the number of cases registered annually in Jalisco.

  6. Characterization and Transcriptome Analysis of Mycobacterium tuberculosis Persisters

    Science.gov (United States)

    Keren, Iris; Minami, Shoko; Rubin, Eric; Lewis, Kim

    2011-01-01

    ABSTRACT Tuberculosis continues to be a major public health problem in many parts of the world. Significant obstacles in controlling the epidemic are the length of treatment and the large reservoir of latently infected people. Bacteria form dormant, drug-tolerant persister cells, which may be responsible for the difficulty in treating both acute and latent infections. We find that in Mycobacterium  tuberculosis, low numbers of drug-tolerant persisters are present in lag and early exponential phases, increasing sharply at late exponential and stationary phases to make up ~1% of the population. This suggests that persister formation is governed by both stochastic and deterministic mechanisms. In order to isolate persisters, an exponentially growing population was treated with d-cycloserine, and cells surviving lysis were collected by centrifugation. A transcriptome of persisters was obtained by using hybridization to an Affymetrix array. The transcriptome shows downregulation of metabolic and biosynthetic pathways, consistent with a certain degree of dormancy. A set of genes was upregulated in persisters, and these are likely involved in persister formation and maintenance. A comparison of the persister transcriptome with transcriptomes obtained for several in vitro dormancy models identified a small number of genes upregulated in all cases, which may represent a core dormancy response. PMID:21673191

  7. Dihydrolipoamide acyltransferase is critical for Mycobacterium tuberculosis pathogenesis.

    Science.gov (United States)

    Shi, Shuangping; Ehrt, Sabine

    2006-01-01

    Mycobacterium tuberculosis has evolved to persist in host macrophages, where it faces a nutrient-poor environment and is exposed to oxidative and nitrosative stress. To defend itself against oxidative/nitrosative stress, M. tuberculosis expresses an NADH-dependent peroxidase and peroxynitrite reductase that is encoded by ahpC, ahpD, lpd, and dlaT. In addition to its central role in the peroxynitrite reductase complex, dlaT (Rv2215) also encodes the E2 component of pyruvate dehydrogenase. Here we demonstrate that inactivation of dlaT in the chromosome of H37Rv resulted in a mutant (H37RvDeltadlaT) that displayed phenotypes associated with DlaT's role in metabolism and in defense against nitrosative stress. The H37RvDeltadlaT strain showed retarded growth in vitro and was highly susceptible to killing by acidified sodium nitrite. Mouse macrophages readily killed intracellular H37RvDeltadlaT organisms, and in mice dlaT was required for full virulence.

  8. Pulsed Field Gel Electrophoresis and Genetic Diversity in Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Mohammad Poyeede

    2013-07-01

    Full Text Available AbstractBackground and objective: Tuberculosis is a considerable public health problem due to its high risk of person-to-person transmission, morbidity, and mortality especially in developing countries. According to the World Health Organization there is the emergence of multi-drug resistant M. tuberculosis and the association of TB with HIV has led to TB being declared. Molecular genotyping methods are important in detecting the dominance of transmission or reinfection in a population. During one year study genotyping of 100 of M. tuberculosis (M.t. isolates from patients referred to Pasteur Institute of Iran were accomplished with PFGE method. Material and methods: After identification of M.t. isolates and performing of antibiotic susceptibility test using standard methods, Melted Incert agarose and lysozyme were mixed with bacterial suspension to prepare PFGE plaques. After lyses and washing process the plaques digested with XbaI restriction enzyme. Finally the digested DNA fragments on 1% agarose with PFGE method were stained with ethidium bromide and analyzed with GelcomparII software.Results: Dendrogram of genetic diversity among 100 M.t. isolates were obtained in comparison of molecular weight marker and revealed two common types. Pulsotype A with 71 isolates and just one MDR and pulsotype B included 29 isolates and 3 MDR cases. No correlation between antibiotypes and pulsotypes were observed.Conclusion: It is very important to know about the existence of any clonal expansion of special M.t. genotypes with resistant strains. Our research shows 3 MDR isolates into the low incidence pulsotype B which could be an alarm for more accurate MDR-TB surveillance program. Probably such observed limited polymorphism may be due to conservation of restriction sites of XbaI enzyme. In order to investigate the genetic relatedness of isolates using other restriction enzymes and different molecular typing methods simultaneously were recommended.

  9. Tuberculosis in Goats and Sheep in Afar Pastoral Region of Ethiopia and Isolation of Mycobacterium tuberculosis from Goat

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    Gezahegne Mamo Kassa

    2012-01-01

    epidemiology of tuberculosis in goats and sheep using comparative intradermal tuberculin skin test, postmortem examination, mycobacteriological culture and molecular typing methods. The overall animal prevalence of TB in small ruminants was 0.5% (95% CI: 0.2%–0.7% at ≥4 mm and 3.8% (95% CI: 3%–4.7% at cutoff ≥2 mm. The herd prevalence was 20% (95% CI: 12–28% and 47% (95% CI: 37–56% at ≥4 mm and ≥2 mm cut-off points, respectively. The overall animal prevalence of Mycobacterium avium complex infection was 2.8% (95% CI: 2.1–3.5% and 6.8% (95% CI: 5.8–7.9% at ≥4 mm and ≥2 mm cut-off points, respectively. Mycobacteriological culture and molecular characterization of isolates from tissue lesions of tuberculin reactor goats resulted in isolation of Mycobacterium tuberculosis (SIT149 and non-tuberculosis mycobacteria as causative agents of tuberculosis and tuberculosis-like diseases in goats, respectively. The isolation of Mycobacterium tuberculosis in goat suggests a potential transmission of the causative agent from human and warrants further investigation in the role of small ruminants in epidemiology of human tuberculosis in the region.

  10. Fast and efficient detection of tuberculosis antigens using liposome encapsulated secretory proteins of Mycobacterium tuberculosis.

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    Tiwari, Dileep; Haque, Shafiul; Tiwari, Ram P; Jawed, Arshad; Govender, Thavendran; Kruger, Hendrik G

    2017-04-01

    A rapid and efficient diagnostic test was developed for the detection of Mycobacterium tuberculosis antigens in serum samples of active tuberculosis (TB) and extrapulmonary TB patients via a liposomal agglutination-based method. A rapid card test has been developed to facilitate the recognition of high-affinity binding rabbit raised purified culture filtrate protein antibodies coupled on the surface of activated liposomal preparation. In the presence of TB antigens, the polyclonal antibodies bound to the liposomal particles demonstrate a visible agglutination reaction. The developed assay was simple, rapid, reliable, sensitive, and specific as a diagnostic test for the detection of antigens in serum samples of clinically confirmed cases of TB within 4-5 minutes' duration. The test was evaluated at different hospitals, medical colleges, and pathology centers, and involved 1483 participants. This investigation was conducted to detect the presence of these antigens during the period of active growth of the microorganism in serum samples for pulmonary TB and processed tissue biopsy for other extrapulmonary TB. Results obtained using this test were compared with acid-fast bacilli smear and culture results. Our study demonstrated that the newly developed liposome tuberculosis antigen card test detected antigens in our study population with approximately 97.48% sensitivity and 95.79% specificity. This is the first study to report the liposomal encapsulation of culture filtrate proteins from M. tuberculosis for diagnostic application. Copyright © 2015. Published by Elsevier B.V.

  11. Mycobacterium tuberculosis strains exhibit differential and strain-specific molecular signatures in pulmonary epithelial cells.

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    Mvubu, Nontobeko Eunice; Pillay, Balakrishna; Gamieldien, Junaid; Bishai, William; Pillay, Manormoney

    2016-12-01

    Although pulmonary epithelial cells are integral to innate and adaptive immune responses during Mycobacterium tuberculosis infection, global transcriptomic changes in these cells remain largely unknown. Changes in gene expression induced in pulmonary epithelial cells infected with M. tuberculosis F15/LAM4/KZN, F11, F28, Beijing and Unique genotypes were investigated by RNA sequencing (RNA-Seq). The Illumina HiSeq 2000 platform generated 50 bp reads that were mapped to the human genome (Hg19) using Tophat (2.0.10). Differential gene expression induced by the different strains in infected relative to the uninfected cells was quantified and compared using Cufflinks (2.1.0) and MeV (4.0.9), respectively. Gene expression varied among the strains with the total number of genes as follows: F15/LAM4/KZN (1187), Beijing (1252), F11 (1639), F28 (870), Unique (886) and H37Rv (1179). A subset of 292 genes was commonly induced by all strains, where 52 genes were down-regulated while 240 genes were up-regulated. Differentially expressed genes were compared among the strains and the number of induced strain-specific gene signatures were as follows: F15/LAM4/KZN (138), Beijing (52), F11 (255), F28 (55), Unique (186) and H37Rv (125). Strain-specific molecular gene signatures associated with functional pathways were observed only for the Unique and H37Rv strains while certain biological functions may be associated with other strain signatures. This study demonstrated that strains of M. tuberculosis induce differential gene expression and strain-specific molecular signatures in pulmonary epithelial cells. Specific signatures induced by clinical strains of M. tuberculosis can be further explored for novel host-associated biomarkers and adjunctive immunotherapies.

  12. Current trends of Mycobacterium tuberculosis molecular epidemiology in Saudi Arabia and associated demographical factors.

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    Al-Hajoj, Sahal; Varghese, Bright; Al-Habobe, Farah; Shoukri, Mohammed M; Mulder, Arnout; van Soolingen, Dick

    2013-06-01

    Data are scarce on demographical factors related to the population structure of Mycobacterium tuberculosis in Saudi Arabia. A study was conducted on 902 clinical isolates to explore current trends in the phylogeography and associated demographical factors of tuberculosis by using spoligotyping and 24 loci based MIRU-VNTR typing. Young male patients (aged 16-29 and 30-44) were predominant in this cohort. The phylogenetic diversity among M. tuberculosis isolates was found high, as almost all known genetic lineages were identified. Delhi/CAS (26.4%), EAI (13.7%) and Haarlem (11.3%) were the most common lineages observed, particularly among the low age groups (16-29 and 30-44 years), whereas elderly patients (>60 years) showed a predominance in the lineages S, Ghana, TUR and Uganda-I. A statistically significant association was observed between gender of the patients and lineages of EAI (p value 0.026) and LAM (p value 0.005). Overall, molecular strain cluster rate was 34.4% with an elevated rate among patients aged below 15 years (43.1%), while cases among the elderly (>60 years) showed the lowest degree of clustering (12.5%). The largest level of clustering was noticed among cases caused by strains of the lineages Haarlem (59.8%), Beijing (55.8%) and LAM (42.8%). The current population structure of M. tuberculosis in Saudi Arabia is highly diverse with significant associations to demography, transmission dynamics and origin of the patients. The difference in genotype distributions among low and high aged patients reflects the ongoing change in the strain population structure in the country.

  13. Drug resistance of Mycobacterium tuberculosis isolates from tuberculosis lymphadenitis patients in Ethiopia

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    Fantahun Biadglegne

    2014-01-01

    Full Text Available Background & objectives: The emergence of drug resistance tuberculosis (TB is a significant challenge for TB control and prevention programmes, and the major problem is multidrug resistant tuberculosis (MDR-TB. The present study was carried out to determine the frequency of drug resistant Mycobacterium tuberculosis isolates among newly and retreated TB lymphadenitis patients and risk factors for acquiring this infection. Methods: Two hundred twenty five M. tuberculosis isolates from TB lymphadenitis patients who were diagnosed as new and retreated tuberculosis cases between April 2012 and May 2012 were included in this study. Isolates were tested for susceptibility to isoniazed (INH, rifampicin (RMP, streptomycin (SM, ethambutol (EMB and pyrazinamide (PZA using the BacT/AlerT 3D system protocol. Results: Among 225 isolates, 15 (6.7% were resistant to at least one first line anti-TB drug. Three (1.3% were MDR-TB. Resistance to INH, RMP, SM, and EMB was found in 8 (3.6%, 4 (1.8%, 10 (4.4%, and 4 (1.8% isolates, respectively. Of the 212 new TB lymphadenitis cases three (1.4% were MDR-TB. A rifampicin resistant M. tuberculosis isolate was diagnosed from smear and culture negative newly treated cases. All isolates were susceptible to PZA. Matted cervical lymph nodes were the prominent sites involved. Newly treated TB lymphadenitis patients had a greater risk for presenting resistance to anti-TB drugs ( p =0.046. Interpretation & conclusions: Our study showed that TB lymphadenitis patients harboured drug resistant TB and MDR-TB, although at a low rate. Resistance was not associated with age, sex, patients′ education and contact history. Further research is required to determine transmission dynamics of drug resistant strains.

  14. Humoral response to low molecular weight antigens of Mycobacterium tuberculosis by tuberculosis patients and contacts

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    Beck S.T.

    2005-01-01

    Full Text Available Much effort has been devoted to the identification of immunologically important antigens of Mycobacterium tuberculosis and to the combination of target antigens to which antibodies from serum of tuberculous patients could react specifically. We searched for IgG antibodies specific for antigens of 45 to 6 kDa obtained after sonication of the well-characterized wild M. tuberculosis strain in order to detect differences in the antibody response to low molecular weight antigens from M. tuberculosis between patients with pulmonary tuberculosis and contacts. Specific IgG antibodies for these antigens were detected by Western blot analysis of 153 serum samples collected from 51 patients with confirmed pulmonary tuberculosis. Three samples were collected from each patient: before therapy, and after 2 and 6 months of treatment. We also analyzed 25 samples obtained from contacts, as well as 30 samples from healthy individuals with known tuberculin status, 50 samples from patients with other lung diseases and 200 samples from healthy blood donors. The positive predictive value for associated IgG reactivity against the 6-kDa and 16-kDa antigens, 6 and 38 kDa, and 16 and 38 kDa was 100% since simultaneous reactivity for these antigens was absent in healthy individuals and individuals with other lung diseases. This association was observed in 67% of the patients, but in only 8% of the contacts. The humoral response against antigens of 16 and 6 kDa seems to be important for the detection of latent tuberculosis since the associated reactivity to these antigens is mainly present in individuals with active disease.

  15. Longitudinal assessment of an ELISPOT test for Mycobacterium tuberculosis infection.

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    Philip C Hill

    2007-06-01

    Full Text Available Very little longitudinal information is available regarding the performance of T cell-based tests for Mycobacterium tuberculosis infection. To address this deficiency, we conducted a longitudinal assessment of the enzyme-linked immunosorbent spot test (ELISPOT test in comparison to the standard tuberculin skin test (TST.In tuberculosis (TB contacts we repeated ELISPOT tests 3 mo (n = 341 and 18 mo (n = 210 after recruitment and TSTs at 18 mo (n = 130. We evaluated factors for association with conversion and reversion and investigated suspected cases of TB. Of 207 ELISPOT-negative contacts, 51 (24.6% had 3-mo ELISPOT conversion, which was associated with a positive recruitment TST (odds ratio [OR] 2.2, 95% confidence interval [CI] 1.0-5.0, p = 0.048 and negatively associated with bacillus Calmette-Guérin (BCG vaccination (OR 0.5, 95% CI 0.2-1.0, p = 0.06. Of 134 contacts, 54 (40.2% underwent 3-mo ELISPOT reversion, which was less likely in those with a positive recruitment TST (OR 0.3, 95% CI 0.1-0.8, p = 0.014. Between 3 and 18 mo, 35/132 (26.5% contacts underwent ELISPOT conversion and 28/78 (35.9% underwent ELISPOT reversion. Of the 210 contacts with complete results, 73 (34.8% were ELISPOT negative at all three time points; 36 (17.1% were positive at all three time points. Between recruitment and 18 mo, 20 (27% contacts had ELISPOT conversion; 37 (50% had TST conversion, which was associated with a positive recruitment ELISPOT (OR 7.2, 95% CI 1.4-37.1, p = 0.019; 18 (32.7% underwent ELISPOT reversion; and five (8.9% underwent TST reversion. Results in 13 contacts diagnosed as having TB were mixed, but suggested higher TST sensitivity.Both ELISPOT conversion and reversion occur after M. tuberculosis exposure. Rapid ELISPOT reversion may reflect M. tuberculosis clearance or transition into dormancy and may contribute to the relatively low reported ELISPOT conversion rate. Therefore, a negative ELISPOT test for M. tuberculosis infection should

  16. Evaluation of the results of Mycobacterium tuberculosis direct test (MTD and Mycobacterial culture in urine samples Avaliação de resultados do Teste Direto para Mycobacterium tuberculosis (MTD e da cultura para micobacterias em amostras de urina

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    Asli Gamze Sener

    2008-12-01

    Full Text Available Tuberculosis remains a public health problem in Turkey. Rapid detection of Mycobacterium tuberculosis plays a key role in control of infection. In this article, the Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test (MTD was evaluated for detection of M. tuberculosis in urine samples. The performance of the MTD was very good and appropriate for routine laboratory diagnosis.A tuberculose continua sendo um problema de saúde pública na Turquia. A detecção rápida de Mycobacterium tuberculosis tem um papel importante no controle da infecção. Nesse artigo, avaliou-se o Gen-Probe Amplified Mycobacterium Tuberculosis Test (MTD para detecção de M. tuberculosis em amostras de urina. O desempenho do MTD foi muito bom e adequado para diagnóstico laboratorial de rotina.

  17. Networked T cell death following macrophage infection by Mycobacterium tuberculosis.

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    Stephen H-F Macdonald

    Full Text Available BACKGROUND: Depletion of T cells following infection by Mycobacterium tuberculosis (Mtb impairs disease resolution, and interferes with clinical test performance that relies on cell-mediated immunity. A number of mechanisms contribute to this T cell suppression, such as activation-induced death and trafficking of T cells out of the peripheral circulation and into the diseased lungs. The extent to which Mtb infection of human macrophages affects T cell viability however, is not well characterised. METHODOLOGY/PRINCIPAL FINDINGS: We found that lymphopenia (<1.5 × 10(9 cells/l was prevalent among culture-positive tuberculosis patients, and lymphocyte counts significantly improved post-therapy. We previously reported that Mtb-infected human macrophages resulted in death of infected and uninfected bystander macrophages. In the current study, we sought to examine the influence of infected human alveolar macrophages on T cells. We infected primary human alveolar macrophages (the primary host cell for Mtb or PMA-differentiated THP-1 cells with Mtb H37Ra, then prepared cell-free supernatants. The supernatants of Mtb-infected macrophages caused dose-dependent, caspase-dependent, T cell apoptosis. This toxic effect of infected macrophage secreted factors did not require TNF-α or Fas. The supernatant cytotoxic signal(s were heat-labile and greater than 50 kDa in molecular size. Although ESAT-6 was toxic to T cells, other Mtb-secreted factors tested did not influence T cell viability; nor did macrophage-free Mtb bacilli or broth from Mtb cultures. Furthermore, supernatants from Mycobacterium bovis Bacille de Calmette et Guerin (BCG- infected macrophages also elicited T cell death suggesting that ESAT-6 itself, although cytotoxic, was not the principal mediator of T cell death in our system. CONCLUSIONS: Mtb-Infected macrophages secrete heat-labile factors that are toxic to T cells, and may contribute to the immunosuppression seen in tuberculosis as well as

  18. Portrait of a pathogen: the Mycobacterium tuberculosis proteome in vivo.

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    Nicole A Kruh

    Full Text Available BACKGROUND: Mycobacterium tuberculosis (Mtb, the causative agent of tuberculosis (TB, is a facultative intracellular pathogen that can persist within the host. The bacteria are thought to be in a state of reduced replication and metabolism as part of the chronic lung infection. Many in vitro studies have dissected the hypothesized environment within the infected lung, defining the bacterial response to pH, starvation and hypoxia. While these experiments have afforded great insight, the picture remains incomplete. The only way to study the combined effects of these environmental factors and the mycobacterial response is to study the bacterial response in vivo. METHODOLOGY/PRINCIPAL FINDINGS: We used the guinea pig model of tuberculosis to examine the bacterial proteome during the early and chronic stages of disease. Lungs were harvested thirty and ninety days after aerosol challenge with Mtb, and analyzed by liquid chromatography-mass spectrometry. To date, in vivo proteomics of the tubercle bacillus has not been described and this work has generated the first large-scale shotgun proteomic data set, comprising over 500 unique protein identifications. Cell wall and cell wall processes, and intermediary metabolism and respiration were the two major functional classes of proteins represented in the infected lung. These classes of proteins displayed the greatest heterogeneity indicating important biological processes for establishment of a productive bacterial infection and its persistence. Proteins necessary for adaptation throughout infection, such as nitrate/nitrite reduction were found at both time points. The PE-PPE protein class, while not well characterized, represented the third most abundant category and showed the most consistent expression during the infection. CONCLUSIONS/SIGNIFICANCE: Cumulatively, the results of this work may provide the basis for rational drug design - identifying numerous Mtb proteins, from essential kinases to

  19. Assessment of trends of ofloxacin resistance in Mycobacterium tuberculosis

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    J S Verma

    2011-01-01

    Full Text Available Purpose: Ofloxacin (OFX is one of the potent fluoroquinolone (FQ recommended to treat MDR-TB. Over a decade, the preexposure of this drug for the treatment of other bacterial infections has resulted in acquisition of FQ resistance among Mycobacterium tuberculosis strains. Considering this possibility, a study was undertaken in a tertiary care center in the capital city (India to assess the drug resistance trends of OFX among susceptible and multidrug resistant (MDR strains of M. tuberculosis. Materials and Methods: A total of 102 M. tuberculosis isolates (47 susceptible to first-line drugs and 55 MDR isolates were screened for susceptibility testing of OFX with a critical concentration of 2 μg/ml by Lowenstein Jensen (LJ proportion method. Results: The results showed 40 (85.1% isolates among 47 susceptible isolates and 34 (61.8% isolates among 55 MDR isolates, were found to be susceptible to OFX. Fisher′s exact test showed significant P-value (0.0136 demonstrating 1.377 fold (95% confidence interval increased risk to become resistant to OFX than susceptible isolates. These finding shows decreased OFX susceptibility is not only limited to MDR isolates but also increasingly seen in susceptible strains as a result of drug abuse. Conclusions: Our finding were not alarming, but highlights the general risk of acquiring resistance to OFX, jeopardizing the potential for these drugs to be used as second-line anti-TB agents in the management of drug-resistant TB and creating incurable TB strains .

  20. Mycobacterium tuberculosis TlyA Protein Negatively Regulates T Helper (Th) 1 and Th17 Differentiation and Promotes Tuberculosis Pathogenesis.

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    Rahman, Md Aejazur; Sobia, Parveen; Dwivedi, Ved Prakash; Bhawsar, Aakansha; Singh, Dhiraj Kumar; Sharma, Pawan; Moodley, Prashini; Van Kaer, Luc; Bishai, William R; Das, Gobardhan

    2015-06-05

    Mycobacterium tuberculosis, the causative agent of tuberculosis, is an ancient pathogen and a major cause of death worldwide. Although various virulence factors of M. tuberculosis have been identified, its pathogenesis remains incompletely understood. TlyA is a virulence factor in several bacterial infections and is evolutionarily conserved in many Gram-positive bacteria, but its function in M. tuberculosis pathogenesis has not been elucidated. Here, we report that TlyA significantly contributes to the pathogenesis of M. tuberculosis. We show that a TlyA mutant M. tuberculosis strain induces increased IL-12 and reduced IL-1β and IL-10 cytokine responses, which sharply contrasts with the immune responses induced by wild type M. tuberculosis. Furthermore, compared with wild type M. tuberculosis, TlyA-deficient M. tuberculosis bacteria are more susceptible to autophagy in macrophages. Consequently, animals infected with the TlyA mutant M. tuberculosis organisms exhibited increased host-protective immune responses, reduced bacillary load, and increased survival compared with animals infected with wild type M. tuberculosis. Thus, M. tuberculosis employs TlyA as a host evasion factor, thereby contributing to its virulence.

  1. Mycobacterium tuberculosis TlyA Protein Negatively Regulates T Helper (Th) 1 and Th17 Differentiation and Promotes Tuberculosis Pathogenesis*

    Science.gov (United States)

    Rahman, Md. Aejazur; Sobia, Parveen; Dwivedi, Ved Prakash; Bhawsar, Aakansha; Singh, Dhiraj Kumar; Sharma, Pawan; Moodley, Prashini; Van Kaer, Luc; Bishai, William R; Das, Gobardhan

    2015-01-01

    Mycobacterium tuberculosis, the causative agent of tuberculosis, is an ancient pathogen and a major cause of death worldwide. Although various virulence factors of M. tuberculosis have been identified, its pathogenesis remains incompletely understood. TlyA is a virulence factor in several bacterial infections and is evolutionarily conserved in many Gram-positive bacteria, but its function in M. tuberculosis pathogenesis has not been elucidated. Here, we report that TlyA significantly contributes to the pathogenesis of M. tuberculosis. We show that a TlyA mutant M. tuberculosis strain induces increased IL-12 and reduced IL-1β and IL-10 cytokine responses, which sharply contrasts with the immune responses induced by wild type M. tuberculosis. Furthermore, compared with wild type M. tuberculosis, TlyA-deficient M. tuberculosis bacteria are more susceptible to autophagy in macrophages. Consequently, animals infected with the TlyA mutant M. tuberculosis organisms exhibited increased host-protective immune responses, reduced bacillary load, and increased survival compared with animals infected with wild type M. tuberculosis. Thus, M. tuberculosis employs TlyA as a host evasion factor, thereby contributing to its virulence. PMID:25847237

  2. Differences in the population of genetics of Mycobacterium tuberculosis between urban migrants and local residents in Beijing, China

    Institute of Scientific and Technical Information of China (English)

    DAI Guang-ming; ZHANG Zhi-guo; DING Peng-ju; ZHANG Qian; WANG Li; WANG Li-xia; Dick van Soolingen

    2013-01-01

    Background Currently,migration has become one of the risk factors of high burden of tuberculosis in China.This study was to explore the influence of mass migration on the dynamics of Mycobacterium (M.) tuberculosis in Beijing,the capital and an urban area of China.Methods Three hundred and thirty-six M.tuberculosis strains from the Changping district,where the problem of urban migrants was more pronounced than in other Beijing regions,were genotyped by Spoligotyping,large sequence polymorphisms (LSPs 105 and 181),and variable number tandem repeat (VNTR) typing.Based on the genotype data,the phylogeny of the isolates was studied.Results In Changping district,the proportion of Beijing lineage M.tuberculosis isolates amounted to 89.0% (299/336),among which 86.6 % (252) belonged to the modern lineage.The frequency of modern Beijing lineage strains is so high (around 75% (252/336)) that associated risk factors affecting the tuberculosis epidemic cannot be determined.The time to the most recent common ancestor (TMRCA) of the Beijing lineage strains was estimated to be 5073 (95% CI:4000-6200) years.There was no significant difference in the genetic variation of Beijing isolates from urban migrants and local residents.Conclusions The clone of modern Beijing lineage M.tuberculosis,which is dominant in the Beijing area,most likely started to expand with the five thousand-year-old Chinese civilization.In the future,with the urbanization in the whole of China,modern Beijing lineage M.tuberculosis may gain the larger geographical spread.

  3. Detección de la expresión génica in vivo de Mycobacterium tuberculosis durante la tuberculosis pulmonar activa Mycobacterium tuberculosis in vivo-expressed genes detection during active pulmonary tuberculosis

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    Alejandra Otazo M

    2012-12-01

    Full Text Available El estudio de la expresión génica de Mycobacterium tuberculosis ha involucrado la experimentación "in vitro ", "ex vivo " e "in vivo " (modelos animales, pero aún sin el éxito esperado. Proponemos que revelar los factores clave de la tuberculosis humana requiere investigar la expresión génica de M. tuberculosis dentro del ser humano ("in vivo ". Para ello, aislamos el mRNA total de M. tuberculosis, desde muestras clínicas respiratorias de pacientes con diagnóstico de tuberculosis pulmonar; posteriormente, sintetizamos el dscDNA y lo analizamos mediante RT-PCR cualitativo. Detectamos la expresión de la secuencia de inserción IS6110 y de los genes "housekeeping " 16SrRNA y sigA en M. tuberculosis creciendo in vivo (tuberculosis pulmonar así como cultivado in vitro. La expresión de los genes mprA y mprB, que codifican el sistema de transducción de señales MprAB, sólo se detectó en M. tuberculosis crecido in vitro. Con nuestros resultados damos el primer paso hacia la implementación de un método no invasivo para el estudio del transcriptoma de M. tuberculosis, dentro de su único hospedero natural, con el fin de analizar la regulación "in vivo" de los determinantes genéticos requeridos para su virulencia y patogénesis.Mycobacterium tuberculosis gene expression studies have involved "in vitro", "ex vivo" and "in vivo" experiments (animal models, but without the expected success. We propose that key features of human tuberculosis could be discovered by studying the M. tuberculosis gene expression within the human host. Therefore, we isolated totalM. tuberculosis mRNA from human clinical respiratory specimens of patients diagnosed with pulmonary tuberculosis; after this, we synthesized the dscDNA and tested it by qualitative RT-PCR assays. We detected the expression of IS6110 insertion sequence and of the "housekeeping" genes 16SrRNA andsigA in M. tuberculosis grown in vivo (pulmonary tuberculosis as well as grown in vitro M

  4. Protein export systems of Mycobacterium tuberculosis: novel targets for drug development?

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    Feltcher, Meghan E; Sullivan, Jonathan Tabb; Braunstein, Miriam

    2010-10-01

    Protein export is essential in all bacteria and many bacterial pathogens depend on specialized protein export systems for virulence. In Mycobacterium tuberculosis, the etiological agent of the disease tuberculosis, the conserved general secretion (Sec) and twin-arginine translocation (Tat) pathways perform the bulk of protein export and are both essential. M. tuberculosis also has specialized export pathways that transport specific subsets of proteins. One such pathway is the accessory SecA2 system, which is important for M. tuberculosis virulence. There are also specialized ESX export systems that function in virulence (ESX-1) or essential physiologic processes (ESX-3). The increasing prevalence of drug-resistant M. tuberculosis strains makes the development of novel drugs for tuberculosis an urgent priority. In this article, we discuss our current understanding of the protein export systems of M. tuberculosis and consider the potential of these pathways to be novel targets for tuberculosis drugs.

  5. Genetic diversity and transmission characteristics of Beijing family strains of Mycobacterium tuberculosis in Peru.

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    Tomotada Iwamoto

    Full Text Available Beijing family strains of Mycobacterium tuberculosis have attracted worldwide attention because of their wide geographical distribution and global emergence. Peru, which has a historical relationship with East Asia, is considered to be a hotspot for Beijing family strains in South America. We aimed to unveil the genetic diversity and transmission characteristics of the Beijing strains in Peru. A total of 200 Beijing family strains were identified from 2140 M. tuberculosis isolates obtained in Lima, Peru, between December 2008 and January 2010. Of them, 198 strains were classified into sublineages, on the basis of 10 sets of single nucleotide polymorphisms (SNPs. They were also subjected to variable number tandem-repeat (VNTR typing using an international standard set of 15 loci (15-MIRU-VNTR plus 9 additional loci optimized for Beijing strains. An additional 70 Beijing family strains, isolated between 1999 and 2006 in Lima, were also analyzed in order to make a longitudinal comparison. The Beijing family was the third largest spoligotyping clade in Peru. Its population structure, by SNP typing, was characterized by a high frequency of Sequence Type 10 (ST10, which belongs to a modern subfamily of Beijing strains (178/198, 89.9%. Twelve strains belonged to the ancient subfamily (ST3 [n=3], ST25 [n=1], ST19 [n=8]. Overall, the polymorphic information content for each of the 24 loci values was low. The 24 loci VNTR showed a high clustering rate (80.3% and a high recent transmission index (RTI(n-1=0.707. These strongly suggest the active and on-going transmission of Beijing family strains in the survey area. Notably, 1 VNTR genotype was found to account for 43.9% of the strains. Comparisons with data from East Asia suggested the genotype emerged as a uniquely endemic clone in Peru. A longitudinal comparison revealed the genotype was present in Lima by 1999.

  6. A Defined Tuberculosis Vaccine Candidate Boosts BCG and Protects Against Multidrug Resistant Mycobacterium tuberculosis

    Science.gov (United States)

    Bertholet, Sylvie; Ireton, Gregory C.; Ordway, Diane J.; Windish, Hillarie Plessner; Pine, Samuel O.; Kahn, Maria; Phan, Tony; Orme, Ian M.; Vedvick, Thomas S.; Baldwin, Susan L.; Coler, Rhea N.; Reed, Steven G.

    2011-01-01

    Despite the widespread use of Mycobacterium bovis bacillus Calmette-Guerin (BCG) childhood vaccine, tuberculosis (TB) remains a serious global health problem. A successful vaccine against TB that replaces or boosts BCG will include antigens that induce or recall appropriate T cell responses. Four Mycobacterium tuberculosis (Mtb) antigens, including members of the virulence factor families PE/PPE and EsX, or antigens associated with latency were produced as a single recombinant fusion protein. When administered with the adjuvant GLA-SE, a stable oil-in-water nanoemulsion, the fusion protein ID93 was immunogenic in mice, guinea pigs, and cynomolgus monkeys. In mice, ID93/GLA-SE combination induced polyfunctional CD4 TH1-cell responses characterized by antigen-specific IFN-gamma, tumor necrosis factor and interleukin-2, as well as a reduction in the number of bacteria in the lungs of animals subsequently infected with virulent or multidrug resistant Mtb strains. Furthermore, boosting BCG-vaccinated guinea pigs with ID93/GLA-SE resulted in reduced pathology and fewer bacilli, and prevented the death of animals challenged with virulent Mtb. Finally, ID93 elicited polyfunctional effector CD4 and CD8 T-cell responses in BCG-vaccinated or Mtb-exposed human peripheral blood mononuclear cells. This study establishes that the protein subunit vaccine ID93/GLA-SE protects against TB and MDR-TB in animals, and is a candidate for boosting the protective efficacy of the childhood BCG vaccine. PMID:20944089

  7. Drug susceptibility patterns of the Mycobacterium tuberculosis isolated from previously treated and new cases of pulmonary tuberculosis at German-Nepal tuberculosis project laboratory, Kathmandu, Nepal

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    Gobinda Thapa

    2016-08-01

    Full Text Available Abstract Background Multidrug resistant tuberculosis (MDR-TB is a serious public health problem in Nepal. It is a major obstacle for the control of the tuberculosis. The main objectives of this study were to determine the prevalence of the multidrug resistant pulmonary tuberculosis and to evaluate the drug susceptibility patterns of Mycobacterium tuberculosis isolated from previously treated and newly diagnosed cases of pulmonary tuberculosis. Methods A cross-sectional study was conducted from March 2013 to August 2013 at German-Nepal tuberculosis project (GENETUP laboratory, Kathmandu, Nepal. For this the sputum samples from total of 153 (49 new and 104 previously treated suspected pulmonary tuberculosis patients were used. The diagnosis of the tuberculosis was performed by using fluorescent microscopy and culture, while the drug susceptibility testing of Mycobacterium tuberculosis was performed by proportion method. Lowenstein-Jensen (L-J medium was used for the culture of Mycobacterium tuberculosis and the colonies grown were identified on the basis of the colony morphology, pigment production and biochemical characteristics. Results The prevalence of MDR-TB among all the cases of culture positive pulmonary tuberculosis was 15.6 %. The rate of MDR-TB among previously treated culture positive tuberculosis patients was 19.4 % and that among newly diagnosed culture positive pulmonary tuberculosis cases was 7.1 %. The highest rate of resistance of Mycobacterium tuberculosis, was toward streptomycin (24.4 % followed by isoniazid (23 %, rifampicin (17.8 % and ethambutol (15.6 %. Among the total of MDR-TB cases among previously treated patients, highest percentage of the cases were relapse (61.1 % followed by chronic (16.7 %. Conclusions The high prevalence of DR/MDR-TB in our study reflects poor implementation of tuberculosis control program. On the basis of the drug susceptibility patterns of M. tuberculosis we found in our study, we

  8. Transcriptional and proteomic analyses of two-component response regulators in multidrug-resistant Mycobacterium tuberculosis.

    Science.gov (United States)

    Zhou, Lei; Yang, Liu; Zeng, Xianfei; Danzheng, Jiacuo; Zheng, Qing; Liu, Jiayun; Liu, Feng; Xin, Yijuan; Cheng, Xiaodong; Su, Mingquan; Ma, Yueyun; Hao, Xiaoke

    2015-07-01

    Two-component systems (TCSs) have been reported to exhibit a sensing and responding role under drug stress that induces drug resistance in several bacterial species. However, the relationship between TCSs and multidrug resistance in Mycobacterium tuberculosis has not been comprehensively analysed to date. In this study, 90 M. tuberculosis clinical isolates were analysed using 15-loci mycobacterial interspersed repetitive unit (MIRU)-variable number tandem repeat (VNTR) typing and repetitive extragenic palindromic (rep)-PCR-based DNA fingerprinting. The results showed that all of the isolates were of the Beijing lineage, and strains with a drug-susceptible phenotype had not diverged into similar genotype clusters. Expression analysis of 13 response regulators of TCSs using real-time PCR and tandem mass spectrometry (MS/MS) proteomic analysis demonstrated that four response regulator genes (devR, mtrA, regX3 and Rv3143) were significantly upregulated in multidrug-resistant (MDR) strains compared with the laboratory strain H37Rv as well as drug-susceptible and isoniazid-monoresistant strains (PMycobacterium bovis BCG did not alter its sensitivity to the four antitubercular drugs. This suggests that upregulation of devR, which is common in MDR-TB strains, might be induced by drug stress and hypoxic adaptation following the acquisition of multidrug resistance.

  9. Clustered regularly interspaced short palindromic repeats (CRISPRs) analysis of members of the Mycobacterium tuberculosis complex.

    Science.gov (United States)

    Botelho, Ana; Canto, Ana; Leão, Célia; Cunha, Mónica V

    2015-01-01

    Typical CRISPR (clustered, regularly interspaced, short palindromic repeat) regions are constituted by short direct repeats (DRs), interspersed with similarly sized non-repetitive spacers, derived from transmissible genetic elements, acquired when the cell is challenged with foreign DNA. The analysis of the structure, in number and nature, of CRISPR spacers is a valuable tool for molecular typing since these loci are polymorphic among strains, originating characteristic signatures. The existence of CRISPR structures in the genome of the members of Mycobacterium tuberculosis complex (MTBC) enabled the development of a genotyping method, based on the analysis of the presence or absence of 43 oligonucleotide spacers separated by conserved DRs. This method, called spoligotyping, consists on PCR amplification of the DR chromosomal region and recognition after hybridization of the spacers that are present. The workflow beneath this methodology implies that the PCR products are brought onto a membrane containing synthetic oligonucleotides that have complementary sequences to the spacer sequences. Lack of hybridization of the PCR products to a specific oligonucleotide sequence indicates absence of the correspondent spacer sequence in the examined strain. Spoligotyping gained great notoriety as a robust identification and typing tool for members of MTBC, enabling multiple epidemiological studies on human and animal tuberculosis.

  10. Spoligotyping of clinical Mycobacterium tuberculosis isolates from the state of Minas Gerais, Brazil

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    Silvana Spíndola de Miranda

    2011-05-01

    Full Text Available We performed spoligotyping on 114 strains of the Mycobacterium tuberculosis (Mtb complex that had been isolated from patients in Minas Gerais Health Units during 2004. A total of 82/114 (72% clinical isolates were clustered and 32/114 (28% were unique. Seven shared types containing nine strains were newly created. A total of nine patterns corresponded to unreported orphan strains, as evaluated against all of the strains recorded in the SITVIT2 proprietary database in the Institut Pasteur de la Guadeloupe. The major clades were composed of isolates that belong to the following genotypes: Latin-America and Mediterranean (63/114, 55.3% (the ill-defined T superfamily (12/114, 10.5%, Haarlem (8/114, 7%, X clade (6/114, 5.3%, S clade (3/114, 2.6% and the East-African Indian and Manu types, each with 1/114 (0.9% isolates. A considerable number of strains (n = 20, 17.5% showed patterns that did not fall within any of the previously described major clades. We conclude the bulk of tuberculosis (TB (92/114, 80.7% in our location is recent evolutionary strains that belong to the principal genetic groups 2/3. Further studies on epidemiology of TB are required to understand Mtb biodiversity and TB transmission in this region.

  11. First insight into the molecular epidemiology of Mycobacterium tuberculosis in Santa Catarina, southern Brazil.

    Science.gov (United States)

    Nogueira, Christiane Lourenço; Prim, Rodrigo Ivan; Senna, Simone Gonçalves; Rovaris, Darcita Büerger; Maurici, Rosemeri; Rossetti, Maria Lúcia; Couvin, David; Rastogi, Nalin; Bazzo, Maria Luiza

    2016-03-01

    Molecular epidemiology of Mycobacterium tuberculosis is useful for understanding disease transmission dynamics, and to establish strategic measures for TB control and prevention. The aim of this study was to analyze clinical, epidemiological and molecular characteristics of MTBC clinical isolates from Santa Catarina state, southern Brazil. During one-year period, 406 clinical isolates of MTBC were collected from Central Laboratory of Public Health and typed by spoligotyping. Demographic and clinical data were collected from the Brazilian National Mandatory Disease Reporting System. The majority of cases occurred in highest population densities regions and about 50% had some condition associated with TB. Among all isolates, 5.7% were MDR, which showed association with drug addiction. LAM was the most predominant lineage with 47.5%, followed by the T superfamily with 25.9% and Haarlem with 12.3%. The MST showed two major groups: the first was formed mainly by the LAM lineage and the second was mainly formed by the T and Haarlem lineages. Others lineages were distributed in peripheral positions. This study provides the first insight into the population structure of M. tuberculosis in SC State. Spoligotyping and other genotyping analyses are important to establish strategic measures for TB control and prevention.

  12. Pyrosequencing for rapid detection of Mycobacterium tuberculosis second-line drugs and ethambutol resistance.

    Science.gov (United States)

    Lacoma, Alicia; Molina-Moya, Barbara; Prat, Cristina; Pimkina, Edita; Diaz, Jessica; Dudnyk, Andriy; García-Sierra, Nerea; Haba, Lucía; Maldonado, Jose; Samper, Sofia; Ruiz-Manzano, Juan; Ausina, Vicente; Dominguez, Jose

    2015-11-01

    The aim of this work was to study the diagnostic accuracy of pyrosequencing to detect resistance to fluoroquinolones, kanamycin, amikacin, capreomycin, and ethambutol (EMB) in Mycobacterium tuberculosis clinical strains. One hundred four clinical isolates previously characterized by BACTEC 460TB/MGIT 960 were included. Specific mutations were targeted in gyrA, rrs, eis promoter, and embB. When there was a discordant result between BACTEC and pyrosequencing, Genotype MTBDRsl (Hain Lifescience, Nehren, Germany) was performed. Sensitivity and specificity of pyrosequencing were 70.6% and 100%, respectively, for fluoroquinolones; 93.3% and 81.7%, respectively, for kanamycin; 94.1% and 95.9%, respectively, for amikacin; 90.0% and 100%, respectively, for capreomycin; and 64.8% and 87.8%, respectively, for EMB. This study shows that pyrosequencing may be a useful tool for making early decisions regarding second-line drugs and EMB resistance. However, for a correct management of patients with suspected extensively drug-resistant tuberculosis, susceptibility results obtained by molecular methods should be confirmed by a phenotypic method.

  13. pncA mutations in clinical Mycobacterium tuberculosis isolates from Korea

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    Kim Cheol Min

    2001-06-01

    Full Text Available Abstract Background Pyrazinamide (PZA is among the first-line drugs for the treatment of tuberculosis. In vitro, it kills semidormant mycobacteria only at low pH. The purpose of this study was to compare PZA resistance with pyrazinamidase (PZase activity and the genotype to better understand the molecular basis of PZA resistance and to expand the profile of pncA mutations worldwide. Results Of the 28 tested strains of Mycobacterium tuberculosis, 6 were susceptible to PZA and positive for PZase activity and had no pncA mutations. Twenty-one strains were resistant to PZA and negative for PZase activity and had mutations in the pncA gene, including 15 point mutations, 5 insertions, and 2 deletions. One strain had no mutation in the pncA gene, even though it was resistant to PZA and negative for PZase activity. Three isolates had adenine to guanine point mutations in the -11 upstream region, making this the most common type of pncA mutations in this study, with at least two different RFLP patterns. Conclusion These data help in the understanding of the molecular basis of PZA resistance. An adenine to guanine point mutation in the -11 upstream region was the most common type of pncA mutation in our isolates. The results of pncA mutation analyses should be carefully interpreted for epidemiologic purposes.

  14. Detection of Mycobacterium tuberculosis and Mycobacterium avium Complexes by Real-Time PCR in Bovine Milk from Brazilian Dairy Farms.

    Science.gov (United States)

    Bezerra, André Vinícius Andrade; Dos Reis, Emily Marques; Rodrigues, Rogério Oliveira; Cenci, Alexander; Cerva, Cristine; Mayer, Fabiana Quoos

    2015-05-01

    Foodborne diseases are a public health problem worldwide. The consumption of contaminated raw milk has been recognized as a major cause of transmission of bovine tuberculosis to humans. Other mycobacteria that may be present in raw milk and may cause diseases are those belonging to the Mycobacterium avium complex. In this study, molecular biology tools were applied to investigate raw milk contamination with Mycobacterium spp. in family dairy farms from Rio Grande do Sul, southern Brazil. Furthermore, different variables related to the source of the milk, herd characteristics, and management were evaluated for their effect on milk contamination. Five hundred and two samples were analyzed, of which 354 were from the Northwest region (102 farms with samples from 93 bulk tanks and 261 animals) and 148 from the South region of the state (22 farms with samples from 23 bulk tanks and 125 animals). Among them, 10 (1.99%) and 7 (1.39%) were positive for Mycobacterium tuberculosis (9 confirmed as Mycobacterium bovis) and M. avium complexes, respectively. There was no difference in the frequencies of positive samples between the regions or the sample sources. Of the positive samples, 4 were collected from a bulk tank (1 positive for M. avium and 3 for M. tuberculosis). Moreover, 1 sample was positive concomitantly for M. tuberculosis and M. avium complexes. On risk analysis, no variable was associated with raw milk contamination by M. tuberculosis complex species. However, washing the udders of all animals and drying them with paper towels were weakly classified as risk factors for M. avium contamination. Positive samples were obtained from both animals and bulk tanks, which emphasizes the importance of tuberculosis control programs and provides evidence that milk monitoring can be used as a control practice. Moreover, the findings of this study reinforce the need for awareness of the problems of raw milk consumption among the general population.

  15. Genomic epidemiology of Lineage 4 Mycobacterium tuberculosis subpopulations in New York City and New Jersey, 1999-2009.

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    Brown, Tyler S; Narechania, Apurva; Walker, John R; Planet, Paul J; Bifani, Pablo J; Kolokotronis, Sergios-Orestis; Kreiswirth, Barry N; Mathema, Barun

    2016-11-21

    Whole genome sequencing (WGS) has rapidly become an important research tool in tuberculosis epidemiology and is likely to replace many existing methods in public health microbiology in the near future. WGS-based methods may be particularly useful in areas with less diverse Mycobacterium tuberculosis populations, such as New York City, where conventional genotyping is often uninformative and field epidemiology often difficult. This study applies four candidate strategies for WGS-based identification of emerging M. tuberculosis subpopulations, employing both phylogenomic and population genetics methods. M. tuberculosis subpopulations in New York City and New Jersey can be distinguished via phylogenomic reconstruction, evidence of demographic expansion and subpopulation-specific signatures of selection, and by determination of subgroup-defining nucleotide substitutions. These methods identified known historical outbreak clusters and previously unidentified subpopulations within relatively monomorphic M. tuberculosis endemic clone groups. Neutrality statistics based on the site frequency spectrum were less useful for identifying M. tuberculosis subpopulations, likely due to the low levels of informative genetic variation in recently diverged isolate groups. In addition, we observed that isolates from New York City endemic clone groups have acquired multiple non-synonymous SNPs in virulence- and growth-associated pathways, and relatively few mutations in drug resistance-associated genes, suggesting that overall pathoadaptive fitness, rather than the acquisition of drug resistance mutations, has played a central role in the evolutionary history and epidemiology of M. tuberculosis subpopulations in New York City. Our results demonstrate that some but not all WGS-based methods are useful for detection of emerging M. tuberculosis clone groups, and support the use of phylogenomic reconstruction in routine tuberculosis laboratory surveillance, particularly in areas with

  16. Dominant modern sublineages and a new modern sublineage of Mycobacterium tuberculosis Beijing family clinical isolates in Heilongjiang Province, China.

    Science.gov (United States)

    Li, Di; Dong, Cai-Bo; Cui, Jia-Yi; Nakajima, Chie; Zhang, Chun-Lei; Pan, Xin-Ling; Sun, Gao-Xiang; Dai, En-Yu; Suzuki, Yasuhiko; Zhuang, Min; Ling, Hong

    2014-10-01

    Mycobacterium tuberculosis Beijing family includes a variety of sublineages. Knowledge of the distribution of a certain sublineage of the Beijing family may help to understand the mechanisms of its rapid spread and to establish an association between a certain genotype and the disease outcome. We have previously found that M. tuberculosis Beijing family clinical isolates represent approximately 90% of the clinical isolates from Heilongjiang Province, China. To clarify the distribution of M. tuberculosis Beijing family sublineages in Heilongjiang Province, China and to investigate the regularity rule for their evolution, we examined single nucleotide polymorphisms (SNPs) of 250 M. tuberculosis Beijing family clinical isolates using 10 SNP loci that have been identified as appropriate for defining Beijing sublineages. After determining the sequence type (ST) of each isolate, the sublineages of all M. tuberculosis Beijing family isolates were determined, and phylogenetic analysis was performed. We found that 9 out of the 10 SNP loci displayed polymorphisms, but locus 1548149 did not. In total, 92.8% of the isolates in Heilongjiang Province are modern sublineages. ST10 is the most prevalent sublineage (ST10 and ST22 accounted for 63.2% and 23.6% of all the Beijing family isolates, respectively). A new ST, accounting for 4% of the Beijing family isolates in this area, was found for the first time. Each new ST isolate showed a unique VNTR pattern, and none were clustered. The present findings suggest that controlling the spread of these modern sublineages is important in Heilongjiang Province and in China.

  17. Molecular Identification of Mycobacterium tuberculosis and Analysis of Its Resistance to Rifampin in Sputa From Tuberculosis Suspected Patients

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    M. Syaifudin

    2010-08-01

    Full Text Available An accurate identification of different species of Mycobacterium provides to allow appropriate treatment for Mycobacterium tuberculosis infection. Beside that, drug resistance of M. tuberculosis strains to rifampin is not clearly understood in contributing to the spread of tuberculosis in Indonesia. To assess the molecular mechanism of rifampin resistance, a number of clinical specimens of M. tuberculosis were analyzed their molecular nature of a part of the rpoB gene using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP methods. DNA’s extracted from sputum samples were amplified and 32P-labeled by PCR with the specific primers and the product was analyzed their mutation conferring resistance by MDE gel electrophoresis. Of the 70 specimens tested, 57 specimens were positive for M. tuberculosis organism only, three specimens contained a mixture of M. tuberculosis and non tuberculosis mycobacteria (NTM, and 10 specimens were negative approved by Duplex PCR. Of these sixty DNA positive samples (thus the sensitivity of PCR was 85.71%, 5 (8.3% of them suspected to contain mutations in rpoB which were associated with rifampin resistance. Even though the frequency of mutation was low, the results from our study clearly indicate that the molecular mechanism of rifampin resistance in M. tuberculosis isolates from Indonesia involves alterations in the rpoB gene. Molecular diagnosis by PCR which is fast and easy to perform is useful for early and rapid detection of TB in sputum specimen.

  18. MOLECULAR CHARACTERIZATION OF MYCOBACTERIUM TUBERCULOSIS STRAINS CIRCULATING IN THE URAL REGION, RUSSIA

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    T. V. Umpeleva

    2013-01-01

    Full Text Available Abstract. Overall 178 Mycobacterium tuberculosis isolates recovered in 2009–2011 from newly diagnosed epidemiologically unlinked to TB patients in the Ural region of Russia have been studied. The absolute concentration method was used for drug susceptibility testing. Mutations katG, inhA and rpoB associated with resistance to isoniazid and rifampicin were detected by microchip technology («TB-Biochip». The isolates were genotyped by real-time PCR for the detection of Beijing/non-Beijing genotypes and 15-locus MIRU-VNTR typing according to «MIRU-VNTRplus» (http://www.miru-vntrplus.org. More than half (55.1% of 178 isolates belonged to the Beijing family, 58.7% of them were multiple drug resistant (MDR mostly due to rpoBSer531→Leu and katGSer315→Thr1 substitutions. Fifty VNTR profiles were found in 98 Beijing isolates; 57 of them grouped into 9 clusters. The largest VNTR cluster included 23 (23.5% Beijing isolates and 21 of them were MDR. The 80 non-Beijing isolates showed 64 distinct VNTR patterns which belonged to 6 genetic families: LAM, Ural, Haarlem, etc. Among LAM and Ural isolates 30.4% and 28.6% were MDR, respectively. The 5 of 7 MDR LAM isolates had specific mutation profile:  rpoBAsp516→Val substitution and mutations katGSer315→Thr1 and inhA_T15. The MDR Ural isolates showed the heterogeneity of mutations in rpoB gene compared to other genotypes. Taken together, these findings suggest the emergence and spread of MDR-TB in the Ural region which is characterized by circulation of MDR strains of different genotypes with the Beijing family genotype to be predominant.

  19. Distribution of Insertion- and Deletion-Associated Genetic Polymorphisms among Four Mycobacterium tuberculosis Phospholipase C Genes and Associations with Extrathoracic Tuberculosis: a Population-Based Study

    OpenAIRE

    Kong, Y.; Cave, M D; Yang, D.; Zhang,L.; Marrs, C. F.; Foxman, B.; Bates, J H; Wilson, F.; Mukasa, L. N.; Yang, Z. H.

    2005-01-01

    The Mycobacterium tuberculosis genome contains four phospholipase C (PLC)-encoding genes, designated plcA, plcB, plcC, and plcD, respectively. Each of the four genes contributes to the overall PLC activity of M. tuberculosis. PLC is hypothesized to contribute to M. tuberculosis virulence. Infection of M. tuberculosis strains carrying a truncated plcD gene is associated with the occurrence of extrathoracic tuberculosis. However, whether the other three plc genes are also associated with extrat...

  20. Mycobacterium bovis infection of cattle and white-tailed deer: Translational research of relevance to human tuberculosis

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    Tuberculosis (TB) is a premier example of a disease complex with pathogens primarily affecting humans (i.e., Mycobacterium tuberculosis) or livestock and wildlife (i.e., Mycobacterium bovis) and with a long history of inclusive collaborations between physicians and veterinarians. Advances with the s...

  1. Surveillance of pyrazinamide susceptibility among multidrug-resistant Mycobacterium tuberculosis isolates from Siriraj Hospital, Thailand

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    Jonmalung Jirarut

    2010-08-01

    Full Text Available Abstract Background Susceptibility testing of pyrazinamide (PZA against Mycobacterium tuberculosis is difficult to perform because the acidity of culture medium that is required for drug activity also inhibits the growth of bacteria. In Thailand, very limited information has been generated on PZA resistance, particularly among multidrug-resistant tuberculosis (MDR-TB isolated from Thailand. Only two studies on PZA susceptibility among Thai M. tuberculosis strains have been reported; one used a pyrazinamidase assay, and the other used the BACTEC 460 TB for PZA susceptibility testing. In this study, we determined the percentage of strains possessing pyrazinamide resistance among pan-susceptible M. tuberculosis and MDR-TB isolates by using the pyrazinamidase assay, BACTEC MGIT 960 PZA method and pncA sequencing, and assessed the correlation in the data generated using these methods. The type and frequency of mutations in pncA were also determined. Results Overall, 150 M. tuberculosis isolates, consisting of 50 susceptible and 100 MDR-TB isolates, were tested for PZA susceptibility by BACTEC MGIT 960 PZA, the pyrazinamidase assay and pncA sequencing. The study indicated PZA resistance in 6% and 49% of susceptible and MDR-TB isolates, respectively. In comparison to the BACTEC MGIT 960 PZA, the PZase assay showed 65.4% sensitivity and 100% specificity, whereas pncA sequencing showed 75% sensitivity and 89.8% specificity. Twenty-four mutation types were found in this study, with the most frequent mutation (16% being His71Asp. Of these mutations, eight have not been previously described. The Ile31Ser and Ile31Thr mutations were found both in PZA susceptible and resistant isolates, suggesting that mutation of this codon might not play a role on PZA resistance. Conclusions Our findings suggest that phenotypic susceptibility testing is still essential for the detection of PZA resistance, especially for MDR-TB isolates. Some mutations were not associated

  2. IS6110限制性片段多态性分析标准方法的建立及其在结核分枝杆菌分子分型中的应用%Establishment and application of a standard IS6110-RFLP method in the study of molecular genotyping analysis on Mycobacterium tuberculosis

    Institute of Scientific and Technical Information of China (English)

    刘敬华; Kristin Kremer; Christine Pourcel; Arnout Mulder; 刘志广; 赵秀芹; 万康林

    2008-01-01

    Objective To develop a standardized IS6110-restriction fragment length polymorphism (RFLP) method, used for evaluating the capacity of genotyping. Methods IS6110-RFLP of 78 Mycobacterium (M.) tuberculosis strains were studied by bio-molecular techniques including DNA isolation, PCR, restriction endonuclease enzyme analysis, southern blotting, agarose gel electrophoresis,together with data analysis by software Gel-Pro analyzer 3.1 and BioNumerics (Version 5.0). Results IS6110-RFLP method was established and standardized successfully, including DNA isolation, PCR,restriction endonuclease enzyme analysis, southern blotting, agarose gel electrophoresis and usage of the analysis software with standard parameters. By this method, 78 M. tuberculosis isolates were classified into 75 genotypes which belonged to 11 different clusters. Of all the isolates, 66.7 % (52/78) belonged to a main cluster. Conclusion Standard IS6110-RFLP method was established successfully. This method had powerful capacity for genotyping and strain level identification and could be used for the surveillance on pathogens of M. tuberculosis in China.%目的 建立IS6110限制性片段多态性分析(IS6110-RFLP)标准方法 并评价该方法 的分型能力.方法 采用核酸提取、PCR、限制性内切酶分析、Southern杂交、琼脂糖凝胶电泳等技术,结合Gel-Pro analyzer 3.1和BioNumeries(Version 5.0)软件,对78株结核分枝杆菌插入序列IS6110-RFLP进行分析.结果 确定标准化的IS6110-RFLP技术,包括核酸提取、PCR、限制性内切酶分析、Southern杂交、琼脂糖凝胶电泳等实验步骤及标化参数的相关数据分析软件的使用;采用该技术,将78株结核分枝杆菌分为75个不同的基因型,分别归属于11个基因簇,其中有52株归属于同一个基因簇,占菌株总数的66.7%(52/78).结论 建立标准化的IS6110-RFLP技术方案,该方法 具有很强的基因分型和株水平鉴定能力,可用于结核病的病原学监测.

  3. SUSCEPTIBILITY OF RIFAMPICIN-ISONIAZID RESISTANT MYCOBACTERIUM TUBERCULOSIS ISOLATES AGAINST LEVOFLOXACIN

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    A. H. Kurniawan

    2016-01-01

    Full Text Available Background: Tuberculosis (TB is a high burden disease in Indonesia with multidrug-resistant (MDR TB incidence started to increase. Treatment success of MDR-TB globally was low in number than it was targeted which was especially caused by fluoroquinolone resistance. One of the fluoroquinolone is levofloxacin, an antibiotic that has been widely used irrationally as antimicrobial treatment. Therefore, this study investigated the sensitivity and MBC of MDR Mycobacterium tuberculosis isolates against Levofloxacin. Method: The susceptibility test for MDR-Mycobacterium tuberculosis on levofloxacin by standard method with levofloxacin were on concentrations 0,5 μg/ml, 1 μg/ml, and 2 μg/ml. Sample of 8 strains MDR-Mycobacterium tuberculosis were cultured with each concentrations on Middlebrook 7H9 for 1 week incubation. Next, each of the incubated concentration was subcultured on solid media Middlebrook 7H10 for 3 weeks incubation. Colonized agar plates after 3 weeks incubation were confirmed with acid-fast stain. Results: On MB 7H10 with levofloxacin concentration 2 μg/ml showed bactericidal effect 100% by no MDR Mycobacterium tuberculosis colony grew (0/8 while the MB 7H10 with levofloxacin concentration 1 μg/ml and 0,5 μg/ml showed the bactericidal effect 37,5% and 25% respectively. The colonized agar plate implied that the MDR Mycobacterium tuberculosis with levofloxacin concentration 1 μg/ml (5/8 and 0,5 μg/ml (6/8 grew well. Conclusion: Levofloxacin concentration 2 μg/ml was susceptible on MDR Mycobacterium tuberculosis. The concentration 2 μg/ml of levofloxacin could be considered as MBC.

  4. Mycobacterium tuberculosis pyrazinamide resistance determinants: a multicenter study.

    Science.gov (United States)

    Miotto, Paolo; Cabibbe, Andrea M; Feuerriegel, Silke; Casali, Nicola; Drobniewski, Francis; Rodionova, Yulia; Bakonyte, Daiva; Stakenas, Petras; Pimkina, Edita; Augustynowicz-Kopeć, Ewa; Degano, Massimo; Ambrosi, Alessandro; Hoffner, Sven; Mansjö, Mikael; Werngren, Jim; Rüsch-Gerdes, Sabine; Niemann, Stefan; Cirillo, Daniela M

    2014-10-21

    Pyrazinamide (PZA) is a prodrug that is converted to pyrazinoic acid by the enzyme pyrazinamidase, encoded by the pncA gene in Mycobacterium tuberculosis. Molecular identification of mutations in pncA offers the potential for rapid detection of pyrazinamide resistance (PZA(r)). However, the genetic variants are highly variable and scattered over the full length of pncA, complicating the development of a molecular test. We performed a large multicenter study assessing pncA sequence variations in 1,950 clinical isolates, including 1,142 multidrug-resistant (MDR) strains and 483 fully susceptible strains. The results of pncA sequencing were correlated with phenotype, enzymatic activity, and structural and phylogenetic data. We identified 280 genetic variants which were divided into four classes: (i) very high confidence resistance mutations that were found only in PZA(r) strains (85%), (ii) high-confidence resistance mutations found in more than 70% of PZA(r) strains, (iii) mutations with an unclear role found in less than 70% of PZA(r) strains, and (iv) mutations not associated with phenotypic resistance (10%). Any future molecular diagnostic assay should be able to target and identify at least the very high and high-confidence genetic variant markers of PZA(r); the diagnostic accuracy of such an assay would be in the range of 89.5 to 98.8%. Importance: Conventional phenotypic testing for pyrazinamide resistance in Mycobacterium tuberculosis is technically challenging and often unreliable. The development of a molecular assay for detecting pyrazinamide resistance would be a breakthrough, directly overcoming both the limitations of conventional testing and its related biosafety issues. Although the main mechanism of pyrazinamide resistance involves mutations inactivating the pncA enzyme, the highly diverse genetic variants scattered over the full length of the pncA gene and the lack of a reliable phenotypic gold standard hamper the development of molecular diagnostic

  5. Management of latent Mycobacterium tuberculosis infection: WHO guidelines for low tuberculosis burden countries

    Science.gov (United States)

    Matteelli, Alberto; Abubakar, Ibrahim; Aziz, Mohamed Abdel; Baddeley, Annabel; Barreira, Draurio; Den Boon, Saskia; Borroto Gutierrez, Susana Marta; Bruchfeld, Judith; Burhan, Erlina; Cavalcante, Solange; Cedillos, Rolando; Chaisson, Richard; Chee, Cynthia Bin-Eng; Chesire, Lucy; Corbett, Elizabeth; Dara, Masoud; Denholm, Justin; de Vries, Gerard; Falzon, Dennis; Ford, Nathan; Gale-Rowe, Margaret; Gilpin, Chris; Girardi, Enrico; Go, Un-Yeong; Govindasamy, Darshini; D. Grant, Alison; Grzemska, Malgorzata; Harris, Ross; Horsburgh Jr, C. Robert; Ismayilov, Asker; Jaramillo, Ernesto; Kik, Sandra; Kranzer, Katharina; Lienhardt, Christian; LoBue, Philip; Lönnroth, Knut; Marks, Guy; Menzies, Dick; Migliori, Giovanni Battista; Mosca, Davide; Mukadi, Ya Diul; Mwinga, Alwyn; Nelson, Lisa; Nishikiori, Nobuyuki; Oordt-Speets, Anouk; Rangaka, Molebogeng Xheedha; Reis, Andreas; Rotz, Lisa; Sandgren, Andreas; Sañé Schepisi, Monica; Schünemann, Holger J.; Sharma, Surender Kumar; Sotgiu, Giovanni; Stagg, Helen R.; Sterling, Timothy R.; Tayeb, Tamara; Uplekar, Mukund; van der Werf, Marieke J.; Vandevelde, Wim; van Kessel, Femke; van't Hoog, Anna; Varma, Jay K.; Vezhnina, Natalia; Voniatis, Constantia; Vonk Noordegraaf-Schouten, Marije; Weil, Diana; Weyer, Karin; Wilkinson, Robert John; Yoshiyama, Takashi; Zellweger, Jean Pierre; Raviglione, Mario

    2015-01-01

    Latent tuberculosis infection (LTBI) is characterised by the presence of immune responses to previously acquired Mycobacterium tuberculosis infection without clinical evidence of active tuberculosis (TB). Here we report evidence-based guidelines from the World Health Organization for a public health approach to the management of LTBI in high risk individuals in countries with high or middle upper income and TB incidence of <100 per 100 000 per year. The guidelines strongly recommend systematic testing and treatment of LTBI in people living with HIV, adult and child contacts of pulmonary TB cases, patients initiating anti-tumour necrosis factor treatment, patients receiving dialysis, patients preparing for organ or haematological transplantation, and patients with silicosis. In prisoners, healthcare workers, immigrants from high TB burden countries, homeless persons and illicit drug users, systematic testing and treatment of LTBI is conditionally recommended, according to TB epidemiology and resource availability. Either commercial interferon-gamma release assays or Mantoux tuberculin skin testing could be used to test for LTBI. Chest radiography should be performed before LTBI treatment to rule out active TB disease. Recommended treatment regimens for LTBI include: 6 or 9 month isoniazid; 12 week rifapentine plus isoniazid; 3–4 month isoniazid plus rifampicin; or 3–4 month rifampicin alone. PMID:26405286

  6. Management of latent Mycobacterium tuberculosis infection: WHO guidelines for low tuberculosis burden countries.

    Science.gov (United States)

    Getahun, Haileyesus; Matteelli, Alberto; Abubakar, Ibrahim; Aziz, Mohamed Abdel; Baddeley, Annabel; Barreira, Draurio; Den Boon, Saskia; Borroto Gutierrez, Susana Marta; Bruchfeld, Judith; Burhan, Erlina; Cavalcante, Solange; Cedillos, Rolando; Chaisson, Richard; Chee, Cynthia Bin-Eng; Chesire, Lucy; Corbett, Elizabeth; Dara, Masoud; Denholm, Justin; de Vries, Gerard; Falzon, Dennis; Ford, Nathan; Gale-Rowe, Margaret; Gilpin, Chris; Girardi, Enrico; Go, Un-Yeong; Govindasamy, Darshini; D Grant, Alison; Grzemska, Malgorzata; Harris, Ross; Horsburgh, C Robert; Ismayilov, Asker; Jaramillo, Ernesto; Kik, Sandra; Kranzer, Katharina; Lienhardt, Christian; LoBue, Philip; Lönnroth, Knut; Marks, Guy; Menzies, Dick; Migliori, Giovanni Battista; Mosca, Davide; Mukadi, Ya Diul; Mwinga, Alwyn; Nelson, Lisa; Nishikiori, Nobuyuki; Oordt-Speets, Anouk; Rangaka, Molebogeng Xheedha; Reis, Andreas; Rotz, Lisa; Sandgren, Andreas; Sañé Schepisi, Monica; Schünemann, Holger J; Sharma, Surender Kumar; Sotgiu, Giovanni; Stagg, Helen R; Sterling, Timothy R; Tayeb, Tamara; Uplekar, Mukund; van der Werf, Marieke J; Vandevelde, Wim; van Kessel, Femke; van't Hoog, Anna; Varma, Jay K; Vezhnina, Natalia; Voniatis, Constantia; Vonk Noordegraaf-Schouten, Marije; Weil, Diana; Weyer, Karin; Wilkinson, Robert John; Yoshiyama, Takashi; Zellweger, Jean Pierre; Raviglione, Mario

    2015-12-01

    Latent tuberculosis infection (LTBI) is characterised by the presence of immune responses to previously acquired Mycobacterium tuberculosis infection without clinical evidence of active tuberculosis (TB). Here we report evidence-based guidelines from the World Health Organization for a public health approach to the management of LTBI in high risk individuals in countries with high or middle upper income and TB incidence of HIV, adult and child contacts of pulmonary TB cases, patients initiating anti-tumour necrosis factor treatment, patients receiving dialysis, patients preparing for organ or haematological transplantation, and patients with silicosis. In prisoners, healthcare workers, immigrants from high TB burden countries, homeless persons and illicit drug users, systematic testing and treatment of LTBI is conditionally recommended, according to TB epidemiology and resource availability. Either commercial interferon-gamma release assays or Mantoux tuberculin skin testing could be used to test for LTBI. Chest radiography should be performed before LTBI treatment to rule out active TB disease. Recommended treatment regimens for LTBI include: 6 or 9 month isoniazid; 12 week rifapentine plus isoniazid; 3-4 month isoniazid plus rifampicin; or 3-4 month rifampicin alone.

  7. Multidrug resistance to Mycobacterium tuberculosis in a tertiary hospital.

    Science.gov (United States)

    Kehinde, Aderemi Oludiran; Obaseki, Felix Ariebuwa; Ishola, Oluponle Christiana; Ibrahim, Kolo Doko

    2007-01-01

    OBJECTIVE: The magnitude of drug-resistant Mycobacterium tuberculosis infection (MDR-TB) in Nigeria, the most populous country in sub-Saharan Africa, is largely unknown. This information would assist policymakers to develop intervention strategies against tuberculosis (TB) in the country. MATERIALS AND METHODS: This is a one-year laboratory-based study. Specimens from suspected new TB patients sent to the TB laboratory of the Department of Medical Microbiology, University College Hospital Ibadan, Nigeria from May 1, 2005 to April 27, 2006 were processed and analyzed. The specimens were stained with Ziehl-Neelsen (Z-N) reagents and cultured on Lowenstein-Jensen medium, incubated at 37 degrees C for 6-8 weeks. Isolates were confirmed as MDR-TB by Z-N reactions and biochemical methods. Drug susceptibility to streptomycin, ethambutol, rifampicin and isoniazid was done using Bactec 460 TB radiometric method. RESULTS: Of the 1,120 specimens processed, 80 (7.1%) were smear positive, while 56 (5.0%) were culture positive, even though the association was not statistically significant (p > 0.05). Culture contamination rate was 8.8%. Thirty (53.6%) of the culture positive isolates were resistant to both isoniazid and rifampicin, while 26 (46.4%) were susceptible. About half--53.3%--of the resistant isolates were from the antiretroviral clinic, while 10 (33.4%) were from peripheral centers. CONCLUSION: This study shows that MDR-TB is emerging in Nigeria. Further studies on MDR-TB are urgently needed in the country to ascertain the magnitude of the problem and to proffer solutions to it. PMID:17987922

  8. Biosynthesis and translocation of unsulfated acyltrehaloses in Mycobacterium tuberculosis.

    Science.gov (United States)

    Belardinelli, Juan Manuel; Larrouy-Maumus, Gérald; Jones, Victoria; Sorio de Carvalho, Luiz Pedro; McNeil, Michael R; Jackson, Mary

    2014-10-03

    A number of species-specific polymethyl-branched fatty acid-containing trehalose esters populate the outer membrane of Mycobacterium tuberculosis. Among them, 2,3-diacyltrehaloses (DAT) and penta-acyltrehaloses (PAT) not only play a structural role in the cell envelope but also contribute to the ability of M. tuberculosis to multiply and persist in the infected host, promoting the intracellular survival of the bacterium and modulating host immune responses. The nature of the machinery, topology, and sequential order of the reactions leading to the biosynthesis, assembly, and export of these complex glycolipids to the cell surface are the object of the present study. Our genetic and biochemical evidence corroborates a model wherein the biosynthesis and translocation of DAT and PAT to the periplasmic space are coupled and topologically split across the plasma membrane. The formation of DAT occurs on the cytosolic face of the plasma membrane through the action of PapA3, FadD21, and Pks3/4; that of PAT occurs on the periplasmic face via transesterification reactions between DAT substrates catalyzed by the acyltransferase Chp2 (Rv1184c). The integral membrane transporter MmpL10 is essential for DAT to reach the cell surface, and its presence in the membrane is required for Chp2 to be active. Disruption of mmpL10 or chp2 leads to an important build-up of DAT inside the cells and to the formation of a novel form of unsulfated acyltrehalose esterified with polymethyl-branched fatty acids normally found in sulfolipids that is translocated to the cell surface.

  9. Predominance of multi-drug-resistant LAM and Beijing family strains among Mycobacterium tuberculosis isolates recovered from prison inmates in Tula Region, Russia.

    Science.gov (United States)

    Ignatova, Anna; Dubiley, Svetlana; Stepanshina, Valentina; Shemyakin, Igor

    2006-10-01

    The genotypic characteristics and drug susceptibility profiles of clinical isolates of Mycobacterium tuberculosis recovered from prison hospital patients in the Tula region (central Russia) during 2001 and 2002 are reported. The emergence of multi-drug-resistant tuberculosis (TB) poses a major health risk to the population, with economic implications for TB control. Prisons serve as a continuous source of TB transmission. The results showed that members of the LAM and Beijing families are major contributors to the epidemiological picture of TB in the population studied. The two families of strains accounted for most of the drug-resistant TB in the population. The genotypic characteristics of the M. tuberculosis predominant LAM strain that was responsible for 31 % of TB cases in this setting are presented.

  10. Cellular immune responses to ESAT-6 discriminate between patients with pulmonary disease due to Mycobacterium avium complex and those with pulmonary disease due to Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Lein, A D; von Reyn, C F; Ravn, P;

    1999-01-01

    ESAT-6 (for 6-kDa early secreted antigenic target) is a secreted antigen found almost exclusively in organisms of the Mycobacterium tuberculosis complex. We compared in vitro gamma interferon (IFN-gamma) responses by peripheral blood mononuclear cells to this antigen in patients with pulmonary...... disease due to either Mycobacterium avium complex (MAC) or Mycobacterium tuberculosis with those in healthy, skin test-negative, control subjects. Significant IFN-gamma responses to ESAT-6 were detected in 16 (59%) of 27 M. tuberculosis pulmonary disease patients, 0 (0%) of 8 MAC disease patients, and 0...... (0%) of 8 controls. Significant IFN-gamma responses to M. tuberculosis purified protein derivative were detected in 23 (85%) of 27 M. tuberculosis disease patients, 2 (25%) of 8 MAC disease patients, and 5 (63%) of 8 healthy controls. M. avium sensitin was recognized in 24 (89%) of 27 M. tuberculosis...

  11. Immunotherapeutical Potential of Mycobacterium Vaccae on M.Tuberculosis Infection in Mice

    Institute of Scientific and Technical Information of China (English)

    Lijun Xu; Yanyan Wang; Xiaodong Zheng; Xiangdong Gui; Lifeng Tao; Haiming Wei

    2009-01-01

    Tuberculosis remains the worldwide infectious disease. To identify the therapeutic potential of M. vaccae in treating tuberculosis, M. vaccae was injected into Mycobacterium tuberculosis (M. tuberculosis) infected mice. The optimal dose of M. vaccae (22.5μg/mouse) treated mice showed lower pathological change index, spleen weight index, lung weight index and vital M. tuberculosis count than those of the untreated group. Treatment with M. vaccae enhanced the percentages of CD3+ and CD4+ T cells, IFN-γ+CD4+ T cells, innate immune cells including NK cells, NK1.1+ T cells and γδ T cells, and reduced the percentage of IL-4+CD4+ T cells. Therefore, M. vaccae could protect the mice from M. tuberculosis infection and improved mouse innate and adaptive ceil-mediated immunity, suggesting that M. vaccae is a potential immunotherapeutic agent in pulmonary tuberculosis.

  12. Whole genome sequencing of Mycobacterium tuberculosis SB24 isolated from Sabah, Malaysia

    Directory of Open Access Journals (Sweden)

    Noraini Philip

    2016-09-01

    Full Text Available Mycobacterium tuberculosis (M. tuberculosis is the causative agent of tuberculosis (TB that causes millions of death every year. We have sequenced the genome of M. tuberculosis isolated from cerebrospinal fluid (CSF of a patient diagnosed with tuberculous meningitis (TBM. The isolated strain was referred as M. tuberculosis SB24. Genomic DNA of the M. tuberculosis SB24 was extracted and subjected to whole genome sequencing using PacBio platform. The draft genome size of M. tuberculosis SB24 was determined to be 4,452,489 bp with a G + C content of 65.6%. The whole genome shotgun project has been deposited in NCBI SRA under the accession number SRP076503.

  13. Using a cDNA microarray to study cellular gene expression altered by Mycobacterium tuberculosis

    Institute of Scientific and Technical Information of China (English)

    徐永忠; 谢建平; 李瑶; 乐军; 陈建平; 淳于利娟; 王洪海

    2003-01-01

    Objective To examine the global effects of Mycobacterium tuberculosis (M.tuberculosis) infection on macrophages. Methods The gene expression profiling of macrophage U937, in response to infection with M.tuberculosis H37Ra, was monitored using a high-density cDNA microarray. Results M.tuberculosis infection caused 463 differentially expressed genes, of which 366 genes are known genes registered in the Gene Bank. These genes function in various cellular processes including intracellular signalling, cytoskeletal rearrangement, apoptosis, transcriptional regulation, cell surface receptors, cell-mediated immunity as well as a variety of cellular metabolic pathways, and may play key roles in M.tuberculosis infection and intracellular survival. Conclusions M.tuberculosis infection alters the expression of host-cell genes, and these genes will provide a foundation for understanding the infection process of M.tuberculosis. The cDNA microarray is a powerful tool for studying pathogen-host cell interaction.

  14. Solution NMR Studies of Mycobacterium tuberculosis Proteins for Antibiotic Target Discovery

    Directory of Open Access Journals (Sweden)

    Do-Hee Kim

    2017-08-01

    Full Text Available Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis, which triggers severe pulmonary diseases. Recently, multidrug/extensively drug-resistant tuberculosis strains have emerged and continue to threaten global health. Because of the development of drug-resistant tuberculosis, there is an urgent need for novel antibiotics to treat these drug-resistant bacteria. In light of the clinical importance of M. tuberculosis, 2067 structures of M. tuberculsosis proteins have been determined. Among them, 52 structures have been solved and studied using solution nuclear magnetic resonance (NMR. The functional details based on structural analysis of M. tuberculosis using NMR can provide essential biochemical data for the development of novel antibiotic drugs. In this review, we introduce diverse structural and biochemical studies on M. tuberculosis proteins determined using NMR spectroscopy.

  15. Validation of a Multiplex Real-Time PCR Assay for Detection of Mycobacterium spp., Mycobacterium tuberculosis Complex, and Mycobacterium avium Complex Directly from Clinical Samples by Use of the BD Max Open System.

    Science.gov (United States)

    Rocchetti, Talita T; Silbert, Suzane; Gostnell, Alicia; Kubasek, Carly; Widen, Raymond

    2016-06-01

    A multiplex real-time PCR was validated on the BD Max open system to detect different Mycobacterium tuberculosis complex, Mycobacterium avium complex, and Mycobacterium spp. directly from clinical samples. The PCR results were compared to those with traditional cultures. The multiplex PCR assay was found to be a specific and sensitive method for the rapid detection of mycobacteria directly from clinical specimens.

  16. T-cell recognition of Mycobacterium tuberculosis culture filtrate fractions in tuberculosis patients and their household contacts

    DEFF Research Database (Denmark)

    Demissie, A; Ravn, P; Olobo, J

    1999-01-01

    We examined the immune responses of patients with active pulmonary tuberculosis (TB) and their healthy household contacts to short-term culture filtrate (ST-CF) of Mycobacterium tuberculosis or molecular mass fractions derived from it. Our goal was to identify fractions strongly recognized......, to secreted mycobacterial antigens is suggestive of an early stage of infection by M. tuberculosis, which could in time result in overt disease or containment of the infection. This possibility is currently being investigated by follow-up studies of the household contacts....

  17. T-cell recognition of Mycobacterium tuberculosis culture filtrate fractions in tuberculosis patients and their household contacts

    DEFF Research Database (Denmark)

    Demissie, A; Ravn, P; Olobo, J

    1999-01-01

    We examined the immune responses of patients with active pulmonary tuberculosis (TB) and their healthy household contacts to short-term culture filtrate (ST-CF) of Mycobacterium tuberculosis or molecular mass fractions derived from it. Our goal was to identify fractions strongly recognized......, to secreted mycobacterial antigens is suggestive of an early stage of infection by M. tuberculosis, which could in time result in overt disease or containment of the infection. This possibility is currently being investigated by follow-up studies of the household contacts....

  18. Confirming Mycobacterium tuberculosis transmission from a cadaver to an embalmer using molecular epidemiology.

    Science.gov (United States)

    Anderson, Janelle A; Meissner, Jeanne Sullivan; Ahuja, Shama Desai; Shashkina, Elena; O'Flaherty, Tholief; Proops, Douglas C

    2015-05-01

    Genotyping results and epidemiologic investigation were used to confirm tuberculosis transmission from a cadaver to an embalmer. This investigation highlights the utility of genotyping in identifying unsuspected epidemiologic links and unusual transmission settings. In addition, the investigation provides additional evidence for the occupational risk of tuberculosis among funeral service workers and indicates a need for education about tuberculosis risk and the importance of adhering to appropriate infection control measures among funeral service workers.

  19. LIGAND-BINDING SITES ON THE MYCOBACTERIUM TUBERCULOSIS UREASE

    Directory of Open Access Journals (Sweden)

    Lisnyak Yu. V.

    2017-10-01

    Full Text Available Introduction. Mycobacterium tuberculosis is the causative agent of tuberculosis that remains a serious medical and social health problem. Despite intensive efforts have been made in the past decade, there are no new efficient anti-tuberculosis drugs today, and that need is growing due to the spread of drug-resistant strains of M.tuberculosis. M. tuberculosis urease (MTU, being an important factor of the bacterium viability and virulence, is an attractive target for anti-tuberculosis drugs acting by inhibition of urease activity. However, the commercially available urease inhibitors are toxic and unstable, that prevent their clinical use. Therefore, new more potent anti-tuberculosis drugs inhibiting new targets are urgently needed. A useful tool for the search of novel inhibitors is a computational drug design. The inhibitor design is significantly easier if binding sites on the enzyme are identified in advance. This paper aimed to determine the probable ligand binding sites on the surface of M. tuberculosis urease. Methods. To identify ligand binding sites on MTU surface, сomputational solvent mapping method FTSite was applied by the use of MTU homology model we have built earlier. The method places molecular probes (small organic molecules containing various functional groups on a dense grid defined around the enzyme, and for each probe finds favorable positions. The selected poses are refined by free energy minimization, the low energy conformations are clustered, and the clusters are ranked on the basis of the average free energy. FTSite server outputs the protein residues delineating a binding sites and the probe molecules representing each cluster. To predict allosteric pockets on MTU, AlloPred and AlloSite servers were applied. AlloPred uses the normal mode analysis (NMA and models how the dynamics of a protein would be altered in the presence of a modulator at a specific pocket. Pockets on the enzyme are predicted using the Fpocket

  20. Inadequate hospital ventilation system increases the risk of nosocomial Mycobacterium tuberculosis.

    Science.gov (United States)

    Hubad, B; Lapanje, A

    2012-01-01

    The ambient air in nine locations in a pulmonary hospital and a tuberculosis diagnostic laboratory was analysed to determine the hazard posed by Mycobacterium tuberculosis. In four locations, between 6 and 20 M. tuberculosis cell equivalents/m(3) were detected. The results indicated that inadequately ventilated corridors had the highest cell equivalents. In these areas healthcare workers were less aware of infection hazard compared to areas with known sources of M. tuberculosis and the wearing of protective masks and passive ventilation were not in place. Based on these results, further infection prevention and control measures need to be implemented.

  1. Protective and therapeutic efficacy of Mycobacterium smegmatis expressing HBHA-hIL12 fusion protein against Mycobacterium tuberculosis in mice.

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    Shanmin Zhao

    Full Text Available Tuberculosis (TB remains a major worldwide health problem. The only vaccine against TB, Mycobacterium bovis Bacille Calmette-Guerin (BCG, has demonstrated relatively low efficacy and does not provide satisfactory protection against the disease. More efficient vaccines and improved therapies are urgently needed to decrease the worldwide spread and burden of TB, and use of a viable, metabolizing mycobacteria vaccine may be a promising strategy against the disease. Here, we constructed a recombinant Mycobacterium smegmatis (rMS strain expressing a fusion protein of heparin-binding hemagglutinin (HBHA and human interleukin 12 (hIL-12. Immune responses induced by the rMS in mice and protection against Mycobacterium tuberculosis (MTB were investigated. Administration of this novel rMS enhanced Th1-type cellular responses (IFN-γ and IL-2 in mice and reduced bacterial burden in lungs as well as that achieved by BCG vaccination. Meanwhile, the bacteria load in M. tuberculosis infected mice treated with the rMS vaccine also was significantly reduced. In conclusion, the rMS strain expressing the HBHA and human IL-12 fusion protein enhanced immunogencity by improving the Th1-type response against TB, and the protective effect was equivalent to that of the conventional BCG vaccine in mice. Furthermore, it could decrease bacterial load and alleviate histopathological damage in lungs of M. tuberculosis infected mice.

  2. Molecular typing methods used in studies of Mycobacterium tuberculosis in Iran: a systematic review

    Science.gov (United States)

    Ravansalar, Hassan; Tadayon3, Keyvan; Ghazvini, Kiarash

    2016-01-01

    Background and Objectives: Molecular typing methods are important and useful tools to assess the transmission, diversity of strains and differentiation between new infections and relapses which can effectively help in controlling infections. The aim of this study was to evaluate the molecular typing methods which have been used in Iran. By evaluating the results and discriminatory power of each method, we can assign appropriate weight to each technique and ultimately offer a common strategy for future epidemiological studies. Method: We searched several databases to identify studies addressing Mycobacterium tuberculosis molecular epidemiology in Iran. Hunter-Gaston discrimination index (HGDI) was used to evaluate the discriminatory power in each method. Relevant articles were selected and analyzed; HGDI index was calculated for each technique. Results: The most common genotyping methods used in the articles were RFLP, MIRU-VNTR, spoligotyping, PFGE and RAPD-PCR. The most frequently techniques were IS6110-RFLP, MIRU-VNTR and spoligotyping alone or in combination. The highest discrimination power (average HGDI: 0.9916) was obtained by RFLP followed by MIRU-VNTR (average HGDI: 0.9638) and spoligotyping (average HGDI: 0.9041) respectively. Conclusion: Combination of MIRU-VNTR with spoligotyping can be recommended for large-scale genotyping in Iran. It seems appropriate to consider spoligotyping as the first technique for screening followed by other techniques with higher discrimination power such as MIRU-VNTR or IS6110-RFLP.

  3. 中国东部农村地区结核分枝杆菌北京基因型菌株的成簇性及耐药特征研究%The cluster and drug-resistant characteristics of Beijing genotype Mycobacterium tuberculosis in eastern rural China