A comparative modeling and molecular docking study on Mycobacterium tuberculosis targets involved in peptidoglycan biosynthesis.

PubMed

Fakhar, Zeynab; Naiker, Suhashni; Alves, Claudio N; Govender, Thavendran; Maguire, Glenn E M; Lameira, Jeronimo; Lamichhane, Gyanu; Kruger, Hendrik G; Honarparvar, Bahareh

2016-11-01

An alarming rise of multidrug-resistant Mycobacterium tuberculosis strains and the continuous high global morbidity of tuberculosis have reinvigorated the need to identify novel targets to combat the disease. The enzymes that catalyze the biosynthesis of peptidoglycan in M. tuberculosis are essential and noteworthy therapeutic targets. In this study, the biochemical function and homology modeling of MurI, MurG, MraY, DapE, DapA, Alr, and Ddl enzymes of the CDC1551 M. tuberculosis strain involved in the biosynthesis of peptidoglycan cell wall are reported. Generation of the 3D structures was achieved with Modeller 9.13. To assess the structural quality of the obtained homology modeled targets, the models were validated using PROCHECK, PDBsum, QMEAN, and ERRAT scores. Molecular dynamics simulations were performed to calculate root mean square deviation (RMSD) and radius of gyration (Rg) of MurI and MurG target proteins and their corresponding templates. For further model validation, RMSD and Rg for selected targets/templates were investigated to compare the close proximity of their dynamic behavior in terms of protein stability and average distances. To identify the potential binding mode required for molecular docking, binding site information of all modeled targets was obtained using two prediction algorithms. A docking study was performed for MurI to determine the potential mode of interaction between the inhibitor and the active site residues. This study presents the first accounts of the 3D structural information for the selected M. tuberculosis targets involved in peptidoglycan biosynthesis.

Meta-analysis of human gene expression in response to Mycobacterium tuberculosis infection reveals potential therapeutic targets.

PubMed

Wang, Zhang; Arat, Seda; Magid-Slav, Michal; Brown, James R

2018-01-10

With the global emergence of multi-drug resistant strains of Mycobacterium tuberculosis, new strategies to treat tuberculosis are urgently needed such as therapeutics targeting potential human host factors. Here we performed a statistical meta-analysis of human gene expression in response to both latent and active pulmonary tuberculosis infections from nine published datasets. We found 1655 genes that were significantly differentially expressed during active tuberculosis infection. In contrast, no gene was significant for latent tuberculosis. Pathway enrichment analysis identified 90 significant canonical human pathways, including several pathways more commonly related to non-infectious diseases such as the LRRK2 pathway in Parkinson's disease, and PD-1/PD-L1 signaling pathway important for new immuno-oncology therapies. The analysis of human genome-wide association studies datasets revealed tuberculosis-associated genetic variants proximal to several genes in major histocompatibility complex for antigen presentation. We propose several new targets and drug-repurposing opportunities including intravenous immunoglobulin, ion-channel blockers and cancer immuno-therapeutics for development as combination therapeutics with anti-mycobacterial agents. Our meta-analysis provides novel insights into host genes and pathways important for tuberculosis and brings forth potential drug repurposing opportunities for host-directed therapies.

Mobile phone-based evaluation of latent tuberculosis infection: Proof of concept for an integrated image capture and analysis system.

PubMed

Naraghi, Safa; Mutsvangwa, Tinashe; Goliath, René; Rangaka, Molebogeng X; Douglas, Tania S

2018-05-08

The tuberculin skin test is the most widely used method for detecting latent tuberculosis infection in adults and active tuberculosis in children. We present the development of a mobile-phone based screening tool for measuring the tuberculin skin test induration. The tool makes use of a mobile application developed on the Android platform to capture images of an induration, and photogrammetric reconstruction using Agisoft PhotoScan to reconstruct the induration in 3D, followed by 3D measurement of the induration with the aid of functions from the Python programming language. The system enables capture of images by the person being screened for latent tuberculosis infection. Measurement precision was tested using a 3D printed induration. Real-world use of the tool was simulated by application to a set of mock skin indurations, created by a make-up artist, and the performance of the tool was evaluated. The usability of the application was assessed with the aid of a questionnaire completed by participants. The tool was found to measure the 3D printed induration with greater precision than the current ruler and pen method, as indicated by the lower standard deviation produced (0.3 mm versus 1.1 mm in the literature). There was high correlation between manual and algorithm measurement of mock skin indurations. The height of the skin induration and the definition of its margins were found to influence the accuracy of 3D reconstruction and therefore the measurement error, under simulated real-world conditions. Based on assessment of the user experience in capturing images, a simplified user interface would benefit wide-spread implementation. The mobile application shows good agreement with direct measurement. It provides an alternative method for measuring tuberculin skin test indurations and may remove the need for an in-person follow-up visit after test administration, thus improving latent tuberculosis infection screening throughput. Copyright © 2018 Elsevier Ltd. All rights reserved.

Identification of new antibacterial targets in RNA polymerase of Mycobacterium tuberculosis by detecting positive selection sites.

PubMed

Wang, QingBiao; Xu, Yiqin; Gu, Zhuoya; Liu, Nian; Jin, Ke; Li, Yao; Crabbe, M James C; Zhong, Yang

2018-04-01

Bacterial RNA polymerase (RNAP) is an effective target for antibacterial treatment. In order to search new potential targets in RNAP of Mycobacterium, we detected adaptive selections of RNAP related genes in 13 strains of Mycobacterium by phylogenetic analysis. We first collected sequences of 17 genes including rpoA, rpoB, rpoC, rpoZ, and sigma factor A-M. Then maximum likelihood trees were constructed, followed by positive selection detection. We found that sigG shows positive selection along the clade (M. tuberculosis, M. bovis), suggesting its important evolutionary role and its potential to be a new antibacterial target. Moreover, the regions near 933Cys and 935His on the rpoB subunit of M. tuberculosis showed significant positive selection, which could also be a new attractive target for anti-tuberculosis drugs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Development of a One-Step Multiplex PCR Assay for Differential Detection of Major Mycobacterium Species

PubMed Central

Chae, Hansong; Han, Seung Jung; Kim, Su-Young; Ki, Chang-Seok; Huh, Hee Jae; Yong, Dongeun

2017-01-01

ABSTRACT The prevalence of tuberculosis continues to be high, and nontuberculous mycobacterial (NTM) infection has also emerged worldwide. Moreover, differential and accurate identification of mycobacteria to the species or subspecies level is an unmet clinical need. Here, we developed a one-step multiplex PCR assay using whole-genome analysis and bioinformatics to identify novel molecular targets. The aims of this assay were to (i) discriminate between the Mycobacterium tuberculosis complex (MTBC) and NTM using rv0577 or RD750, (ii) differentiate M. tuberculosis (M. tuberculosis) from MTBC using RD9, (iii) selectively identify the widespread M. tuberculosis Beijing genotype by targeting mtbk_20680, and (iv) simultaneously detect five clinically important NTM (M. avium, M. intracellulare, M. abscessus, M. massiliense, and M. kansasii) by targeting IS1311, DT1, mass_3210, and mkan_rs12360. An initial evaluation of the multiplex PCR assay using reference strains demonstrated 100% specificity for the targeted Mycobacterium species. Analytical sensitivity ranged from 1 to 10 pg for extracted DNA and was 103 and 104 CFU for pure cultures and nonhomogenized artificial sputum cultures, respectively, of the targeted species. The accuracy of the multiplex PCR assay was further evaluated using 55 reference strains and 94 mycobacterial clinical isolates. Spoligotyping, multilocus sequence analysis, and a commercial real-time PCR assay were employed as standard assays to evaluate the multiplex PCR assay with clinical M. tuberculosis and NTM isolates. The PCR assay displayed 100% identification agreement with the standard assays. Our multiplex PCR assay is a simple, convenient, and reliable technique for differential identification of MTBC, M. tuberculosis, M. tuberculosis Beijing genotype, and major NTM species. PMID:28659320

Development of a One-Step Multiplex PCR Assay for Differential Detection of Major Mycobacterium Species.

PubMed

Chae, Hansong; Han, Seung Jung; Kim, Su-Young; Ki, Chang-Seok; Huh, Hee Jae; Yong, Dongeun; Koh, Won-Jung; Shin, Sung Jae

2017-09-01

The prevalence of tuberculosis continues to be high, and nontuberculous mycobacterial (NTM) infection has also emerged worldwide. Moreover, differential and accurate identification of mycobacteria to the species or subspecies level is an unmet clinical need. Here, we developed a one-step multiplex PCR assay using whole-genome analysis and bioinformatics to identify novel molecular targets. The aims of this assay were to (i) discriminate between the Mycobacterium tuberculosis complex (MTBC) and NTM using rv0577 or RD750, (ii) differentiate M. tuberculosis ( M. tuberculosis ) from MTBC using RD9, (iii) selectively identify the widespread M. tuberculosis Beijing genotype by targeting mtbk_20680 , and (iv) simultaneously detect five clinically important NTM ( M. avium , M. intracellulare , M. abscessus , M. massiliense , and M. kansasii ) by targeting IS 1311 , DT1, mass_3210 , and mkan_rs12360 An initial evaluation of the multiplex PCR assay using reference strains demonstrated 100% specificity for the targeted Mycobacterium species. Analytical sensitivity ranged from 1 to 10 pg for extracted DNA and was 10 3 and 10 4 CFU for pure cultures and nonhomogenized artificial sputum cultures, respectively, of the targeted species. The accuracy of the multiplex PCR assay was further evaluated using 55 reference strains and 94 mycobacterial clinical isolates. Spoligotyping, multilocus sequence analysis, and a commercial real-time PCR assay were employed as standard assays to evaluate the multiplex PCR assay with clinical M. tuberculosis and NTM isolates. The PCR assay displayed 100% identification agreement with the standard assays. Our multiplex PCR assay is a simple, convenient, and reliable technique for differential identification of MTBC, M. tuberculosis , M. tuberculosis Beijing genotype, and major NTM species. Copyright © 2017 American Society for Microbiology.

Evaluating clinicians' user experience and acceptability of LearnTB, a smartphone application for tuberculosis in India.

PubMed

Pande, Tripti; Saravu, Kavitha; Temesgen, Zelalem; Seyoum, Al; Rai, Shipra; Rao, Raghavendra; Mahadev, Deekshith; Pai, Madhukar; Gagnon, Marie-Pierre

2017-01-01

Tuberculosis (TB) is the leading infectious killer, and India accounts for 2.8 of the 10.4 million TB cases that occur each year, making it the highest TB burden country worldwide. Poor quality of TB care is a major driver of the epidemic in India. India's large private, unregulated sector manages over 50% of the TB patients, with studies showing suboptimal diagnosis and treatment in the private sector. Better education of doctors using mobile applications (apps) is a possible solution. While India has seen an explosion of mobile phone services, and while the use of mobile health interventions has been gaining interest, little is known about mHealth around tuberculosis in India. Our study aimed to understand the user experience and acceptability of a smartphone application, LearnTB , amongst private sector academic clinicians in India. This study was conducted amongst 101 clinicians at Kasturba Hospital, Manipal, India. The user experience of participants (part 1) and acceptability (part 2) were evaluated with the use of two valid, English, paper-based questionnaires. The first questionnaire was based on the System Usability Scale (SUS); the second questionnaire was based on the Technology Acceptance Model (TAM). Data were collected during February and March 2017 and were analyzed using descriptive statistics, multiple linear regression as well as logistic regression analysis. A response rate of 99% was achieved; 100 participants responded to the second questionnaire and 100% of the participants responded to the first questionnaire. User experience was very high [mean SUS score =94.4 (92.07-96.76)]. Perceived usefulness (PU) was significantly correlated to intention to use (IU) (r=0.707, P<0.0001), and perceived ease of use (PEU) was significantly correlated to PU (r=0.466, P<0.0001). Path analysis confirmed the direct relationship between PU and IU (0.936, P<0.0001), and the indirect relationship between PEU and IU (0.5102, P<0.0001). Logistic regression analysis helped target items strongly influencing IU, such as "The use of the LearnTB application is compatible with my work habits" [OR =3.20 (1.04-9.84), P=0.004] and "The use of the LearnTB application could promote good clinical practice" [OR =5.23 (1.35-20.29); P=0.016]. The first part of the study indicated high user experience of the LearnTB application. The TAM questionnaire (second part) explained a significant portion of the variance in clinicians' IU the LearnTB application. The PU of the application has the highest impact on the clinicians' IU the Learn TB application. This study provides a preliminary analysis of mobile health interventions for tuberculosis in India, and emphasizes the need for future research in this domain.

Aspartate decarboxylase (PanD) as a new target of pyrazinamide in Mycobacterium tuberculosis.

PubMed

Shi, Wanliang; Chen, Jiazhen; Feng, Jie; Cui, Peng; Zhang, Shuo; Weng, Xinhua; Zhang, Wenhong; Zhang, Ying

2014-08-01

Pyrazinamide (PZA) is a frontline anti-tuberculosis drug that plays a crucial role in the treatment of both drug-susceptible and multidrug-resistant tuberculosis (MDR-TB). PZA is a prodrug that is converted to its active form, pyrazinoic acid (POA), by a nicotinamidase/pyrazinamidase encoded by the pncA gene, the mutation of which is the major cause of PZA resistance. Although RpsA (ribosomal protein S1, involved in trans-translation) has recently been shown to be a target of POA/PZA, whole-genome sequencing has identified mutations in the panD gene encoding aspartate decarboxylase in PZA-resistant strains lacking pncA and rpsA mutations. To gain more insight into a possible new target of PZA, we isolated 30 POA-resistant mutants lacking mutations in pncA and rpsA from M. tuberculosis in vitro, and whole-genome sequencing of 3 mutants identified various mutations in the panD gene. Additionally, sequencing analysis revealed that the remaining 27 POA-resistant mutants all harbored panD mutations affecting the C-terminus of the PanD protein, with PanD M117I being the most frequent mutation (24/30, 80%). Conditional overexpression of panD from M. tuberculosis, M. smegmatis or E. coli, or of M. tuberculosis mutant PanD M117I, all conferred resistance to POA and PZA in M. tuberculosis. β-alanine and pantothenate, which are downstream products of PanD, were found to antagonize the antituberculosis activity of POA. In addition, the activity of the M. tuberculosis PanD enzyme was inhibited by POA at therapeutically relevant concentrations in a concentration-dependent manner but was not inhibited by the prodrug PZA or the control compound nicotinamide. These findings suggest that PanD represents a new target of PZA/POA. These results have implications for a better understanding of this peculiar persister drug and for the design of new drugs targeting M. tuberculosis persisters for improved treatment.

Pathogenic Gene Screening of Mycobacterium tuberculosis by Literature Data Mining and Information Pathway Enrichment Analysis.

PubMed

Xu, Guangyu; Wen, Simin; Pan, Yuchen; Zhang, Nan; Wang, Yuanyi

2018-05-01

Recent studies have unraveled mutations which have led to changes in the original conformation of functional proteins targeted by frontline drugs against Mycobacterium tuberculosis. These mutations are likely responsible for the emergence of drug-resistant strains of M. tuberculosis. Identification of new therapeutic targets is fundamental to the development of novel anti-TB drugs. Boost evolution analysis of interactome data with use of high-throughput biological experimental technologies provides opportunities for identification of pathogenic genes and for screening out novel therapeutic targets. In this study, we identified 584 proven pathogenic genes of M. tuberculosis and new pathogenic genes via bibliometrics and relevant websites such as PubMed, KEGG, and DOOR websites. We identified 13 new genes that are most likely to be pathogenic. This study may contribute to the discovery of new pathogenic genes and help unravel new functions of known pathogenic genes of M. tuberculosis.

Dataset of potential targets for Mycobacterium tuberculosis H37Rv through comparative genome analysis.

PubMed

Asif, Siddiqui M; Asad, Amir; Faizan, Ahmad; Anjali, Malik S; Arvind, Arya; Neelesh, Kapoor; Hirdesh, Kumar; Sanjay, Kumar

2009-12-31

Mycobacterium tuberculosis is the causative agent of the disease, tuberculosis and H37Rv is the most studied clinical strain. We use comparative genome analysis of Mycobacterium tuberculosis H37Rv and human for the identification of potential targets dataset. We used DEG (Database of Essential Genes) to identify essential genes in the H37Rv strain. The analysis shows that 628 of the 3989 genes in Mycobacterium tuberculosis H37Rv were found to be essential of which 324 genes lack similarity to the human genome. Subsequently hypothetical proteins were removed through manual curation. This further resulted in a dataset of 135 proteins with essential function and no homology to human.

First Baseline of Circulating Genotypic Lineages of Mycobacterium tuberculosis in Patients from the Brazilian Borders with Argentina and Paraguay

PubMed Central

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

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. PMID:25202909

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

PubMed

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.

Towards host-directed therapies for tuberculosis.

PubMed

Zumla, Alimuddin; Maeurer, Markus; Chakaya, Jeremiah; Hoelscher, Michael; Ntoumi, Francine; Rustomjee, Roxana; Vilaplana, Cristina; Yeboah-Manu, Dorothy; Rasolof, Voahangy; Munderi, Paula; Singh, Nalini; Aklillu, Eleni; Padayatchi, Nesri; Macete, Eusebio; Kapata, Nathan; Mulenga, Modest; Kibiki, Gibson; Mfinanga, Sayoki; Nyirenda, Thomas; Maboko, Leonard; Garcia-Basteiro, Alberto; Rakotosamimanana, Niaina; Bates, Matthew; Mwaba, Peter; Reither, Klaus; Gagneux, Sebastien; Edwards, Sarah; Mfinanga, Elirehema; Abdulla, Salim; Cardona, Pere-Joan; Russell, James B W; Gant, Vanya; Noursadeghi, Mahdad; Elkington, Paul; Bonnet, Maryline; Menendez, Clara; Dieye, Tandakha N; Diarra, Bassirou; Maiga, Almoustapha; Aseffa, Abraham; Parida, Shreemanta; Wejse, Christian; Petersen, Eskild; Kaleebu, Pontiano; Oliver, Matt; Craig, Gill; Corrah, Tumena; Tientcheu, Leopold; Antonio, Martin; Rao, Martin; McHugh, Timothy D; Sheikh, Aziz; Ippolito, Giuseppe; Ramjee, Gita; Kaufmann, Stefan H E; Churchyard, Gavin; Steyn, Andrie; Grobusch, Martin; Sanne, Ian; Martinson, Neil; Madansein, Rajhmun; Wilkinson, Robert J; Mayosi, Bongani; Schito, Marco; Wallis, Robert S

2015-08-01

The treatment of tuberculosis is based on combinations of drugs that directly target Mycobacterium tuberculosis. A new global initiative is now focusing on a complementary approach of developing adjunct host-directed therapies.

Antibodies and tuberculosis: finally coming of age?

PubMed

Li, Hao; Javid, Babak

2018-06-05

Are antibodies important for protection against tuberculosis? The jury has been out for more than 100 years. B cell depletion in experimental Mycobacterium tuberculosis infection failed to identify a major role for these cells in immunity to tuberculosis. However, recent identification of naturally occurring antibodies in humans that are protective during M. tuberculosis infection has reignited the debate. Here, we discuss the evidence for a protective role for antibodies in tuberculosis and consider the feasibility of designing novel tuberculosis vaccines targeting humoral immunity.

Monitoring Therapy Adherence of Tuberculosis Patients by using Video-Enabled Electronic Devices

PubMed Central

Story, Alistair; Garfein, Richard S.; Hayward, Andrew; Rusovich, Valiantsin; Dadu, Andrei; Soltan, Viorel; Oprunenco, Alexandru; Collins, Kelly; Sarin, Rohit; Quraishi, Subhi; Sharma, Mukta; Migliori, Giovanni Battista; Varadarajan, Maithili

2016-01-01

A recent innovation to help patients adhere to daily tuberculosis (TB) treatment over many months is video (or virtually) observed therapy (VOT). VOT is becoming increasingly feasible as mobile telephone applications and tablet computers become more widely available. Studies of the effectiveness of VOT in improving TB patient outcomes are being conducted. PMID:26891363

Targeted delivery of anti-tuberculosis drugs to macrophages: targeting mannose receptors

NASA Astrophysics Data System (ADS)

Filatova, L. Yu; Klyachko, N. L.; Kudryashova, E. V.

2018-04-01

The development of systems for targeted delivery of anti-tuberculosis drugs is a challenge of modern biotechnology. Currently, these drugs are encapsulated in a variety of carriers such as liposomes, polymers, emulsions and so on. Despite successful in vitro testing of these systems, virtually no success was achieved in vivo, because of low accessibility of the foci of infection located in alveolar macrophage cells. A promising strategy for increasing the efficiency of therapeutic action of anti-tuberculosis drugs is to encapsulate the agents into mannosylated carriers targeting the mannose receptors of alveolar macrophages. The review addresses the methods for modification of drug substance carriers, such as liposomes and biodegradable polymers, with mannose residues. The use of mannosylated carriers to deliver anti-tuberculosis agents increases the drug circulation time in the blood stream and increases the drug concentration in alveolar macrophage cells. The bibliography includes 113 references.

Defining the Needs for Next Generation Assays for Tuberculosis

PubMed Central

Denkinger, Claudia M.; Kik, Sandra V.; Cirillo, Daniela Maria; Casenghi, Martina; Shinnick, Thomas; Weyer, Karin; Gilpin, Chris; Boehme, Catharina C.; Schito, Marco; Kimerling, Michael; Pai, Madhukar

2015-01-01

To accelerate the fight against tuberculosis, major diagnostic challenges need to be addressed urgently. Post-2015 targets are unlikely to be met without the use of novel diagnostics that are more accurate and can be used closer to where patients first seek care in affordable diagnostic algorithms. This article describes the efforts by the stakeholder community that led to the identification of the high-priority diagnostic needs in tuberculosis. Subsequently target product profiles for the high-priority diagnostic needs were developed and reviewed in a World Health Organization (WHO)-led consensus meeting. The high-priority diagnostic needs included (1) a sputum-based replacement test for smear-microscopy; (2) a non-sputum-based biomarker test for all forms of tuberculosis, ideally suitable for use at levels below microscopy centers; (3) a simple, low cost triage test for use by first-contact care providers as a rule-out test, ideally suitable for use by community health workers; and (4) a rapid drug susceptibility test for use at the microscopy center level. The developed target product profiles, along with complimentary work presented in this supplement, will help to facilitate the interaction between the tuberculosis community and the diagnostics industry with the goal to lead the way toward the post-2015 global tuberculosis targets. PMID:25765104

Identification of KasA as the cellular target of an anti-tubercular scaffold

PubMed Central

Abrahams, Katherine A.; Chung, Chun-wa; Ghidelli-Disse, Sonja; Rullas, Joaquín; Rebollo-López, María José; Gurcha, Sudagar S.; Cox, Jonathan A. G.; Mendoza, Alfonso; Jiménez-Navarro, Elena; Martínez-Martínez, María Santos; Neu, Margarete; Shillings, Anthony; Homes, Paul; Argyrou, Argyrides; Casanueva, Ruth; Loman, Nicholas J.; Moynihan, Patrick J.; Lelièvre, Joël; Selenski, Carolyn; Axtman, Matthew; Kremer, Laurent; Bantscheff, Marcus; Angulo-Barturen, Iñigo; Izquierdo, Mónica Cacho; Cammack, Nicholas C.; Drewes, Gerard; Ballell, Lluis; Barros, David; Besra, Gurdyal S.; Bates, Robert H.

2016-01-01

Phenotypic screens for bactericidal compounds are starting to yield promising hits against tuberculosis. In this regard, whole-genome sequencing of spontaneous resistant mutants generated against an indazole sulfonamide (GSK3011724A) identifies several specific single-nucleotide polymorphisms in the essential Mycobacterium tuberculosis β-ketoacyl synthase (kas) A gene. Here, this genomic-based target assignment is confirmed by biochemical assays, chemical proteomics and structural resolution of a KasA-GSK3011724A complex by X-ray crystallography. Finally, M. tuberculosis GSK3011724A-resistant mutants increase the in vitro minimum inhibitory concentration and the in vivo 99% effective dose in mice, establishing in vitro and in vivo target engagement. Surprisingly, the lack of target engagement of the related β-ketoacyl synthases (FabH and KasB) suggests a different mode of inhibition when compared with other Kas inhibitors of fatty acid biosynthesis in bacteria. These results clearly identify KasA as the biological target of GSK3011724A and validate this enzyme for further drug discovery efforts against tuberculosis. PMID:27581223

Bringing state-of-the-art diagnostics to vulnerable populations: The use of a mobile screening unit in active case finding for tuberculosis in Palawan, the Philippines.

PubMed

Morishita, Fukushi; Garfin, Anna Marie Celina Gonzales; Lew, Woojin; Oh, Kyung Hyun; Yadav, Rajendra-Prasad; Reston, Janeth Cuencaho; Infante, Lenie Lucio; Acala, Maria Rebethia Crueldad; Palanca, Dean Lim; Kim, Hee Jin; Nishikiori, Nobuyuki

2017-01-01

Globally, case detection of tuberculosis (TB) has stabilized in recent years. Active case finding (ACF) has regained an increased attention as a complementary strategy to fill the case detection gap. In the Philippines, the DetecTB project implemented an innovative ACF strategy that offered a one-stop diagnostic service with a mobile unit equipped with enhanced diagnostic tools including chest X-ray (CXR) and Xpert®MTB/RIF (Xpert). The project targeted the rural poor, the urban poor, prison inmates, indigenous population and high school students. This is a retrospective review of TB screening data from 25,103 individuals. A descriptive analysis was carried out to compare screening and treatment outcomes across target populations. Univariate and multivariate analyses were performed to identify predictors of TB for each population. The composition of bacteriologically-confirmed cases by smear and symptom status was further investigated. The highest yield with lowest number needed to screen (NNS) was found in prison (6.2%, NNS: 16), followed by indigenous population (2.9%, NNS: 34), the rural poor (2.2%, NNS: 45), the urban poor (2.1%, NNS: 48), and high school (0.2%, NNS: 495). The treatment success rate for all populations was high with 89.5% in rifampicin-susceptible patients and 83.3% in rifampicin-resistant patients. A relatively higher loss to follow-up rate was observed in indigenous population (7.5%) and the rural poor (6.4%). Only cough more than two weeks showed a significant association with TB diagnosis in all target populations (Adjusted Odds Ratio ranging from 1.71 to 6.73) while other symptoms and demographic factors varied in their strength of association. The urban poor had the highest proportion of smear-positive patients with cough more than two weeks (72.0%). The proportion of smear-negative (Xpert-positive) patients without cough more than two weeks was the highest in indigenous population (39.3%), followed by prison inmates (27.7%), and the rural poor (22.8%). The innovative ACF strategy using mobile unit yielded a substantial number of TB patients and achieved successful treatment outcomes. TB screening in prison, indigenous population, and urban and rural poor communities was found to be effective. The combined use of CXR and Xpert largely contributed to increased case detection.

Bringing state-of-the-art diagnostics to vulnerable populations: The use of a mobile screening unit in active case finding for tuberculosis in Palawan, the Philippines

PubMed Central

Morishita, Fukushi; Garfin, Anna Marie Celina Gonzales; Lew, Woojin; Oh, Kyung Hyun; Yadav, Rajendra-Prasad; Reston, Janeth Cuencaho; Infante, Lenie Lucio; Acala, Maria Rebethia Crueldad; Palanca, Dean Lim; Kim, Hee Jin; Nishikiori, Nobuyuki

2017-01-01

Background Globally, case detection of tuberculosis (TB) has stabilized in recent years. Active case finding (ACF) has regained an increased attention as a complementary strategy to fill the case detection gap. In the Philippines, the DetecTB project implemented an innovative ACF strategy that offered a one-stop diagnostic service with a mobile unit equipped with enhanced diagnostic tools including chest X-ray (CXR) and Xpert®MTB/RIF (Xpert). The project targeted the rural poor, the urban poor, prison inmates, indigenous population and high school students. Methods This is a retrospective review of TB screening data from 25,103 individuals. A descriptive analysis was carried out to compare screening and treatment outcomes across target populations. Univariate and multivariate analyses were performed to identify predictors of TB for each population. The composition of bacteriologically-confirmed cases by smear and symptom status was further investigated. Results The highest yield with lowest number needed to screen (NNS) was found in prison (6.2%, NNS: 16), followed by indigenous population (2.9%, NNS: 34), the rural poor (2.2%, NNS: 45), the urban poor (2.1%, NNS: 48), and high school (0.2%, NNS: 495). The treatment success rate for all populations was high with 89.5% in rifampicin-susceptible patients and 83.3% in rifampicin-resistant patients. A relatively higher loss to follow-up rate was observed in indigenous population (7.5%) and the rural poor (6.4%). Only cough more than two weeks showed a significant association with TB diagnosis in all target populations (Adjusted Odds Ratio ranging from 1.71 to 6.73) while other symptoms and demographic factors varied in their strength of association. The urban poor had the highest proportion of smear-positive patients with cough more than two weeks (72.0%). The proportion of smear-negative (Xpert-positive) patients without cough more than two weeks was the highest in indigenous population (39.3%), followed by prison inmates (27.7%), and the rural poor (22.8%). Conclusions The innovative ACF strategy using mobile unit yielded a substantial number of TB patients and achieved successful treatment outcomes. TB screening in prison, indigenous population, and urban and rural poor communities was found to be effective. The combined use of CXR and Xpert largely contributed to increased case detection. PMID:28152082

The Cyclic Peptide Ecumicin Targeting ClpC1 Is Active against Mycobacterium tuberculosis In Vivo

PubMed Central

Gao, Wei; Kim, Jin-Yong; Anderson, Jeffrey R.; Akopian, Tatos; Hong, Seungpyo; Jin, Ying-Yu; Kandror, Olga; Kim, Jong-Woo; Lee, In-Ae; Lee, Sun-Young; McAlpine, James B.; Mulugeta, Surafel; Sunoqrot, Suhair; Wang, Yuehong; Yang, Seung-Hwan; Yoon, Tae-Mi; Goldberg, Alfred L.; Pauli, Guido F.; Cho, Sanghyun

2014-01-01

Drug-resistant tuberculosis (TB) has lent urgency to finding new drug leads with novel modes of action. A high-throughput screening campaign of >65,000 actinomycete extracts for inhibition of Mycobacterium tuberculosis viability identified ecumicin, a macrocyclic tridecapeptide that exerts potent, selective bactericidal activity against M. tuberculosis in vitro, including nonreplicating cells. Ecumicin retains activity against isolated multiple-drug-resistant (MDR) and extensively drug-resistant (XDR) strains of M. tuberculosis. The subcutaneous administration to mice of ecumicin in a micellar formulation at 20 mg/kg body weight resulted in plasma and lung exposures exceeding the MIC. Complete inhibition of M. tuberculosis growth in the lungs of mice was achieved following 12 doses at 20 or 32 mg/kg. Genome mining of lab-generated, spontaneous ecumicin-resistant M. tuberculosis strains identified the ClpC1 ATPase complex as the putative target, and this was confirmed by a drug affinity response test. ClpC1 functions in protein breakdown with the ClpP1P2 protease complex. Ecumicin markedly enhanced the ATPase activity of wild-type (WT) ClpC1 but prevented activation of proteolysis by ClpC1. Less stimulation was observed with ClpC1 from ecumicin-resistant mutants. Thus, ClpC1 is a valid drug target against M. tuberculosis, and ecumicin may serve as a lead compound for anti-TB drug development. PMID:25421483

Active case-finding for tuberculosis by mobile teams in Myanmar: yield and treatment outcomes.

PubMed

Myint, Ohnmar; Saw, Saw; Isaakidis, Petros; Khogali, Mohammed; Reid, Anthony; Hoa, Nguyen Binh; Kyaw, Thi Thi; Zaw, Ko Ko; Khaing, Tin Mi Mi; Aung, Si Thu

2017-06-02

Since 2005, the Myanmar National Tuberculosis Programme (NTP) has been implementing active case finding (ACF) activities involving mobile teams in hard-to-reach areas. This study revealed the contribution of mobile team activities to total tuberculosis (TB) case detection, characteristics of TB patients detected by mobile teams and their treatment outcomes. This was a descriptive study using routine programme data between October 2014 and December 2014. Mobile team activities were a one-stop service and included portable digital chest radiography (CXR) and microscopy of two sputum samples. The algorithm of the case detection included screening patients by symptoms, then by CXR followed by sputum microscopy for confirmation. Diagnosed patients were started on treatment and followed until a final outcome was ascertained. A total of 9 349 people with symptoms suggestive of TB were screened by CXR, with an uptake of 96.6%. Of those who were meant to undergo sputum smear microscopy, 51.4% had sputum examinations. Finally, 504 TB patients were identified by the mobile teams and the overall contribution to total TB case detection in the respective townships was 25.3%. Among total cases examined by microscopy, 6.4% were sputum smear positive TB. Treatment success rate was high as 91.8% in study townships compared to national rate 85% (2014 cohort). This study confirmed the feasibility and acceptability of ACF by mobile teams in hard-to-reach contexts, especially when equipped with portable, digital CXR machines that provided immediate results. However, the follow-up process of sputum examination created a significant barrier to confirmation of the diagnosis. In order to optimize the ACF through mobile team activity, future ACF activities were needed to be strengthened one stop service including molecular diagnostics or provision of sputum cups to all presumptive TB cases prior to CXR and testing if CXR suggestive of TB.

Protective activity of Lentinan in experimental tuberculosis.

PubMed

Markova, Nadya; Kussovski, Vesselin; Drandarska, Ivanka; Nikolaeva, Sascha; Georgieva, Neli; Radoucheva, Tatyana

2003-10-01

Protective effects of Lentinan (Ajinomoto, Japan) against Mycobacterium tuberculosis infection were studied by in vitro and in vivo mouse models. The effectiveness of Lentinan administrated intraperitoneally (i.p.) before infection at a dose of 1 mg/kg three times at 2-day intervals was monitored in vivo by several parameters (body temperature; spleen weight; CFU counts of M. tuberculosis in spleen, liver and lung; and histomorphological observations). Peritoneal macrophages obtained from animals treated with Lentinan were greatly stimulated, as assayed by establishing their number, acid phosphatase activity, H2O2 production and killing ability against M. tuberculosis in vitro. The in vivo model demonstrated that administration of Lentinan before infection can mobilize host defense potential and reduce mycobacterial infection.

Can Australia eliminate TB? Modelling immigration strategies for reaching MDG targets in a low-transmission setting.

PubMed

Denholm, Justin T; McBryde, Emma S

2014-02-01

The 2050 Millennium Development Goals (MDG) for tuberculosis (TB) aim for elimination of TB as a public health issue. We used a mathematical modelling approach to evaluate the feasibility of this target in a low-prevalence setting with immigration-related strategies directed at latent tuberculosis. We used a stochastic individual-based model to simulate tuberculosis disease among immigrants to Victoria, Australia; a representative low-transmission setting. A variety of screening and treatment approaches aimed at preventing reactivation of latent infection were applied to evaluate overall tuberculosis incidence reduction and rates of multidrug resistant disease. Without additional intervention, tuberculosis incidence was predicted to reach 34.5 cases/million by 2050. Strategies involving the introduction of an available screening/treatment combination reduced TB incidence to between 16.9-23.8 cases/million, and required screening of 136-427 new arrivals for each case of TB prevented. Limiting screening to higher incidence regions of origin was less effective but more efficient. Public health strategies targeting latent tuberculosis infection in immigrants may substantially reduce tuberculosis incidence in a low prevalence region. However, immigration-focused strategies cannot achieve the 2050 MDG and alternative or complementary approaches are required. © 2014 The Authors. ANZJPH © 2014 Public Health Association of Australia.

Targeting neutrophils for host-directed therapy to treat tuberculosis.

PubMed

Dallenga, Tobias; Linnemann, Lara; Paudyal, Bhesh; Repnik, Urska; Griffiths, Gareth; Schaible, Ulrich E

2017-10-07

M. tuberculosis is one of the prime killers from infectious diseases worldwide. Infections with multidrug-resistant variants counting for almost half a million new cases per year are steadily on the rise. Tuberculosis caused by extensively drug-resistant variants that are even resistant against newly developed or last resort antibiotics have to be considered untreaTable Susceptible tuberculosis already requires a six-months combinational therapy which requires further prolongation to treat drug-resistant infections. Such long treatment schedules are often accompanied by serious adverse effects causing patients to stop therapy. To tackle the global tuberculosis emergency, novel approaches for treatment need to be urgently explored. Host-directed therapies that target components of the defense system represent such a novel approach. In this review, we put a spotlight on neutrophils and neutrophil-associated effectors as promising targets for adjunct host-directed therapies to improve antibiotic efficacy and reduce both, treatment time and long-term pathological sequelae. Copyright © 2017 Elsevier GmbH. All rights reserved.

Could Killing Bacterial Subpopulations Hit Tuberculosis out of the Park?

PubMed

Baranowski, Catherine; Rubin, Eric J

2016-07-14

One hurdle to treating tuberculosis could be that it is so difficult to kill nonreplicating subpopulations of the causative pathogens. This work describes two new cephalosporin derivatives that specifically target this population of Mycobacterium tuberculosis.

Biochemical and structural investigations on phosphoribosylpyrophosphate synthetase from Mycobacterium smegmatis

PubMed Central

Donini, Stefano; Garavaglia, Silvia; Ferraris, Davide M.; Miggiano, Riccardo; Mori, Shigetarou; Shibayama, Keigo

2017-01-01

Mycobacterium smegmatis represents one model for studying the biology of its pathogenic relative Mycobacterium tuberculosis. The structural characterization of a M. tuberculosis ortholog protein can serve as a valid tool for the development of molecules active against the M. tuberculosis target. In this context, we report the biochemical and structural characterization of M. smegmatis phosphoribosylpyrophosphate synthetase (PrsA), the ortholog of M. tuberculosis PrsA, the unique enzyme responsible for the synthesis of phosphoribosylpyrophosphate (PRPP). PRPP is a key metabolite involved in several biosynthetic pathways including those for histidine, tryptophan, nucleotides and decaprenylphosphoryl-arabinose, an essential precursor for the mycobacterial cell wall biosynthesis. Since M. tuberculosis PrsA has been validated as a drug target for the development of antitubercular agents, the data presented here will add to the knowledge of the mycobacterial enzyme and could contribute to the development of M. tuberculosis PrsA inhibitors of potential pharmacological interest. PMID:28419153

Biochemical and structural investigations on phosphoribosylpyrophosphate synthetase from Mycobacterium smegmatis.

PubMed

Donini, Stefano; Garavaglia, Silvia; Ferraris, Davide M; Miggiano, Riccardo; Mori, Shigetarou; Shibayama, Keigo; Rizzi, Menico

2017-01-01

Mycobacterium smegmatis represents one model for studying the biology of its pathogenic relative Mycobacterium tuberculosis. The structural characterization of a M. tuberculosis ortholog protein can serve as a valid tool for the development of molecules active against the M. tuberculosis target. In this context, we report the biochemical and structural characterization of M. smegmatis phosphoribosylpyrophosphate synthetase (PrsA), the ortholog of M. tuberculosis PrsA, the unique enzyme responsible for the synthesis of phosphoribosylpyrophosphate (PRPP). PRPP is a key metabolite involved in several biosynthetic pathways including those for histidine, tryptophan, nucleotides and decaprenylphosphoryl-arabinose, an essential precursor for the mycobacterial cell wall biosynthesis. Since M. tuberculosis PrsA has been validated as a drug target for the development of antitubercular agents, the data presented here will add to the knowledge of the mycobacterial enzyme and could contribute to the development of M. tuberculosis PrsA inhibitors of potential pharmacological interest.

SMS reminders to improve the tuberculosis cure rate in developing countries (TB-SMS Cameroon): a protocol of a randomised control study

PubMed Central

2014-01-01

Background Tuberculosis is a public health problem in Cameroon, just like in many other countries in the world. The National Tuberculosis Control Programme (PNLT) put in place by the state, aims to fight tuberculosis through the implementation of international directives (Directly Observed Treatment Short, DOTS). Despite the deployment of this strategy across the world, its implementation is difficult in the context of low-resource countries. Some expected results are not achieved. In Cameroon, the cure rate for patients with sputum positive pulmonary tuberculosis (TPM+) after 6 months is only about 65%, 20% below the target. This is mainly due to poor patient adherence to treatment. By relying on the potential of mobile Health, the objective of this study is to evaluate the effect of SMS reminders on the cure rate of TPM + patients, measured using 6-month bacilloscopy. Methods/design This is a blinded, randomised controlled multicentre study carried out in Cameroon. The research hypothesis is that sending daily SMS messages to remind patients to take their prescribed tuberculosis medication, together with the standard DOTS strategy, will increase the cure rate from 65% (control group: DOTS, no SMS intervention) to 85% (intervention group: DOTS, with SMS intervention) in a group of new TPM + patients. In accordance with each treatment centre, the participants will be randomly allocated into the two groups using a computer program: the intervention group and the control group. A member of the research team will send daily SMS messages. Study data will be collected by health professionals involved in the care of patients. Data analysis will be done by the intention-to-treat method. Discussion The achieving of expected outcomes by the PNLT through implementation of DOTS requires several challenges. Although it has been demonstrated that the DOTS strategy is effective in the fight against tuberculosis, its application remains difficult in developing countries. This study explores the potential of mHealth to support DOTS strategy. It will gather new evidence on the effectiveness of mHealth-based interventions and SMS reminders in the improvement of treatment adherence and the cure rate of tuberculosis patients, especially in a low-resource country such as Cameroon. Trial registration The trial is registered on the Pan-African Clinical Trials Registry (http://www.pactr.org) under unique identification number: PACTR201307000583416. PMID:24460827

Novel Multiplex Real-Time PCR Diagnostic Assay for Identification and Differentiation of Mycobacterium tuberculosis, Mycobacterium canettii, and Mycobacterium tuberculosis Complex Strains▿†

PubMed Central

Reddington, Kate; O'Grady, Justin; Dorai-Raj, Siobhan; Maher, Majella; van Soolingen, Dick; Barry, Thomas

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 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. PMID:21123525

Eliminating Tuberculosis One Neighborhood at a Time

PubMed Central

Griffith, David E.; McGaha, Paul K.; Wolfgang, Melanie; Robinson, Celia B.; Clark, Patricia A.; Hassell, Willis L.; Robison, Valerie A.; Walker, Kerfoot P.; Wallace, Charles

2014-01-01

Objectives. We evaluated a strategy for preventing tuberculosis (TB) in communities most affected by it. Methods. In 1996, we mapped reported TB cases (1985–1995) and positive tuberculin skin test (TST) reactors (1993–1995) in Smith County, Texas. We delineated the 2 largest, densest clusters, identifying 2 highest-incidence neighborhoods (180 square blocks, 3153 residents). After extensive community preparation, trained health care workers went door-to-door offering TST to all residents unless contraindicated. TST-positive individuals were escorted to a mobile clinic for radiography, clinical evaluation, and isoniazid preventive treatment (IPT) as indicated. To assess long-term impact, we mapped all TB cases in Smith County during the equivalent time period after the project. Results. Of 2258 eligible individuals, 1291 (57.1%) were tested, 229 (17.7%) were TST positive, and 147 were treated. From 1996 to 2006, there were no TB cases in either project neighborhood, in contrast with the preintervention decade and the continued occurrence of TB in the rest of Smith County. Conclusions. Targeting high-incidence neighborhoods for active, community-based screening and IPT may hasten TB elimination in the United States. PMID:24899457

Eliminating Tuberculosis One Neighborhood at a Time

PubMed Central

Griffith, David E.; McGaha, Paul K.; Wolfgang, Melanie; Robinson, Celia B.; Clark, Patricia A.; Hassell, Willis L.; Robison, Valerie A.; Walker, Kerfoot P.; Wallace, Charles

2013-01-01

Objectives. We evaluated a strategy for preventing tuberculosis (TB) in communities most affected by it. Methods. In 1996, we mapped reported TB cases (1985–1995) and positive tuberculin skin test (TST) reactors (1993–1995) in Smith County, Texas. We delineated the 2 largest, densest clusters, identifying 2 highest-incidence neighborhoods (180 square blocks, 3153 residents). After extensive community preparation, trained health care workers went door-to-door offering TST to all residents unless contraindicated. TST-positive individuals were escorted to a mobile clinic for radiography, clinical evaluation, and isoniazid preventive treatment (IPT) as indicated. To assess long-term impact, we mapped all TB cases in Smith County during the equivalent time period after the project. Results. Of 2258 eligible individuals, 1291 (57.1%) were tested, 229 (17.7%) were TST positive, and 147 were treated. From 1996 to 2006, there were no TB cases in either project neighborhood, in contrast with the preintervention decade and the continued occurrence of TB in the rest of Smith County. Conclusions. Targeting high-incidence neighborhoods for active, community-based screening and IPT may hasten TB elimination in the United States. PMID:23078465

Impact of mobile teams on tuberculosis treatment outcomes, Riyadh Region, Kingdom of Saudi Arabia, 2013-2015.

PubMed

Alqahtani, Sami; Kashkary, Abdulhameed; Asiri, Abdullah; Kamal, Heba; Binongo, Jose; Castro, Kenneth; McNabb, Scott

2018-03-01

The objective of this study was to evaluate the impact of the tuberculosis (TB) mobile teams on treatment outcomes in Riyadh Region by comparing patients who received treatment under mobile teams and those who did not, from 2013 to 2015. This was a retrospective descriptive study using National TB Control and Prevention Program data from 2013 to 2015 from Riyadh, Kingdom of Saudi Arabia. Descriptive analyses were used to summarize characteristics of TB case-patients served by mobile teams and those who were not served. The χ 2 test measured the significant differences between mobile-served and non-mobile-served case-patients. Exposure was whether or not the TB case-patient was under the care of the mobile team; the outcome of interest was whether or not treatment was successful, defined as treatment completed and cured. We found that the ratio of treatment success among mobile team case-patients was 1.28 greater than among those not served by mobile teams. The χ 2 test showed a statistically significant finding (probability ratio=1.28; 95% confidence interval=1.21-1.35, p<0.01). Mobile teams increased the treatment success rate to 92%, compared to 71.77% among those not served by mobile teams. This study shows that community mobilization of mobile teams is an effective strategy to enhance TB treatment, reduced mortality and loss to follow-up and improve TB treatment outcomes. Copyright © 2017 Ministry of Health, Saudi Arabia. Published by Elsevier Ltd. All rights reserved.

Identification and characterization of potential druggable targets among hypothetical proteins of extensively drug resistant Mycobacterium tuberculosis (XDR KZN 605) through subtractive genomics approach.

PubMed

Uddin, Reaz; Siddiqui, Quratulain Nehal; Azam, Syed Sikander; Saima, Bibi; Wadood, Abdul

2018-03-01

Among the resistant isolates of tuberculosis (TB), the multidrug resistance tuberculosis (MDR-TB) and extensively drug resistant tuberculosis (XDR-TB) are the areas of growing concern for which the front-line antibiotics are no more effective. As a result, the search of new therapeutic targets against TB is an imperative need of time. On the other hand, the target identification is an a priori step in drug discovery based research. Furthermore, the availability of the complete proteomic data of extensively drug resistant Mycobacterium tuberculosis (XDR-MTB) made it possible to carry out in silico analysis for the discovery of new drug targets. In the current study, we aimed to prioritize the potential drug targets among the hypothetical proteins of XDR-TB via subtractive genomics approach. In the subtractive genomics, we stepwise reduced the complete proteome of XDR-MTB to only two hypothetical proteins and evidently proposed them as new therapeutic targets. The 3D structure of one of the two target proteins was predicted via homology modeling and later on, validated by various analysis tools. Our study suggested that the domains identified and the motif hits found in the sequences of the shortlisted drug targets are crucial for the survival of the XDR-MTB. To the best of our knowledge, the current study is the first attempt in which the complete proteomic data of XDR-MTB was subjected to the computational subtractive genomics approach and therefore, would provide an opportunity to identify the unique therapeutic targets against deadly XDR-MTB. Copyright © 2017 Elsevier B.V. All rights reserved.

Evaluating clinicians’ user experience and acceptability of LearnTB, a smartphone application for tuberculosis in India

PubMed Central

Pande, Tripti; Saravu, Kavitha; Temesgen, Zelalem; Seyoum, Al; Rai, Shipra; Rao, Raghavendra; Mahadev, Deekshith; Pai, Madhukar

2017-01-01

Background Tuberculosis (TB) is the leading infectious killer, and India accounts for 2.8 of the 10.4 million TB cases that occur each year, making it the highest TB burden country worldwide. Poor quality of TB care is a major driver of the epidemic in India. India’s large private, unregulated sector manages over 50% of the TB patients, with studies showing suboptimal diagnosis and treatment in the private sector. Better education of doctors using mobile applications (apps) is a possible solution. While India has seen an explosion of mobile phone services, and while the use of mobile health interventions has been gaining interest, little is known about mHealth around tuberculosis in India. Methods Our study aimed to understand the user experience and acceptability of a smartphone application, LearnTB, amongst private sector academic clinicians in India. This study was conducted amongst 101 clinicians at Kasturba Hospital, Manipal, India. The user experience of participants (part 1) and acceptability (part 2) were evaluated with the use of two valid, English, paper-based questionnaires. The first questionnaire was based on the System Usability Scale (SUS); the second questionnaire was based on the Technology Acceptance Model (TAM). Data were collected during February and March 2017 and were analyzed using descriptive statistics, multiple linear regression as well as logistic regression analysis. Results A response rate of 99% was achieved; 100 participants responded to the second questionnaire and 100% of the participants responded to the first questionnaire. User experience was very high [mean SUS score =94.4 (92.07–96.76)]. Perceived usefulness (PU) was significantly correlated to intention to use (IU) (r=0.707, P<0.0001), and perceived ease of use (PEU) was significantly correlated to PU (r=0.466, P<0.0001). Path analysis confirmed the direct relationship between PU and IU (0.936, P<0.0001), and the indirect relationship between PEU and IU (0.5102, P<0.0001). Logistic regression analysis helped target items strongly influencing IU, such as “The use of the LearnTB application is compatible with my work habits” [OR =3.20 (1.04–9.84), P=0.004] and “The use of the LearnTB application could promote good clinical practice” [OR =5.23 (1.35–20.29); P=0.016]. Conclusion The first part of the study indicated high user experience of the LearnTB application. The TAM questionnaire (second part) explained a significant portion of the variance in clinicians’ IU the LearnTB application. The PU of the application has the highest impact on the clinicians’ IU the Learn TB application. This study provides a preliminary analysis of mobile health interventions for tuberculosis in India, and emphasizes the need for future research in this domain. PMID:28828377

A Study on the Role of Mobile Phone Communication in Tuberculosis DOTS Treatment

PubMed Central

Elangovan, R; Arulchelvan, S

2013-01-01

Background: Every year, a lot of Tuberculosis (TB) patients undergo Directly Observed Treatment Short-course (DOTS) in Salem city, one of the high TB districts in South India. Mobile phone usage among these patients and health workers is common. Mobile phone communication has a great potential in TB treatment. Objectives: To analyze the mobile phone usage and its effectiveness in TB DOTS treatment. Materials and Methods: A cross-sectional survey with 150 TB patients was followed by a focus group discussion with treatment supervisors, DOTS providers, and health workers. Results: Majority of patients use mobile phones to make calls to health workers to clarify their doubts on side effects, food, and symptoms of the disease. TB treatment supervisors effectively use mobile phones to counsel patients to adhere to the treatment regimen. Patients see mobile phones as a useful communication tool in TB treatment though they prefer direct interpersonal communication with health workers. Though the mobile ownership is 68% among the TB patients, many of them are not able to send text messages or read messages in English. Conclusion: Mobile phone possession and usage is high among the patients. Patients need to be trained to use mobile phone features such as alarm, voice mail, and interactive voice response. Incentives like free talk time and short message service (SMS) will encourage patients to communicate frequently with health workers, thereby, increasing the chances of better adherence to DOTS. SMS could be made available in the regional languages. PMID:24302824

A Study on the Role of Mobile Phone Communication in Tuberculosis DOTS Treatment.

PubMed

Elangovan, R; Arulchelvan, S

2013-10-01

Every year, a lot of Tuberculosis (TB) patients undergo Directly Observed Treatment Short-course (DOTS) in Salem city, one of the high TB districts in South India. Mobile phone usage among these patients and health workers is common. Mobile phone communication has a great potential in TB treatment. To analyze the mobile phone usage and its effectiveness in TB DOTS treatment. A cross-sectional survey with 150 TB patients was followed by a focus group discussion with treatment supervisors, DOTS providers, and health workers. Majority of patients use mobile phones to make calls to health workers to clarify their doubts on side effects, food, and symptoms of the disease. TB treatment supervisors effectively use mobile phones to counsel patients to adhere to the treatment regimen. Patients see mobile phones as a useful communication tool in TB treatment though they prefer direct interpersonal communication with health workers. Though the mobile ownership is 68% among the TB patients, many of them are not able to send text messages or read messages in English. Mobile phone possession and usage is high among the patients. Patients need to be trained to use mobile phone features such as alarm, voice mail, and interactive voice response. Incentives like free talk time and short message service (SMS) will encourage patients to communicate frequently with health workers, thereby, increasing the chances of better adherence to DOTS. SMS could be made available in the regional languages.

Insights from the docking and molecular dynamics simulation of the Phosphopantetheinyl transferase (PptT) structural model from Mycobacterium tuberculosis.

PubMed

Rohini, Karunakaran; Srikumar, Padmalayam Sadanandan

2013-01-01

A great challenge is posed to the treatment of tuberculosis due to the evolution of multidrug-resistant (MDR) and extensively drugresistant (XDR) strains of Mycobacterium tuberculosis in recent times. The complex cell envelope of the bacterium contains unusual structures of lipids which protects the bacterium from host enzymes and escape immune response. To overcome the drug resistance, targeting "drug targets" which have a critical role in growth and virulence factor is a novel approach for better tuberculosis treatment. The enzyme Phosphopantetheinyl transferase (PptT) is an attractive drug target as it is primarily involved in post translational modification of various types-I polyketide synthases and assembly of mycobactin, which is required for lipid virulence factors. Our in silico studies reported that the structural model of M.tuberculosis PptT characterizes the structure-function activity. The refinement of the model was carried out with molecular dynamics simulations and was analyzed with root mean square deviation (RMSD), and radius of gyration (Rg). This confirmed the structural behavior of PptT in dynamic system. Molecular docking with substrate coenzyme A (CoA) identified the binding pocket and key residues His93, Asp114 and Arg169 involved in PptT-CoA binding. In conclusion, our results show that the M.tuberculosis PptT model and critical CoA binding pocket initiate the inhibitor design of PptT towards tuberculosis treatment.

Impact of community tracer teams on treatment outcomes among tuberculosis patients in South Africa.

PubMed

Bronner, Liza E; Podewils, Laura J; Peters, Annatjie; Somnath, Pushpakanthi; Nshuti, Lorna; van der Walt, Martie; Mametja, Lerole David

2012-08-07

Tuberculosis (TB) indicators in South Africa currently remain well below global targets. In 2008, the National Tuberculosis Program (NTP) implemented a community mobilization program in all nine provinces to trace TB patients that had missed a treatment or clinic visit. Implementation sites were selected by TB program managers and teams liaised with health facilities to identify patients for tracing activities. The objective of this analysis was to assess the impact of the TB Tracer Project on treatment outcomes among TB patients. The study population included all smear positive TB patients registered in the Electronic TB Registry from Quarter 1 2007-Quarter 1 2009 in South Africa. Subdistricts were used as the unit of analysis, with each designated as either tracer (standard TB program plus tracer project) or non-tracer (standard TB program only). Mixed linear regression models were utilized to calculate the percent quarterly change in treatment outcomes and to compare changes in treatment outcomes from Quarter 1 2007 to Quarter 1 2009 between tracer and non-tracer subdistricts. For all provinces combined, the percent quarterly change decreased significantly for default treatment outcomes among tracer subdistricts (-0.031%; p < 0.001) and increased significantly for successful treatment outcomes among tracer subdistricts (0.003%; p = 0.03). A significant decrease in the proportion of patient default was observed for all provinces combined over the time period comparing tracer and non-tracer subdistricts (p = 0.02). Examination in stratified models revealed the results were not consistent across all provinces; significant differences were observed between tracer and non-tracer subdistricts over time in five of nine provinces for treatment default. Community mobilization of teams to trace TB patients that missed a clinic appointment or treatment dose may be an effective strategy to mitigate default rates and improve treatment outcomes. Additional information is necessary to identify best practices and elucidate discrepancies across provinces; these findings will help guide the NTP in optimizing the adoption of tracing activities for TB control.

Identification of new benzamide inhibitor against α-subunit of tryptophan synthase from Mycobacterium tuberculosis through structure-based virtual screening, anti-tuberculosis activity and molecular dynamics simulations.

PubMed

Naz, Sadia; Farooq, Umar; Ali, Sajid; Sarwar, Rizwana; Khan, Sara; Abagyan, Ruben

2018-03-13

Multi-drug-resistant tuberculosis and extensively drug-resistant tuberculosis has emerged as global health threat, causing millions of deaths worldwide. Identification of new drug candidates for tuberculosis (TB) by targeting novel and less explored protein targets will be invaluable for antituberculosis drug discovery. We performed structure-based virtual screening of eMolecules database against a homology model of relatively unexplored protein target: the α-subunit of tryptophan synthase (α-TRPS) from Mycobacterium tuberculosis essential for bacterial survival. Based on physiochemical properties analysis and molecular docking, the seven candidate compounds were selected and evaluated through whole cell-based activity against the H37Rv strain of M. tuberculosis. A new Benzamide inhibitor against α-subunit of tryptophan synthase (α-TRPS) from M. tuberculosis has been identified causing 100% growth inhibition at 25 μg/ml and visible bactericidal activity at 6 μg/ml. This benzamide inhibitor displayed a good predicted binding score (-48.24 kcal/mol) with the α-TRPS binding pocket and has logP value (2.95) comparable to Rifampicin. Further refinement of docking results and evaluation of inhibitor-protein complex stability were investigated through Molecular dynamic (MD) simulations studies. Following MD simulations, Root mean square deviation, Root mean square fluctuation and secondary structure analysis confirmed that protein did not unfold and ligand stayed inside the active pocket of protein during the explored time scale. This identified benzamide inhibitor against the α-subunit of TRPS from M. tuberculosis could be considered as candidate for drug discovery against TB and will be further evaluated for enzyme-based inhibition in future studies.

Financing tuberculosis control: the role of a global financial monitoring system.

PubMed

Floyd, Katherine; Pantoja, Andrea; Dye, Christopher

2007-05-01

Control of tuberculosis (TB), like health care in general, costs money. To sustain TB control at current levels, and to make further progress so that global targets can be achieved, information about funding needs, sources of funding, funding gaps and expenditures is important at global, regional, national and sub-national levels. Such data can be used for resource mobilization efforts; to document how funding requirements and gaps are changing over time; to assess whether increases in funding can be translated into increased expenditures and whether increases in expenditure are producing improvements in programme performance; and to identify which countries or regions have the greatest needs and funding gaps. In this paper, we discuss a global system for financial monitoring of TB control that was established in WHO in 2002. By early 2007, this system had accounted for actual or planned expenditures of more than US$ 7 billion and was systematically reporting financial data for countries that carry more than 90% of the global burden of TB. We illustrate the value of this system by presenting major findings that have been produced for the period 2002-2007, including results that are relevant to the achievement of global targets for TB control set for 2005 and 2015. We also analyse the strengths and limitations of the system and its relevance to other health-care programmes.

Inflammation responses in patients with pulmonary tuberculosis in an intensive care unit

PubMed Central

Liu, Qiu-Yue; Han, Fen; Pan, Li-Ping; Jia, Hong-Yan; Li, Qi; Zhang, Zong-De

2018-01-01

Pulmonary tuberculosis caused by Mycobacterium tuberculosis remains a global problem. Inflammatory responses are the primary characteristics of patients with pulmonary tuberculosis in intensive care units (ICU). The aim of the present study was to investigate the clinical importance of inflammatory cells and factors for patients with pulmonary tuberculosis in ICU. A total of 124 patients with pulmonary tuberculosis in ICU were recruited for the present study. The inflammatory responses in patients with pulmonary tuberculosis in ICU were examined by changes in inflammatory cells and factors in the serum. The results indicated that serum levels of lymphocytes, plasma cells, granulocytes and monocytes were increased in patients with pulmonary tuberculosis in ICU compared with healthy controls. The serum levels of inflammatory factors interleukin (IL)-1, IL-6, IL-10, IL-12, and IL-4 were upregulated in patients with pulmonary tuberculosis in ICU. Lower plasma concentrations of IL-2, IL-15 and interferon-γ were detected in patients with pulmonary tuberculosis compared with healthy controls. It was demonstrated that high mobility group box-1 protein expression levels were higher in the serum of patients with pulmonary tuberculosis compared with healthy controls. Notably, an imbalance of T-helper cell (Th)1/Th2 cytokines was observed in patients with pulmonary tuberculosis. Pulmonary tuberculosis caused by M. tuberculosis also upregulated expression of matrix metalloproteinase (MMP)-1 and MMP-9 in hPMCs. In conclusion, these outcomes demonstrated that inflammatory responses and inflammatory factors are associated with the progression of pulmonary tuberculosis, suggesting that inhibition of inflammatory responses and inflammatory factors may be beneficial for the treatment of patients with pulmonary tuberculosis in ICU. PMID:29456674

Validation of CoaBC as a Bactericidal Target in the Coenzyme A Pathway of Mycobacterium tuberculosis.

PubMed

Evans, Joanna C; Trujillo, Carolina; Wang, Zhe; Eoh, Hyungjin; Ehrt, Sabine; Schnappinger, Dirk; Boshoff, Helena I M; Rhee, Kyu Y; Barry, Clifton E; Mizrahi, Valerie

2016-12-09

Mycobacterium tuberculosis relies on its own ability to biosynthesize coenzyme A to meet the needs of the myriad enzymatic reactions that depend on this cofactor for activity. As such, the essential pantothenate and coenzyme A biosynthesis pathways have attracted attention as targets for tuberculosis drug development. To identify the optimal step for coenzyme A pathway disruption in M. tuberculosis, we constructed and characterized a panel of conditional knockdown mutants in coenzyme A pathway genes. Here, we report that silencing of coaBC was bactericidal in vitro, whereas silencing of panB, panC, or coaE was bacteriostatic over the same time course. Silencing of coaBC was likewise bactericidal in vivo, whether initiated at infection or during either the acute or chronic stages of infection, confirming that CoaBC is required for M. tuberculosis to grow and persist in mice and arguing against significant CoaBC bypass via transport and assimilation of host-derived pantetheine in this animal model. These results provide convincing genetic validation of CoaBC as a new bactericidal drug target.

Tuberculosis Screening and Targeted Testing of College and University Students

ERIC Educational Resources Information Center

Journal of American College Health, 2011

2011-01-01

Screening and targeted testing for tuberculosis (TB) is a key strategy for controlling and preventing infection on college and university campuses. Early detection provides an opportunity to promote the health of affected individuals through prompt diagnosis and treatment while preventing potential spread to others. Implementation of a screening…

Defining the needs for next generation assays for tuberculosis.

PubMed

Denkinger, Claudia M; Kik, Sandra V; Cirillo, Daniela Maria; Casenghi, Martina; Shinnick, Thomas; Weyer, Karin; Gilpin, Chris; Boehme, Catharina C; Schito, Marco; Kimerling, Michael; Pai, Madhukar

2015-04-01

To accelerate the fight against tuberculosis, major diagnostic challenges need to be addressed urgently. Post-2015 targets are unlikely to be met without the use of novel diagnostics that are more accurate and can be used closer to where patients first seek care in affordable diagnostic algorithms. This article describes the efforts by the stakeholder community that led to the identification of the high-priority diagnostic needs in tuberculosis. Subsequently target product profiles for the high-priority diagnostic needs were developed and reviewed in a World Health Organization (WHO)-led consensus meeting. The high-priority diagnostic needs included (1) a sputum-based replacement test for smear-microscopy; (2) a non-sputum-based biomarker test for all forms of tuberculosis, ideally suitable for use at levels below microscopy centers; (3) a simple, low cost triage test for use by first-contact care providers as a rule-out test, ideally suitable for use by community health workers; and (4) a rapid drug susceptibility test for use at the microscopy center level. The developed target product profiles, along with complimentary work presented in this supplement, will help to facilitate the interaction between the tuberculosis community and the diagnostics industry with the goal to lead the way toward the post-2015 global tuberculosis targets. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Impact of a Daily SMS Medication Reminder System on Tuberculosis Treatment Outcomes: A Randomized Controlled Trial.

PubMed

Mohammed, Shama; Glennerster, Rachel; Khan, Aamir J

2016-01-01

The rapid uptake of mobile phones in low and middle-income countries over the past decade has provided public health programs unprecedented access to patients. While programs have used text messages to improve medication adherence, there have been no high-powered trials evaluating their impact on tuberculosis treatment outcomes. To measure the impact of Zindagi SMS, a two-way SMS reminder system, on treatment success of people with drug-sensitive tuberculosis. We conducted a two-arm, parallel design, effectiveness randomized controlled trial in Karachi, Pakistan. Individual participants were randomized to either Zindagi SMS or the control group. Zindagi SMS sent daily SMS reminders to participants and asked them to respond through SMS or missed (unbilled) calls after taking their medication. Non-respondents were sent up to three reminders a day. Public and private sector tuberculosis clinics in Karachi, Pakistan. Newly-diagnosed patients with smear or bacteriologically positive pulmonary tuberculosis who were on treatment for less than two weeks; 15 years of age or older; reported having access to a mobile phone; and intended to live in Karachi throughout treatment were eligible to participate. We enrolled 2,207 participants, with 1,110 randomized to Zindagi SMS and 1,097 to the control group. The primary outcome was clinically recorded treatment success based upon intention-to-treat. We found no significant difference between the Zindagi SMS or control groups for treatment success (719 or 83% vs. 903 or 83%, respectively, p = 0·782). There was no significant program effect on self-reported medication adherence reported during unannounced visits during treatment. In this large-scale randomized controlled effectiveness trial of SMS medication reminders for tuberculosis treatment, we found no significant impact. The trial was registered with ClinicalTrials.gov, NCT01690754.

Structural and functional features of enzymes of Mycobacterium tuberculosis peptidoglycan biosynthesis as targets for drug development

PubMed Central

Moraes, Gleiciane Leal; Gomes, Guelber Cardoso; de Sousa, Paulo Robson Monteiro; Alves, Cláudio Nahum; Govender, Thavendran; Kruger, Hendrik G.; Maguire, Glenn E.M.; Lamichhane, Gyanu; Lameira, Jerônimo

2015-01-01

SUMMARY Tuberculosis (TB) is the second leading cause of human mortality from infectious diseases worldwide. The WHO reported 1.3 million deaths and 8.6 million new cases of TB in 2012. Mycobacterium tuberculosis (M. tuberculosis), the infectious bacteria that causes TB, is encapsulated by a thick and robust cell wall. The innermost segment of the cell wall is comprised of peptidoglycan, a layer that is required for survival and growth of the pathogen. Enzymes that catalyse biosynthesis of the peptidoglycan are essential and are therefore attractive targets for discovery of novel antibiotics as humans lack similar enzymes making it possible to selectively target bacteria only. In this paper, we have reviewed the structures and functions of enzymes GlmS, GlmM, GlmU, MurA, MurB, MurC, MurD, MurE and MurF from M. tuberculosis that are involved in peptidoglycan biosynthesis. In addition, we report homology modelled 3D structures of those key enzymes from M. tuberculosis of which the structures are still unknown. We demonstrated that natural substrates can be successfully docked into the active sites of the GlmS and GlmU respectively. It is therefore expected that the models and the data provided herein will facilitate translational research to develop new drugs to treat TB. PMID:25701501

Targeting Drug-Sensitive and -Resistant Strains of Mycobacterium tuberculosis by Inhibition of Src Family Kinases Lowers Disease Burden and Pathology.

PubMed

Chandra, Pallavi; Rajmani, R S; Verma, Garima; Bhavesh, Neel Sarovar; Kumar, Dhiraj

2016-01-01

In view of emerging drug resistance among bacterial pathogens, including Mycobacterium tuberculosis, the development of novel therapeutic strategies is increasingly being sought. A recent paradigm in antituberculosis (anti-TB) drug development is to target the host molecules that are crucial for intracellular survival of the pathogen. We previously showed the importance of Src tyrosine kinases in mycobacterial pathogenesis. Here, we report that inhibition of Src significantly reduced survival of H37Rv as well as multidrug-resistant (MDR) and extremely drug-resistant (XDR) strains of M. tuberculosis in THP-1 macrophages. Src inhibition was also effective in controlling M. tuberculosis infection in guinea pigs. In guinea pigs, reduced M. tuberculosis burden due to Src inhibition also led to a marked decline in the disease pathology. In agreement with the theoretical framework of host-directed approaches against the pathogen, Src inhibition was equally effective against an XDR strain in controlling infection in guinea pigs. We propose that Src inhibitors could be developed into effective host-directed anti-TB drugs, which could be indiscriminately used against both drug-sensitive and drug-resistant strains of M. tuberculosis. IMPORTANCE The existing treatment regimen for tuberculosis (TB) suffers from deficiencies like high doses of antibiotics, long treatment duration, and inability to kill persistent populations in an efficient manner. Together, these contribute to the emergence of drug-resistant tuberculosis. Recently, several host factors were identified which help intracellular survival of Mycobacterium tuberculosis within the macrophage. These factors serve as attractive targets for developing alternate therapeutic strategies against M. tuberculosis. This strategy promises to be effective against drug-resistant strains. The approach also has potential to considerably lower the risk of emergence of new drug-resistant strains. We explored tyrosine kinase Src as a host factor exploited by virulent M. tuberculosis for intracellular survival. We show that Src inhibition can effectively control tuberculosis in infected guinea pigs. Moreover, Src inhibition ameliorated TB-associated pathology in guinea pigs. Thus, Src inhibitors have strong potential to be developed as possible anti-TB drugs.

Socioeconomic Factors Associated with Knowledge on Tuberculosis among Adults in Ethiopia.

PubMed

Gelaw, Sifrash Meseret

2016-01-01

Background. Ethiopia is among highly tuberculosis affected countries. This might be related to low level of awareness on the disease in the population. The objective of the study was to determine the level of tuberculosis knowledge and socioeconomic factors associated with it. Methods. The 2011 Ethiopia health and demographic survey data were used. Overall tuberculosis knowledge score was computed to evaluate the outcome variable. Multivariable logistic regression was employed to identify independent socioeconomic factors associated with low tuberculosis knowledge. Results. The overall tuberculosis knowledge was low, 44.05% (95% CI: 42.05-46.24%) among women and 32.3% (95% CI: 30.34-34.32%) among men. Rural women (AOR = 1.22) and youth, no formal education (women: AOR = 3.28, men: AOR = 7.42), attending only primary education (women: AOR = 1.95, men: AOR = 3.49), lowest wealth quintiles (women: AOR = 1.4, Men: AOR = 1.28), unskilled female manual workers (AOR = 4.15), female agricultural employee (AOR = 2.28), and lack of access to media (women: AOR = 1.52, men: AOR = 1.71) are significantly associated with low tuberculosis knowledge. Conclusion. The level of tuberculosis knowledge among adults in Ethiopia is low and varied by socioeconomic groups. Tuberculosis control programs should consider appropriate strategies for tuberculosis education, promotion, communication, and social mobilization to address the rural women, youths, the poor, less educated people, and unskilled workers.

Radiolabelling and positron emission tomography of PT70, a time-dependent inhibitor of InhA, the Mycobacterium tuberculosis enoyl-ACP reductase

DOE PAGES

Wang, Hui; Liu, Li; Lu, Yang; ...

2015-07-14

PT70 is a diaryl ether inhibitor of InhA, the enoyl-ACP reductase in the Mycobacterium tuberculosis fatty acid biosynthesis pathway. It has a residence time of 24 min on the target, and also shows antibacterial activity in a mouse model of tuberculosis infection. Due to the interest in studying target tissue pharmacokinetics of PT70, we developed a method to radiolabel PT70 with carbon-11 and have studied its pharmacokinetics in mice and baboons using positron emission tomography.

Radiolabelling and positron emission tomography of PT70, a time-dependent inhibitor of InhA, the Mycobacterium tuberculosis enoyl-ACP reductase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Hui; Liu, Li; Lu, Yang

PT70 is a diaryl ether inhibitor of InhA, the enoyl-ACP reductase in the Mycobacterium tuberculosis fatty acid biosynthesis pathway. It has a residence time of 24 min on the target, and also shows antibacterial activity in a mouse model of tuberculosis infection. Due to the interest in studying target tissue pharmacokinetics of PT70, we developed a method to radiolabel PT70 with carbon-11 and have studied its pharmacokinetics in mice and baboons using positron emission tomography.

Cholesterol catabolism as a therapeutic target in Mycobacterium tuberculosis

PubMed Central

Ouellet, Hugues; Johnston, Jonathan B.; Ortiz de Montellano, Paul R.

2011-01-01

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that infects 10 million worldwide and kills 2 million people every year. The uptake and utilization of nutrients by Mtb within the host cell is still poorly understood, although lipids play an important role in Mtb persistence. The recent identification of a large regulon of cholesterol catabolic genes suggests that Mtb can use host sterol for infection and persistence. In this review, we report on recent progress in elucidation of the Mtb cholesterol catabolic reactions and their potential utility as targets for tuberculosis therapeutic agents. PMID:21924910

Drug discovery in tuberculosis. New drug targets and antimycobacterial agents.

PubMed

Campaniço, André; Moreira, Rui; Lopes, Francisca

2018-04-25

Tuberculosis (TB) remains a major health problem worldwide. The infectious agent, Mycobacterium tuberculosis, has a unique ability to survive within the host, alternating between active and latent disease states, and escaping the immune system defences. The extended duration of anti-TB regimens and the increasing prevalence of multidrug- (MDR) and extensively drug-resistant (XDR) M. tuberculosis strains have created an urgent need for new antibiotics active against drug-resistant organisms and that can shorten standard therapy. However, despite success in identifying active compounds through phenotypic screens, the conversion of hits into novel chemical series and ultimately into clinical candidates is hampered by the poor efficacy in eliminating M. tuberculosis within different host compartments, including macrophages, as well as a lack of knowledge about the specific target(s) inhibited and/or upregulated. The current status of anti-TB lead generation has much improved over the last decade, as exemplified by the recent approval of bedaquiline and delamanid to treat MDR-TB and XDR-TB. This review provides a critical analysis on the strategies used to progress hit compounds into viable lead candidates, and how emerging targets may play a role in TB drug discovery in the near future. Four new relevant targets are addressed: the enoyl-acyl carrier protein reductase, InhA; the transmembrane transport protein large, MmpL3; the decaprenylphospho-beta-d-ribofuranose 2-oxidase, DprE1; and the ubiquinol-cytochrome C reductase, QcrB. Validated hit compounds for each target are presented and explored, and the medicinal chemistry strategies to expand SAR around novel chemotypes analyzed. In addition, very recent emerging targets are also discussed. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Nano-biosensing approaches on tuberculosis: Defy of aptamers.

PubMed

Golichenari, Behrouz; Nosrati, Rahim; Farokhi-Fard, Aref; Abnous, Khalil; Vaziri, Farzam; Behravan, Javad

2018-06-11

Tuberculosis is a major global health problem caused by the bacterium Mycobacterium tuberculosis (Mtb) complex. According to WHO reports, 53 million TB patients died from 2000 to 2016. Therefore, early diagnosis of the disease is of great importance for global health care programs. The restrictions of traditional methods have encouraged the development of innovative methods for rapid, reliable, and cost-effective diagnosis of tuberculosis. In recent years, aptamer-based biosensors or aptasensors have drawn great attention to sensitive and accessible detection of tuberculosis. Aptamers are small short single-stranded molecules of DNA or RNA that fold to a unique form and bind to targets. Once combined with nanomaterials, nano-scale aptasensors provide powerful analytical platforms for diagnosing of tuberculosis. Various groups designed and studied aptamers specific for the whole cells of M. tuberculosis, mycobacterial proteins and IFN-γ for early diagnosis of TB. Advantages such as high specificity and strong affinity, potential for binding to a larger variety of targets, increased stability, lower costs of synthesis and storage requirements, and lower probability of contamination make aptasensors pivotal alternatives for future TB diagnostics. In recent years, the concept of SOMAmer has opened new horizons in high precision detection of tuberculosis biomarkers. This review article provides a description of the research progresses of aptamer-based and SOMAmer-based biosensors and nanobiosensors for the detection of tuberculosis. Copyright © 2018 Elsevier B.V. All rights reserved.

Tuberculosis Diagnostics in 2015: Landscape, Priorities, Needs, and Prospects

PubMed Central

Pai, Madhukar; Schito, Marco

2015-01-01

In 2015, tuberculosis remains a major global health problem, and drug-resistant tuberculosis is a growing threat. Although tuberculosis diagnosis in many countries is still reliant on older tools, new diagnostics are changing the landscape. Stimulated, in part, by the success and roll out of Xpert MTB/RIF, there is now considerable interest in new technologies. The landscape looks promising, with a robust pipeline of new tools, particularly molecular diagnostics, and well over 50 companies actively engaged in product development. However, new diagnostics are yet to reach scale, and there needs to be greater convergence between diagnostics development and development of shorter-duration tuberculosis drug regimens. Another concern is the relative absence of non–sputum-based diagnostics in the pipeline for children and of biomarker tests for triage, cure, and progression of latent Mycobacterium tuberculosis infection. Several initiatives, described in this supplement, have been launched to further stimulate product development and policy, including assessment of needs and priorities, development of target product profiles, compilation of data on resistance-associated mutations, and assessment of market size and potential for new diagnostics. Advocacy is needed to increase funding for tuberculosis research and development, and governments in high-burden countries must invest more in tuberculosis control to meet post-2015 targets for care, control, and prevention. PMID:25765103

Diagnostic Performance of Tuberculosis-Specific IgG Antibody Profiles in Patients with Presumptive Tuberculosis from Two Continents.

PubMed

Broger, Tobias; Basu Roy, Robindra; Filomena, Angela; Greef, Charles H; Rimmele, Stefanie; Havumaki, Joshua; Danks, David; Schneiderhan-Marra, Nicole; Gray, Christen M; Singh, Mahavir; Rosenkrands, Ida; Andersen, Peter; Husar, Gregory M; Joos, Thomas O; Gennaro, Maria L; Lochhead, Michael J; Denkinger, Claudia M; Perkins, Mark D

2017-04-01

Development of rapid diagnostic tests for tuberculosis is a global priority. A whole proteome screen identified Mycobacterium tuberculosis antigens associated with serological responses in tuberculosis patients. We used World Health Organization (WHO) target product profile (TPP) criteria for a detection test and triage test to evaluate these antigens. Consecutive patients presenting to microscopy centers and district hospitals in Peru and to outpatient clinics at a tuberculosis reference center in Vietnam were recruited. We tested blood samples from 755 HIV-uninfected adults with presumptive pulmonary tuberculosis to measure IgG antibody responses to 57 M. tuberculosis antigens using a field-based multiplexed serological assay and a 132-antigen bead-based reference assay. We evaluated single antigen performance and models of all possible 3-antigen combinations and multiantigen combinations. Three-antigen and multiantigen models performed similarly and were superior to single antigens. With specificity set at 90% for a detection test, the best sensitivity of a 3-antigen model was 35% (95% confidence interval [CI], 31-40). With sensitivity set at 85% for a triage test, the specificity of the best 3-antigen model was 34% (95% CI, 29-40). The reference assay also did not meet study targets. Antigen performance differed significantly between the study sites for 7/22 of the best-performing antigens. Although M. tuberculosis antigens were recognized by the IgG response during tuberculosis, no single antigen or multiantigen set performance approached WHO TPP criteria for clinical utility among HIV-uninfected adults with presumed tuberculosis in high-volume, urban settings in tuberculosis-endemic countries. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.

Associations between national tuberculosis program budgets and tuberculosis outcomes: an ecological study.

PubMed

Chapple, Will; Katz, Alan Roy; Li, Dongmei

2012-01-01

The objective of this study is to explore the associations between national tuberculosis program (NTP) budget allocation and tuberculosis related outcomes in the World Health Organization's 22 high burden countries from 2007-2009. This ecological study used mixed effects and generalized estimating equation models to identify independent associations between NTP budget allocations and various tuberculosis related outcomes. Models were adjusted for a number of independent variables previously noted to be associated with tuberculosis incidence. Increasing the percent of the NTP budget for advocacy, communication and social mobilization was associated with an increase in the case detection rate. Increasing TB-HIV funding was associated with an increase in HIV testing among TB patients. Increasing the percent of the population covered by the Directly Observed Therapy (DOT) program was associated with an increase in drug susceptibility testing. Laboratory funding was positively associated with tuberculosis notification. Increasing the budgets for first line drugs, management and multi-drug resistant tuberculosis (MDR-TB) was associated with a decrease in smear positive deaths. Effective TB control is a complex and multifaceted challenge. This study revealed a number of budget allocation related factors associated with improved TB outcome parameters. If confirmed with future longitudinal studies, these findings could help guide NTP managers with allocation decisions.

Associations between national tuberculosis program budgets and tuberculosis outcomes: an ecological study

PubMed Central

Chapple, Will; Katz, Alan Roy; Li, Dongmei

2012-01-01

Introduction The objective of this study is to explore the associations between national tuberculosis program (NTP) budget allocation and tuberculosis related outcomes in the World Health Organization's 22 high burden countries from 2007–2009. Methods This ecological study used mixed effects and generalized estimating equation models to identify independent associations between NTP budget allocations and various tuberculosis related outcomes. Models were adjusted for a number of independent variables previously noted to be associated with tuberculosis incidence. Results Increasing the percent of the NTP budget for advocacy, communication and social mobilization was associated with an increase in the case detection rate. Increasing TB-HIV funding was associated with an increase in HIV testing among TB patients. Increasing the percent of the population covered by the Directly Observed Therapy (DOT) program was associated with an increase in drug susceptibility testing. Laboratory funding was positively associated with tuberculosis notification. Increasing the budgets for first line drugs, management and multi-drug resistant tuberculosis (MDR-TB) was associated with a decrease in smear positive deaths. Conclusion Effective TB control is a complex and multifaceted challenge. This study revealed a number of budget allocation related factors associated with improved TB outcome parameters. If confirmed with future longitudinal studies, these findings could help guide NTP managers with allocation decisions. PMID:23024825

Novel inhibitors of Mycobacterium tuberculosis growth based on modified pyrimidine nucleosides and their analogues

NASA Astrophysics Data System (ADS)

Shmalenyuk, E. R.; Kochetkov, S. N.; Alexandrova, L. A.

2013-09-01

The review summarizes data on the synthesis and antituberculosis activity of pyrimidine nucleoside derivatives and their analogues. Enzymes from M. tuberculosis as promising targets for prototypes of new-generation drugs are considered. Nucleosides as inhibitors of drug-resistant M. tuberculosis strains are characterized. The bibliography includes 101 references.

Structural and functional features of enzymes of Mycobacterium tuberculosis peptidoglycan biosynthesis as targets for drug development.

PubMed

Moraes, Gleiciane Leal; Gomes, Guelber Cardoso; Monteiro de Sousa, Paulo Robson; Alves, Cláudio Nahum; Govender, Thavendran; Kruger, Hendrik G; Maguire, Glenn E M; Lamichhane, Gyanu; Lameira, Jerônimo

2015-03-01

Tuberculosis (TB) is the second leading cause of human mortality from infectious diseases worldwide. The WHO reported 1.3 million deaths and 8.6 million new cases of TB in 2012. Mycobacterium tuberculosis (M. tuberculosis), the infectious bacteria that causes TB, is encapsulated by a thick and robust cell wall. The innermost segment of the cell wall is comprised of peptidoglycan, a layer that is required for survival and growth of the pathogen. Enzymes that catalyse biosynthesis of the peptidoglycan are essential and are therefore attractive targets for discovery of novel antibiotics as humans lack similar enzymes making it possible to selectively target bacteria only. In this paper, we have reviewed the structures and functions of enzymes GlmS, GlmM, GlmU, MurA, MurB, MurC, MurD, MurE and MurF from M. tuberculosis that are involved in peptidoglycan biosynthesis. In addition, we report homology modelled 3D structures of those key enzymes from M. tuberculosis of which the structures are still unknown. We demonstrated that natural substrates can be successfully docked into the active sites of the GlmS and GlmU respectively. It is therefore expected that the models and the data provided herein will facilitate translational research to develop new drugs to treat TB. Copyright © 2015. Published by Elsevier Ltd.

Novel Inhibitors of Mycobacterium tuberculosis dTDP-6-deoxy-L-lyxo-4-hexulose Reductase (RmlD) Identified by Virtual Screening

PubMed Central

Wang, Yi; Hess, Tamara Noelle; Jones, Victoria; Zhou, Joe Zhongxiang; McNeil, Michael R.; McCammon, J. Andrew

2011-01-01

The complex and highly impermeable cell wall of Mycobacterium tuberculosis (Mtb) is largely responsible for the ability of the mycobacterium to resist the action of chemical therapeutics. An L-rhamnosyl residue, which occupies an important anchoring position in the Mtb cell wall, is an attractive target for novel anti-tuberculosis drugs. In this work, we report a virtual screening (VS) study targeting Mtb dTDP-deoxy-L-lyxo-4-hexulose reductase (RmlD), the last enzyme in the L-rhamnosyl synthesis pathway. Through two rounds of VS, we have identified four RmlD inhibitors with half inhibitory concentrations of 0.9-25 μM, and whole-cell minimum inhibitory concentrations of 20-200 μg/ml. Compared with our previous high throughput screening targeting another enzyme involved in L-rhamnosyl synthesis, virtual screening produced higher hit rates, supporting the use of computational methods in future anti-tuberculosis drug discovery efforts. PMID:22014548

Latent Tuberculosis Infection in an Urban Cohort: Screening and Treatment for Latent TB in an Urban Setting

PubMed Central

Morano, Jamie P.; Walton, Mary R.; Zelenev, Alexei; Bruce, R. Douglas; Altice, Frederick L.

2013-01-01

Background Despite its benefit for treating active tuberculosis, directly observed therapy (DOT) for latent tuberculosis infection (LTBI) has been largely understudied among challenging inner city populations. Methods Utilizing questionnaire data from a comprehensive mobile healthcare clinic in New Haven, CT from 2003 to July 2011, a total of 2523 completed tuberculin skin tests (TST’s) resulted in 357 new LTBIs. Multivariate logistic regression correlated covariates of the two outcomes 1) initiation of isoniazid preventative therapy (IPT) and 2) completion of 9-months IPT. Results Of 357 new LTBIs, 86.3% (n=308) completed screening CXRs: 90.3% (n=278) were normal and 0.3% (n=1) with active tuberculosis. Of those completing CXR screening, 44.0% (n=135) agreed to IPT: 69.6% (n=94) selected DOT, and 30.4% (n=41) selected SAT. Initiating IPT was correlated with undocumented status (AOR=3.43; p<0.001) and being born in a country of highest and third highest tuberculosis prevalence (AOR=14.09; p=0.017 and AOR=2.25; p=0.005, respectively). Those selecting DOT were more likely to be Hispanic (83.0% vs 53.7%; p<0.0001), undocumented (57.4% vs 41.5%; p=0.012), have stable housing (p=0.002), employed (p<0.0001), uninsured (p=0.014), no prior cocaine or crack use (p=0.013) and no recent incarceration (p=0.001). Completing 9-months of IPT was correlated with no recent incarceration (AOR 5.95; p=0.036) and younger age (AOR 1.03; p=0.031). SAT and DOT participants did not significantly differ for IPT duration (6.54 vs 5.68 months; p=0.216) nor 9-month completion (59.8% vs 46.3%; p=0.155). Conclusions In an urban mobile healthcare sample, screening completion for LTBI was high with nearly half initiating IPT. Undocumented, Hispanic immigrants from high prevalence tuberculosis countries were more likely to self-select DOT at the mobile outreach clinic, potentially because of more culturally, linguistically, and logistically accessible services and self-selection optimization phenomena (SSOP). Within a diverse, urban environment, DOT and SAT IPT models for LTBI treatment resulted in similar outcomes, yet outcomes were hampered by differential measurement bias between DOT and SAT participants. PMID:23728822

Urine colorimetry for therapeutic drug monitoring of pyrazinamide during tuberculosis treatment.

PubMed

Zentner, Isaac; Modongo, Chawangwa; Zetola, Nicola M; Pasipanodya, Jotam G; Srivastava, Shashikant; Heysell, Scott K; Mpagama, Stellah; Schlect, Hans P; Gumbo, Tawanda; Bisson, Gregory P; Vinnard, Christopher

2018-03-01

Pyrazinamide is a key drug in the first-line treatment regimen for tuberculosis, with a potent sterilizing effect. Although low pyrazinamide peak serum concentrations (C max ) are associated with poor treatment outcomes, many resource-constrained settings do not have sufficient laboratory capacity to support therapeutic drug monitoring (TDM). The objective of this study was to determine whether a colorimetric test of urine can identify tuberculosis patients with adequate pyrazinamide exposures, as defined by serum C max above a target threshold. In the derivation study of healthy volunteers, three dose sizes of pyrazinamide were evaluated, and intensive pharmacokinetic blood sampling was performed over an 8-h period, with a timed urine void at 4h post-dosing. Pyrazinamide in urine was isolated by spin column centrifugation with an exchange resin, followed by colorimetric analysis; the absorbance peak at 495nm was measured. The urine assay was then evaluated in a study of 39 HIV/tuberculosis patients in Botswana enrolled in an intensive pharmacokinetic study. Receiver operating characteristics (ROC) curves were used to measure diagnostic accuracy. The guideline-recommended pyrazinamide serum C max target of 35mg/l was evaluated in the primary analysis; this target was found to be predictive of favorable outcomes in a clinical study. Following this, a higher serum C max target of 58mg/l was evaluated in the secondary analysis. At the optimal cut-off identified in the derivation sample, the urine colorimetric assay was 97% sensitive and 50% specific to identify 35 of 39 HIV/tuberculosis patients with pharmacokinetic target attainment, with an area under the ROC curve of 0.81 (95% confidence interval 0.60-0.97). Diagnostic accuracy was lower at the 58mg/l serum C max target, with an area under the ROC curve of 0.68 (95% confidence interval 0.48-0.84). Men were less likely than women to attain either serum pharmacokinetic target. The urine colorimetric assay was sensitive but not specific for the detection of adequate pyrazinamide pharmacokinetic exposures among HIV/tuberculosis patients in a high-burden setting. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Information architecture considerations in designing a comprehensive tuberculosis enterprise system in Western Kenya.

PubMed

Gichoya, Judy; Pearce, Chris; Wickramasinghe, Nilmini

2013-01-01

Kenya ranks among the twenty-two countries that collectively contribute about 80% of the world's Tuberculosis cases; with a 50-200 fold increased risk of tuberculosis in HIV infected persons versus non-HIV hosts. Contemporaneously, there is an increase in mobile penetration and its use to support healthcare throughout Africa. Many are skeptical that such m-health solutions are unsustainable and not scalable. We seek to design a scalable, pervasive m-health solution for Tuberculosis care to become a use case for sustainable and scalable health IT in limited resource settings. We combine agile design principles and user-centered design to develop the architecture needed for this initiative. Furthermore, the architecture runs on multiple devices integrated to deliver functionality critical for successful Health IT implementation in limited resource settings. It is anticipated that once fully implemented, the proposed m-health solution will facilitate superior monitoring and management of Tuberculosis and thereby reduce the alarming statistic regarding this disease in this region.

Health information needs of professional nurses required at the point of care.

PubMed

Ricks, Esmeralda; ten Ham, Wilma

2015-06-11

Professional nurses work in dynamic environments and need to keep up to date with relevant information for practice in nursing to render quality patient care. Keeping up to date with current information is often challenging because of heavy workload, diverse information needs and the accessibility of the required information at the point of care. The aim of the study was to explore and describe the information needs of professional nurses at the point of care in order to make recommendations to stakeholders to develop a mobile library accessible by means of smart phones when needed. The researcher utilised a quantitative, descriptive survey design to conduct this study. The target population comprised 757 professional nurses employed at a state hospital. Simple random sampling was used to select a sample of the wards, units and departments for inclusion in the study. A convenience sample of 250 participants was selected. Two hundred and fifty structured self-administered questionnaires were distributed amongst the participants. Descriptive statistics were used to analyse the data. A total of 136 completed questionnaires were returned. The findings highlighted the types and accessible sources of information. Information needs of professional nurses were identified such as: extremely drug-resistant tuberculosis, multi-drug-resistant tuberculosis, HIV, antiretrovirals and all chronic lifestyle diseases. This study has enabled the researcher to identify the information needs required by professional nurses at the point of care to enhance the delivery of patient care. The research results were used to develop a mobile library that could be accessed by professional nurses.

Scaling up interventions to achieve global tuberculosis control: progress and new developments.

PubMed

Raviglione, Mario; Marais, Ben; Floyd, Katherine; Lönnroth, Knut; Getahun, Haileyesus; Migliori, Giovanni B; Harries, Anthony D; Nunn, Paul; Lienhardt, Christian; Graham, Steve; Chakaya, Jeremiah; Weyer, Karin; Cole, Stewart; Kaufmann, Stefan H E; Zumla, Alimuddin

2012-05-19

Tuberculosis is still one of the most important causes of death worldwide. The 2010 Lancet tuberculosis series provided a comprehensive overview of global control efforts and challenges. In this update we review recent progress. With improved control efforts, the world and most regions are on track to achieve the Millennium Development Goal of decreasing tuberculosis incidence by 2015, and the Stop TB Partnership target of halving 1990 mortality rates by 2015; the exception is Africa. Despite these advances, full scale-up of tuberculosis and HIV collaborative activities remains challenging and emerging drug-resistant tuberculosis is a major threat. Recognition of the effect that non-communicable diseases--such as smoking-related lung disease, diet-related diabetes mellitus, and alcohol and drug misuse--have on individual vulnerability, as well as the contribution of poor living conditions to community vulnerability, shows the need for multidisciplinary approaches. Several new diagnostic tests are being introduced in endemic countries and for the first time in 40 years a coordinated portfolio of promising new tuberculosis drugs exists. However, none of these advances offer easy solutions. Achievement of international tuberculosis control targets and maintenance of these gains needs optimum national health policies and services, with ongoing investment into new approaches and strategies. Despite growing funding in recent years, a serious shortfall persists. International and national financial uncertainty places gains at serious risk. Perseverance and renewed commitment are needed to achieve global control of tuberculosis, and ultimately, its elimination. Copyright © 2012 Elsevier Ltd. All rights reserved.

In-silico Metabolome Target Analysis Towards PanC-based Antimycobacterial Agent Discovery.

PubMed

Khoshkholgh-Sima, Baharak; Sardari, Soroush; Izadi Mobarakeh, Jalal; Khavari-Nejad, Ramezan Ali

2015-01-01

Mycobacterium tuberculosis, the main cause of tuberculosis (TB), has still remained a global health crisis especially in developing countries. Tuberculosis treatment is a laborious and lengthy process with high risk of noncompliance, cytotoxicity adverse events and drug resistance in patient. Recently, there has been an alarming rise of drug resistant in TB. In this regard, it is an unmet need to develop novel antitubercular medicines that target new or more effective biochemical pathways to prevent drug resistant Mycobacterium. Integrated study of metabolic pathways through in-silico approach played a key role in antimycobacterial design process in this study. Our results suggest that pantothenate synthetase (PanC), anthranilate phosphoribosyl transferase (TrpD) and 3-isopropylmalate dehydratase (LeuD) might be appropriate drug targets. In the next step, in-silico ligand analysis was used for more detailed study of chemical tractability of targets. This was helpful to identify pantothenate synthetase (PanC, Rv3602c) as the best target for antimycobacterial design procedure. Virtual library screening on the best ligand of PanC was then performed for inhibitory ligand design. At the end, five chemical intermediates showed significant inhibition of Mycobacterium bovis with good selectivity indices (SI) ≥10 according to Tuberculosis Antimicrobial Acquisition & Coordinating Facility of US criteria for antimycobacterial screening programs.

Lead selection and characterization of antitubercular compounds using the Nested Chemical Library.

PubMed

Sipos, Anna; Pató, János; Székely, Rita; Hartkoorn, Ruben C; Kékesi, László; Őrfi, László; Szántai-Kis, Csaba; Mikušová, Katarína; Svetlíková, Zuzana; Korduláková, Jana; Nagaraja, Valakunja; Godbole, Adwait Anand; Bush, Natassja; Collin, Frédéric; Maxwell, Anthony; Cole, Stewart T; Kéri, György

2015-06-01

Discovering new drugs to treat tuberculosis more efficiently and to overcome multidrug resistance is a world health priority. To find novel antitubercular agents several approaches have been used in various institutions worldwide, including target-based approaches against several validated mycobacterial enzymes and phenotypic screens. We screened more than 17,000 compounds from Vichem's Nested Chemical Library™ using an integrated strategy involving whole cell-based assays with Corynebacterium glutamicum and Mycobacterium tuberculosis, and target-based assays with protein kinases PknA, PknB and PknG as well as other targets such as PimA and bacterial topoisomerases simultaneously. With the help of the target-based approach we have found very potent hits inhibiting the selected target enzymes, but good minimal inhibitory concentrations (MIC) against M. tuberculosis were not achieved. Focussing on the whole cell-based approach several potent hits were found which displayed minimal inhibitory concentrations (MIC) against M. tuberculosis below 10 μM and were non-mutagenic, non-cytotoxic and the targets of some of the hits were also identified. The most active hits represented various scaffolds. Medicinal chemistry-based lead optimization was performed applying various strategies and, as a consequence, a series of novel potent compounds were synthesized. These efforts resulted in some effective potential antitubercular lead compounds which were confirmed in phenotypic assays. Copyright © 2015 Elsevier Ltd. All rights reserved.

The Role of High Mobility Group Box 1 Protein (HMGB1) in the Immunopathology of Experimental Pulmonary Tuberculosis.

PubMed

Hernández-Pando, Rogelio; Barrios-Payán, Jorge; Mata-Espinosa, Dulce; Marquina-Castillo, Brenda; Hernández-Ramírez, Diego; Bottasso, Oscar Adelmo; Botasso, Oscar Adelmo; Bini, Estela Isabel

2015-01-01

The high mobility group box 1 (HMGB1) is the prototype of alarmin protein released by stressed or dying cells. The redox state of this protein confers different functions in the regulation of inflammation and immune response. Determine the kinetics, cellular sources and function of HMGB1 in experimental tuberculosis. BALB/c mice were infected with Mycobacterium tuberculosis strain H37Rv. At different time points, HMGB1 was quantified in bronchial lavage fluid (BALF) and in lungs was determined its cellular sources by immunohistochemistry. HMGB1 was blocked with specific antibodies or recombinant HMGB1 was administered during early or late infection. Bacilli burdens, inflammation and cytokines expression were determined. The maximal concentration of HMGB1 in BALF was at day one of infection. Bronchial epithelium and macrophages were the most important sources. At day 7 to 21 the oxidized HMGB1 was predominant, while during late infection only the reduced form was seen. Blocking HMGB1 during early infection produced significant decrease of bacilli burdens and high production of pro-inflammatory cytokines, while the opposite was seen when HMGB1 was administered. Blocking HMGB1 activity or administrated it in high amounts during late infection worsening the disease. HMGB1 is liberated during experimental tuberculosis and promotes or suppress the immune response and inflammation depending on the redox state.

Establishment of a Community Advisory Board (CAB) for tuberculosis control and research in the Inanda, Ntuzuma and KwaMashu (INK) area of KwaZulu-Natal, South Africa.

PubMed

Ntshanga, Sbongile P; Ngcobo, Paulos S; Mabaso, Musawenkosi L H

2010-05-01

To strengthen community mobilization, education, awareness and involvement in research to improve Tuberculosis (TB) control, by building partnership between communities and the health sector through the establishment of a Community Advisory Board (CAB) in the Inanda, Ntuzuma and KwaMashu (INK) area. Key stakeholders from multiple sectors of the target community were identified. Two workshops were held with the aim of communicating the need for CAB and selecting its members. A 22 member INK CAB with representatives from different sectors in the community was selected. CAB members were trained through a 2-day workshop on TB and research. Various activities were successfully initiated and spearheaded by the INK CAB, and these involved training of Traditional Healers, recruiting of more TB tracer teams, organizing awareness campaigns during TB World Day, establishing TB school peer educators and links with NGOs to support impoverished TB patients all with positive outcomes. The INK CAB is a successful example of providing a mechanism for community consultation and participation that contributes to promoting and facilitating relevant research and TB control activities. Copyright (c) 2009 Elsevier Ireland Ltd. All rights reserved.

Bim is a crucial regulator of apoptosis induced by Mycobacterium tuberculosis

PubMed Central

Aguiló, N; Uranga, S; Marinova, D; Martín, C; Pardo, J

2014-01-01

Mycobacterium tuberculosis, the causative agent of tuberculosis, induces apoptosis in infected macrophages in vitro and in vivo. However, the molecular mechanism controlling this process is not known. In order to study the involvement of the mitochondrial apoptotic pathway in M. tuberculosis-induced apoptosis, we analysed cell death in M. tuberculosis-infected embryonic fibroblasts (MEFs) derived from different knockout mice for genes involved in this route. We found that apoptosis induced by M. tuberculosis is abrogated in the absence of Bak and Bax, caspase 9 or the executioner caspases 3 and 7. Notably, we show that MEF deficient in the BH3-only BCL-2-interacting mediator of cell death (Bim) protein were also resistant to this process. The relevance of these results has been confirmed in the mouse macrophage cell line J774, where cell transfection with siRNA targeting Bim impaired apoptosis induced by virulent mycobacteria. Notably, only infection with a virulent strain, but not with attenuated ESX-1-defective strains, such as Bacillus Calmette-Guerin and live-attenuated M. tuberculosis vaccine strain MTBVAC, induced Bim upregulation and apoptosis, probably implicating virulence factor early secreted antigenic target 6-kDa protein in this process. Our results suggest that Bim upregulation and apoptosis is mediated by the p38MAPK-dependent pathway. Our findings show that Bim is a master regulator of apoptosis induced by M. tuberculosis. PMID:25032866

Predictive modeling targets thymidylate synthase ThyX in Mycobacterium tuberculosis.

PubMed

Djaout, Kamel; Singh, Vinayak; Boum, Yap; Katawera, Victoria; Becker, Hubert F; Bush, Natassja G; Hearnshaw, Stephen J; Pritchard, Jennifer E; Bourbon, Pauline; Madrid, Peter B; Maxwell, Anthony; Mizrahi, Valerie; Myllykallio, Hannu; Ekins, Sean

2016-06-10

There is an urgent need to identify new treatments for tuberculosis (TB), a major infectious disease caused by Mycobacterium tuberculosis (Mtb), which results in 1.5 million deaths each year. We have targeted two essential enzymes in this organism that are promising for antibacterial therapy and reported to be inhibited by naphthoquinones. ThyX is an essential thymidylate synthase that is mechanistically and structurally unrelated to the human enzyme. DNA gyrase is a DNA topoisomerase present in bacteria and plants but not animals. The current study set out to understand the structure-activity relationships of these targets in Mtb using a combination of cheminformatics and in vitro screening. Here, we report the identification of new Mtb ThyX inhibitors, 2-chloro-3-(4-methanesulfonylpiperazin-1-yl)-1,4-dihydronaphthalene-1,4-dione) and idebenone, which show modest whole-cell activity and appear to act, at least in part, by targeting ThyX in Mtb.

Improved Phenoxyalkylbenzimidazoles with Activity against Mycobacterium tuberculosis Appear to Target QcrB

PubMed Central

2017-01-01

The phenoxy alkyl benzimidazoles (PABs) have good antitubercular activity. We expanded our structure–activity relationship studies to determine the core components of PABs required for activity. The most potent compounds had minimum inhibitory concentrations against Mycobacterium tuberculosis in the low nanomolar range with very little cytotoxicity against eukaryotic cells as well as activity against intracellular bacteria. We isolated resistant mutants against PAB compounds, which had mutations in either Rv1339, of unknown function, or qcrB, a component of the cytochrome bc1 oxidase of the electron transport chain. QcrB mutant strains were resistant to all PAB compounds, whereas Rv1339 mutant strains were only resistant to a subset, suggesting that QcrB is the target. The discovery of the target for PAB compounds will allow for the improved design of novel compounds to target intracellular M. tuberculosis. PMID:29035551

Investigating the metabolic capabilities of Mycobacterium tuberculosis H37Rv using the in silico strain iNJ661 and proposing alternative drug targets

PubMed Central

Jamshidi, Neema; Palsson, Bernhard Ø

2007-01-01

Background: Mycobacterium tuberculosis continues to be a major pathogen in the third world, killing almost 2 million people a year by the most recent estimates. Even in industrialized countries, the emergence of multi-drug resistant (MDR) strains of tuberculosis hails the need to develop additional medications for treatment. Many of the drugs used for treatment of tuberculosis target metabolic enzymes. Genome-scale models can be used for analysis, discovery, and as hypothesis generating tools, which will hopefully assist the rational drug development process. These models need to be able to assimilate data from large datasets and analyze them. Results: We completed a bottom up reconstruction of the metabolic network of Mycobacterium tuberculosis H37Rv. This functional in silico bacterium, iNJ661, contains 661 genes and 939 reactions and can produce many of the complex compounds characteristic to tuberculosis, such as mycolic acids and mycocerosates. We grew this bacterium in silico on various media, analyzed the model in the context of multiple high-throughput data sets, and finally we analyzed the network in an 'unbiased' manner by calculating the Hard Coupled Reaction (HCR) sets, groups of reactions that are forced to operate in unison due to mass conservation and connectivity constraints. Conclusion: Although we observed growth rates comparable to experimental observations (doubling times ranging from about 12 to 24 hours) in different media, comparisons of gene essentiality with experimental data were less encouraging (generally about 55%). The reasons for the often conflicting results were multi-fold, including gene expression variability under different conditions and lack of complete biological knowledge. Some of the inconsistencies between in vitro and in silico or in vivo and in silico results highlight specific loci that are worth further experimental investigations. Finally, by considering the HCR sets in the context of known drug targets for tuberculosis treatment we proposed new alternative, but equivalent drug targets. PMID:17555602

Drug resistance mechanisms and novel drug targets for tuberculosis therapy.

PubMed

Islam, Md Mahmudul; Hameed, H M Adnan; Mugweru, Julius; Chhotaray, Chiranjibi; Wang, Changwei; Tan, Yaoju; Liu, Jianxiong; Li, Xinjie; Tan, Shouyong; Ojima, Iwao; Yew, Wing Wai; Nuermberger, Eric; Lamichhane, Gyanu; Zhang, Tianyu

2017-01-20

Drug-resistant tuberculosis (TB) poses a significant challenge to the successful treatment and control of TB worldwide. Resistance to anti-TB drugs has existed since the beginning of the chemotherapy era. New insights into the resistant mechanisms of anti-TB drugs have been provided. Better understanding of drug resistance mechanisms helps in the development of new tools for the rapid diagnosis of drug-resistant TB. There is also a pressing need in the development of new drugs with novel targets to improve the current treatment of TB and to prevent the emergence of drug resistance in Mycobacterium tuberculosis. This review summarizes the anti-TB drug resistance mechanisms, furnishes some possible novel drug targets in the development of new agents for TB therapy and discusses the usefulness using known targets to develop new anti-TB drugs. Whole genome sequencing is currently an advanced technology to uncover drug resistance mechanisms in M. tuberculosis. However, further research is required to unravel the significance of some newly discovered gene mutations in their contribution to drug resistance. Copyright © 2016 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

Investments in tuberculosis research - what are the gaps?

PubMed

Khan, Mishal S; Fletcher, Helen; Coker, Richard

2016-08-25

Through decades of research, numerous studies have generated robust evidence about effective interventions for tuberculosis control. Yet, the global annual decline in incidence of approximately 1 % is evidence that current approaches and investment strategies are not sufficient. In this article, we assess recent tuberculosis research funding and discuss two critical gaps in funding and in scientific evidence from topics that have been left off the research priority agenda.We first examine research and development funding goals in the 2011-2015 Global Plan to Stop Tuberculosis and analyze disbursements to different research areas by funders worldwide in 2014. We then summarize, through a compilation of published literature and consultation with 35 researchers across multiple disciplines in the London School of Hygiene and Tropical Medicine TB Centre, priorities identified by the tuberculosis research community. Finally, we compare researchers' priority areas to the global funding agendas and activities.Our analysis shows that, among the five key research areas defined in the 2011-2015 Global Plan - namely drugs, basic science, vaccines, diagnostics and operational research - drug discovery and basic science on Mycobacterium tuberculosis accounted for 60 % of the $2 billion annual funding target. None of the research areas received the recommended level of funding. Operational research, which had the lowest target, received 66 % of its target funding, whereas new diagnostics received only 19 %. Although many of the priority research questions identified by researchers fell within the Global Plan categories, our analysis highlights important areas that are not explicitly mentioned in the current plan. These priority research areas included improved understanding of tuberculosis transmission dynamics, the role of social protection and social determinants, and health systems and policy research.While research priorities are increasingly important in light of the limited funding for tuberculosis, there is a risk that we neglect important research areas and encourage the formation of research silos. To ensure that funding priorities, researchers' agendas and national tuberculosis control policies are better coordinated, there should be more, and wider, dialogue between stakeholders in high tuberculosis burden countries, researchers, international policymakers and funders.

Proteomic analysis of drug-resistant Mycobacterium tuberculosis by one-dimensional gel electrophoresis and charge chromatography.

PubMed

Yari, Shamsi; Hadizadeh Tasbiti, Alireza; Ghanei, Mostafa; Shokrgozar, Mohammad Ali; Fateh, Abolfazl; Mahdian, Reza; Yari, Fatemeh; Bahrmand, Ahmadreza

2017-01-01

Multidrug-resistant tuberculosis (MDR-TB) is a form of TB caused by Mycobacterium tuberculosis (M. tuberculosis) that do not respond to, at least, isoniazid and rifampicin, the two most powerful, first-line (or standard) anti-TB drugs. Novel intervention strategies for eliminating this disease were based on finding proteins that can be used for designing new drugs or new and reliable kits for diagnosis. The aim of this study was to compare the protein profiles of MDR-TB with sensitive isolates. Proteomic analysis of M. tuberculosis MDR-TB and sensitive isolates was obtained with ion exchange chromatography coupled with MALDI-TOF-TOF (matrix-assisted laser desorption/ionization) in order to identify individual proteins that have different expression in MDR-TB to be used as a drug target or diagnostic marker for designing valuable TB vaccines or TB rapid tests. We identified eight proteins in MDR-TB isolates, and analyses showed that these proteins are absent in M. tuberculosis-sensitive isolates: (Rv2140c, Rv0009, Rv1932, Rv0251c, Rv2558, Rv1284, Rv3699 and MMP major membrane proteins). These data will provide valuable clues in further investigation for suitable TB rapid tests or drug targets against drug-resistant and sensitive M. tuberculosis isolates.

Pili of Mycobacterium tuberculosis: current knowledge and future prospects.

PubMed

Ramsugit, Saiyur; Pillay, Manormoney

2015-08-01

Many pathogenic bacteria express filamentous appendages, termed pili, on their surface. These organelles function in several important bacterial processes, including mediating bacterial interaction with, and colonization of the host, signalling events, locomotion, DNA uptake, electric conductance, and biofilm formation. In the last decade, it has been established that the tuberculosis-causing bacterium, Mycobacterium tuberculosis, produces two pili types: curli and type IV pili. In this paper, we review studies on M. tuberculosis pili, highlighting their structure and biological significance to M. tuberculosis pathogenesis, and discuss their potential as targets for therapeutic intervention and diagnostic test development.

Tracking and Treating Mobile Populations. The TB Net System. Migrant Clinicians Network Monograph Series. = El Sistema de Red para la TB.

ERIC Educational Resources Information Center

Migrant Clinicians Network, Inc., Austin, TX.

A comprehensive tracking and referral network that helps provide continuity of care for mobile populations with active tuberculosis (TB) or TB infection is considered essential for effective treatment of TB. However, the interstate referral system that exists between state health departments has been highly inefficient for serving migrant…

Targeting Mycobacterium tuberculosis nucleoid-associated protein HU with structure-based inhibitors

NASA Astrophysics Data System (ADS)

Bhowmick, Tuhin; Ghosh, Soumitra; Dixit, Karuna; Ganesan, Varsha; Ramagopal, Udupi A.; Dey, Debayan; Sarma, Siddhartha P.; Ramakumar, Suryanarayanarao; Nagaraja, Valakunja

2014-06-01

The nucleoid-associated protein HU plays an important role in maintenance of chromosomal architecture and in global regulation of DNA transactions in bacteria. Although HU is essential for growth in Mycobacterium tuberculosis (Mtb), there have been no reported attempts to perturb HU function with small molecules. Here we report the crystal structure of the N-terminal domain of HU from Mtb. We identify a core region within the HU-DNA interface that can be targeted using stilbene derivatives. These small molecules specifically inhibit HU-DNA binding, disrupt nucleoid architecture and reduce Mtb growth. The stilbene inhibitors induce gene expression changes in Mtb that resemble those induced by HU deficiency. Our results indicate that HU is a potential target for the development of therapies against tuberculosis.

Using mobile phones to ensure that referred tuberculosis patients reach their treatment facilities: a call that makes a difference.

PubMed

Choun, Kimcheng; Achanta, Shanta; Naik, Balaji; Tripathy, Jaya Prasad; Thai, Sopheak; Lorent, Natalie; Khun, Kim Eam; van Griensven, Johan; Kumar, Ajay M V; Zachariah, Rony

2017-08-22

Over the last decade, the availability and use of mobile phones have grown exponentially globally and in Cambodia. In the Sihanouk Hospital Centre of Hope(SHCH) in Cambodia about half of all tuberculosis patients referred out to peripheral health facilities for TB treatment initiation or continuation were lost to contact after referral ranging from 19 to 69% between 2008 and 2013. To address this, we implemented a mobile phone-based patient tracking intervention. Here, we report the number and proportion of referred TB patients who could be contacted through a mobile phone and retained in care after the introduction of mobile phone tracking. A descriptive study involving follow-up of TB patients referred out from SHCH to peripheral health facilities during May-October 2014. Standard operating procedures were used to contact individual patients and/or health facilities using a mobile phone. Among 109 TB patients referred to peripheral health facilities, 107(98%) had access to a mobile phone of whom, 103(97%) could be contacted directly while 5(2%) were contacted through their health care providers. A total of 108(99%) of 109 referred TB patients in intervention period were thus placed on TB treatment. This study provides preliminary, but promising evidence that using mobile phones was accompanied with improved retention of referred TB patients compared to historical cohorts. Given the limitations associated with historical controls, we need better designed studies with larger sample size to strengthen the evidence before national scale-up.

Anti-Tuberculosis Policy of the Government General of Korea during Japanese-Colonial Period (1910-1945): From Simple Restriction to Active Enlightenment.

PubMed

Choi, Eun Kyung

2013-12-01

In this paper, I tried to examine the characteristic of anti-tuberculosis policy in colonial Korea and find out internal constraint of hygienic administration by Japanese government during Japanese-Colonial Period. Despite of high prevalence of tuberculosis among Japanese in Korea, the Japanese Government General of Korea had done almost nothing until 1936. Japan's hygienic administration was highly dependent upon hygienic police, and mainly with compulsory isolation and disinfection. It was inefficient in tuberculosis problem. In 1918, Japanese Government General enacted 'Ordinance of Prevention of Tuberculosis', solely based upon naive tuberculosis etiology in sputum; consisted of simple crackdown and isolation and had no effect due to the limit of anti-tuberculosis and health budget. Also the ordinance actually set limitation upon the tuberculosis facilities, only a few health care facilities could be affordable for tuberculosis patients. Since 1936, the Japanese Government General of Korea began tuberculosis prevention measures in earnest. Due to the Second Sino- Japanese War and World War II, there was urgent need to make Korean society and population as "safe, and healthy rear area". The Government organized 'Chosen Anti-tuberculosis Association' and highly pursued enlightment campaign. It was almost temporary measures of enlightenment and publicity. Also various types of health screening and tuberculosis prevalence research were introduced to Korean people. But it was not so effective to identify tuberculosis problem in Korea. Mass tuberculin test and X-ray test was introduced, but it was not well organized and scientifically designed. Besides, tuberculosis treatment facility was extremely rare because of strict isolation and high standard policy. Japanese Governemtn set numerous tuberculosis-counseling centers and mobilized public doctor for consulting tuberculosis, but the accessibility of centers was very low. Moreover, there was no source to establish facilities like sanatorium. The Japanese Government General of Korea was constantly suffered from limit of budget and a lot of Japanese in Korea had no inherent motive for installing sanatorium and anti-tuberculosis measures. As the result, the effort made by Japanese Government General of Korea to diminish tuberculosis in Korea failed during the wartime.

Identification of shikimate kinase inhibitors among anti-Mycobacterium tuberculosis compounds by LC-MS.

PubMed

Simithy, Johayra; Reeve, Nathaniel; Hobrath, Judith V; Reynolds, Robert C; Calderón, Angela I

2014-03-01

Increasing drug resistance has challenged the control and treatment of tuberculosis, sparking recent interest in finding new antitubercular agents with different chemical scaffolds and mechanisms of action. Mycobacterium tuberculosis shikimate kinase (MtSK), an enzyme present in the shikimate pathway in bacteria, is essential for the survival of the tubercle bacillus, representing an ideal target for therapeutic intervention given its absence in mammals. In this study, a small library of 404 synthetic antimycobacterial compounds identified and supplied through the NIH Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF) high throughput screening program against whole cell M. tuberculosis H37Rv was further screened using a mass spectrometry-based functional assay in order to identify a potential enzymatic target. Fourteen compounds containing an oxadiazole-amide or a 2-aminobenzothiazole core scaffold showed MtSK inhibitory activity at 50 μM, with the lowest giving an IC50 of 1.94 μM. Induced fit docking studies suggested that the scaffolds shared by these compounds fit well in the shikimate binding pocket of MtSK. In summary, we report new early discovery stage lead scaffolds targeting the essential protein MtSK that can be further pursued in a rational drug design program for the discovery of more selective antitubercular drugs. Copyright © 2014 Elsevier Ltd. All rights reserved.

Can penicillins and other beta-lactam antibiotics be used to treat tuberculosis?

PubMed Central

Chambers, H F; Moreau, D; Yajko, D; Miick, C; Wagner, C; Hackbarth, C; Kocagöz, S; Rosenberg, E; Hadley, W K; Nikaido, H

1995-01-01

An increase in the number of tuberculosis cases caused by multiple-drug-resistant strains of Mycobacterium tuberculosis has stimulated search for new antituberculous agents. Beta-lactam antibiotics, traditionally regarded as ineffective against tuberculosis, merit consideration. Four major penicillin-binding proteins (PBPs) with approximate molecular sizes of 94, 82, 52, and 37 kDa were detected by fluorography of [3H]penicillin-radiolabeled membrane proteins prepared from M. tuberculosis H37Ra. The presence of membrane-associated beta-lactamase precluded the use of membranes for assaying the binding affinities of beta-lactam antibiotics. Therefore, ampicillin affinity chromatography was used to purify these four PBPs from crude membranes in order to assay the binding affinities of beta-lactam antibiotics. Ampicillin, amoxicillin, and imipenem, beta-lactam antibiotics previously reported to be active in vitro against M. tuberculosis, bound to M. tuberculosis PBPs at therapeutically achievable concentrations. Binding of the 94-, 82-, and 52-kDa PBPs, but not the 37-kDa PBP, was associated with antibacterial activity, suggesting that these PBPs are the critical targets. Studies of mycobacterial cell wall permeability, which was assayed with a panel of reference cephalosporins and penicillins with different charge positivities, indicated that the rate of penetration of beta-lactam antibiotics to the target PBPs could not account for resistance. Resistance could be reversed with the beta-lactamase inhibitors clavulanate or sulbactam or could be circumvented by the use of a beta-lactamase-stable drug, imipenem, indicating that mycobacterial beta-lactamase, probably in conjunction with slow penetration, is a major determinant of M. tuberculosis resistance to beta-lactam antibiotics. These findings confirm in vitro data that M. tuberculosis is susceptible to some beta-lactam antibiotics. Further evaluation of these drugs for the treatment of tuberculosis in animal models and in clinical trials is warranted. PMID:8592990

[Expression of high mobility group box-1 in the lung tissue and serum of patients with pulmonary tuberculosis].

PubMed

Yang, Xiao-min; Yang, Hua

2013-07-01

To explore the expression of high mobility group box-1 (HMGB1) in the lung tissue and serum of patients with pulmonary tuberculosis and to explore its relationship with tumor necrosis factor (TNF)-α and interleukin(IL)-1β. Sixty samples of lung tissues were obtained from patients with pulmonary tuberculosis who had underwent pneumonectomy in Department of Chest Surgery, First Affiliated Hospital of Zunyi Medical College from June 2010 to December 2011. At the same period, 40 normal lung samples were also obtained from patients with pulmonary contusion and lung cancer by surgical resections as the control group. The mRNA expressions of HMGB1 was detected by reverse transcription-polymerase chain reaction (RT-PCR), and the protein level of HMGB1 was measured by immunohistochemical staining of tissue microarrays in lung tissue. Blood samples were taken from 89 patients with active pulmonary tuberculosis (pulmonary tuberculosis group), including hematogenous disseminated pulmonary tuberculosis (type II) in 35 cases and secondary pulmonary tuberculosis (type III) in 54 cases, and 50 healthy volunteers (control group). Furthermore, the 54 patients with secondary pulmonary tuberculosis were divided into different subgroups according to cavity formation and the lung fields involved: patients without lung cavity (35 cases) vs those with lung cavity (19 cases), patients with involvement of <2 lung fields (31 cases) vs ≥ 2 lung fields (23 cases). Serum concentration of HMGB1, TNF-α and IL-1β were detected by ELISA. Two sample t-test was used to compare date among groups, liner correlation analysis was established for correlation analysis. The average optical density of HMGB1 in pulmonary tuberculosis (69 ± 29) was significantly higher than that in normal lung tissue (22 ± 12) (t = 2.389, P < 0.05). The mRNA relative transcript levels of HMGB1 in pulmonary tuberculosis (786 ± 86) was significantly higher than that in normal lung tissue (202 ± 60) (t = 3.872, P < 0.01). The serum concentration of HMGB1, TNF-α and IL-1β in the pulmonary tuberculosis group were (5.0 ± 3.2) µg/L, (118 ± 77) ng/L and (33 ± 20) ng/L, respectively, which were significantly higher than those in the control group [(1.7 ± 1.0) µg/L, (40 ± 11) ng/L and (18 ± 12) ng/L, respectively], the respective t values being -0.928, 4.268 and 11.064, all P < 0.01. In the subgroup of patients with hematogenous disseminated pulmonary tuberculosis, the serum concentration of HMGB1 and TNF-α[ (6.4 ± 3.3) µg/L, (147 ± 89) ng/L] were significantly higher than those in patients with secondary pulmonary tuberculosis [(4.1 ± 2.7) µg/L, (85 ± 37) ng/L] (t = 3.643 and t = 3.111, both P < 0.01). HMGB1 were correlated positively with TNF-α and IL-1β (r = 0.722 and r = 0.620, P < 0.01, respectively, n = 89) in the pulmonary tuberculosis group. Overexpression of HMGB1 in the lung tissue and serum of patients with pulmonary tuberculosis may play an important role in the inflammatory response of pulmonary tuberculosis. The measurement of serum HMGB1 is useful to evaluate the severity of disease.

CE: Tuberculosis: A New Screening Recommendation and an Expanded Approach to Elimination in the United States.

PubMed

Parmer, John; Allen, Leeanna; Walton, Wanda

2017-08-01

: Nurses play a critical role in the diagnosis and treatment of tuberculosis and in the prevention of tuberculosis transmission through infection control practices. To eliminate tuberculosis in the United States, however, an expanded approach to testing and treating people with latent tuberculosis infection must be implemented. Recently, the U.S. Preventive Services Task Force (USPSTF) issued a new recommendation statement on latent tuberculosis infection testing that expands nurses' opportunities to identify at-risk populations for tuberculosis prevention. In combination with newer testing methodologies and shorter treatment regimens, implementation of the USPSTF recommendation has the potential to remove previously existing barriers to screening and treatment of both patients and health care providers. This article provides a general overview of tuberculosis transmission, pathogenesis, and epidemiology; presents preventive care recommendations for targeted testing among high-risk groups; and discusses the USPSTF recommendation's applicability to public health and primary care practice in the United States.

DNA topoisomerase I and DNA gyrase as targets for TB therapy.

PubMed

Nagaraja, Valakunja; Godbole, Adwait A; Henderson, Sara R; Maxwell, Anthony

2017-03-01

Tuberculosis (TB) is the deadliest bacterial disease in the world. New therapeutic agents are urgently needed to replace existing drugs for which resistance is a significant problem. DNA topoisomerases are well-validated targets for antimicrobial and anticancer chemotherapies. Although bacterial topoisomerase I has yet to be exploited as a target for clinical antibiotics, DNA gyrase has been extensively targeted, including the highly clinically successful fluoroquinolones, which have been utilized in TB therapy. Here, we review the exploitation of topoisomerases as antibacterial targets and summarize progress in developing new agents to target DNA topoisomerase I and DNA gyrase from Mycobacterium tuberculosis. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Depression comorbid with tuberculosis and its impact on health status: cross-sectional analysis of community-based data from 48 low- and middle-income countries.

PubMed

Koyanagi, Ai; Vancampfort, Davy; Carvalho, André F; DeVylder, Jordan E; Haro, Josep Maria; Pizzol, Damiano; Veronese, Nicola; Stubbs, Brendon

2017-11-28

Depression in tuberculosis increases the risk for adverse health outcomes. However, little is known about comorbid depression and tuberculosis in the general population. Thus, we assessed the association between depression and tuberculosis, and the decrements in health status associated with this comorbidity in 48 low- and middle-income countries. Cross-sectional, community-based data from the World Health Survey on 242,952 individuals aged ≥ 18 years were analyzed. Based on the World Mental Health Survey version of the Composite International Diagnostic Interview, past 12-month depression was categorized into depressive episode, brief depressive episode, subsyndromal depression, and no depression. Health status across six domains (cognition, interpersonal activities, sleep/energy, self-care, mobility, pain/discomfort) was assessed. Multivariable logistic and linear regression analyses were performed to assess the associations. The prevalence of depressive episode among those with and without tuberculosis was 23.7% and 6.8%, respectively (P < 0.001). Tuberculosis was associated with a 1.98 (95% CI 1.47-2.67), 1.75 (95% CI 1.26-2.42), and 3.68 (95% CI 3.01-4.50) times higher odds for subsyndromal depression, brief depressive episode, and depressive episode, respectively. Depressive episode co-occurring with tuberculosis was associated with significantly worse health status across all six domains compared to tuberculosis alone. Interaction analysis showed that depression significantly amplifies the association between TB and difficulties in self-care but not in other health domains. Depression is highly prevalent in adults with tuberculosis, and is associated with worse health status compared to tuberculosis without depression. Public health efforts directed to the recognition and management of depression in people with tuberculosis may lead to better outcomes.

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

PubMed

Machado, Diana; Coelho, Tatiane S; Perdigão, João; Pereira, Catarina; Couto, Isabel; Portugal, Isabel; Maschmann, Raquel De Abreu; Ramos, Daniela F; von Groll, Andrea; Rossetti, Maria L R; Silva, Pedro A; Viveiros, Miguel

2017-01-01

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 that is often disregarded by the tuberculosis specialists in favor of the almost undisputed importance of antibiotic target-gene mutations for the resistance in M. tuberculosis .

Strategies to Improve Vaccine Efficacy against Tuberculosis by Targeting Innate Immunity

PubMed Central

Schaible, Ulrich E.; Linnemann, Lara; Redinger, Natalja; Patin, Emmanuel C.; Dallenga, Tobias

2017-01-01

The global tuberculosis epidemic is the most common cause of death after infectious disease worldwide. Increasing numbers of infections with multi- and extensively drug-resistant variants of the Mycobacterium tuberculosis complex, resistant even to newly discovered and last resort antibiotics, highlight the urgent need for an efficient vaccine. The protective efficacy to pulmonary tuberculosis in adults of the only currently available vaccine, M. bovis BCG, is unsatisfactory and geographically diverse. More importantly, recent clinical studies on new vaccine candidates did not prove to be better than BCG, yet. Here, we propose and discuss novel strategies to improve efficacy of existing anti-tuberculosis vaccines. Modulation of innate immune responses upon vaccination already provided promising results in animal models of tuberculosis. For instance, neutrophils have been shown to influence vaccine efficacy, both, positively and negatively, and stimulate specific antibody secretion. Modulating immune regulatory properties after vaccination such as induction of different types of innate immune cell death, myeloid-derived suppressor or regulatory T cells, production of anti-inflammatory cytokines such as IL-10 may have beneficial effects on protection efficacy. Incorporation of lipid antigens presented via CD1 molecules to T cells have been discussed as a way to enhance vaccine efficacy. Finally, concepts of dendritic cell-based immunotherapies or training the innate immune memory may be exploitable for future vaccination strategies against tuberculosis. In this review, we put a spotlight on host immune networks as potential targets to boost protection by old and new tuberculosis vaccines. PMID:29312298

Strategies to Improve Vaccine Efficacy against Tuberculosis by Targeting Innate Immunity.

PubMed

Schaible, Ulrich E; Linnemann, Lara; Redinger, Natalja; Patin, Emmanuel C; Dallenga, Tobias

2017-01-01

The global tuberculosis epidemic is the most common cause of death after infectious disease worldwide. Increasing numbers of infections with multi- and extensively drug-resistant variants of the Mycobacterium tuberculosis complex, resistant even to newly discovered and last resort antibiotics, highlight the urgent need for an efficient vaccine. The protective efficacy to pulmonary tuberculosis in adults of the only currently available vaccine, M. bovis BCG, is unsatisfactory and geographically diverse. More importantly, recent clinical studies on new vaccine candidates did not prove to be better than BCG, yet. Here, we propose and discuss novel strategies to improve efficacy of existing anti-tuberculosis vaccines. Modulation of innate immune responses upon vaccination already provided promising results in animal models of tuberculosis. For instance, neutrophils have been shown to influence vaccine efficacy, both, positively and negatively, and stimulate specific antibody secretion. Modulating immune regulatory properties after vaccination such as induction of different types of innate immune cell death, myeloid-derived suppressor or regulatory T cells, production of anti-inflammatory cytokines such as IL-10 may have beneficial effects on protection efficacy. Incorporation of lipid antigens presented via CD1 molecules to T cells have been discussed as a way to enhance vaccine efficacy. Finally, concepts of dendritic cell-based immunotherapies or training the innate immune memory may be exploitable for future vaccination strategies against tuberculosis. In this review, we put a spotlight on host immune networks as potential targets to boost protection by old and new tuberculosis vaccines.

Risk of developing tuberculosis disease among persons diagnosed with latent tuberculosis infection in the Netherlands.

PubMed

Erkens, Connie G M; Slump, Erika; Verhagen, Maurits; Schimmel, Henrieke; Cobelens, Frank; van den Hof, Susan

2016-11-01

Diagnosis and preventive treatment of latent tuberculosis infection (LTBI) among high-risk groups is recommended to achieve tuberculosis (TB) elimination in low-incidence countries.We studied TB incidence rates among those notified with LTBI in the Netherlands from 2005 to 2013 and analysed associated risk factors. We stratified analyses by target group for screening, and by initiation and completion of preventive treatment.The incidence for those completing, stopping and not receiving preventive treatment was 187, 436 and 355 per 100 000 person-years for contacts of TB patients, respectively, and 63, 96 and 110 per 100 000 person-years for other target groups. The rate ratio for TB development among contacts compared to other target groups was 3.1 (95% CI 2.0-4.9). In both groups, incidence was highest in the first year after diagnosis. Independent factors associated with progression to TB among contacts were age <5 years and stopping preventive treatment within 28 days compared to those not receiving preventive treatment. Among other target groups, being foreign born was the only risk factor associated with the risk of developing TB.We conclude that the epidemiological impact of preventive treatment is highest in contacts of TB patients and limited in other target groups for LTBI management in the Netherlands. Copyright ©ERS 2016.

Mobile phone text messaging for promoting adherence to anti-tuberculosis treatment: a systematic review

PubMed Central

2013-01-01

Background Mobile phone text messaging (SMS) has the potential to promote adherence to tuberculosis treatment. This systematic review aims to synthesize current evidence on the effectiveness of SMS interventions in improving patients’ adherence to tuberculosis treatment. Methods We searched electronic databases (PubMed, EMBASE, Science Citation Index), reference lists of relevant articles, conference proceedings, and selected websites for eligible studies available by 15 February 2013; regardless of language or publication status. Two authors independently screened selected eligible studies, and assessed risk of bias in included studies; resolving discrepancies by discussion and consensus. Results We identified four studies that compared the outcomes of the SMS intervention group with controls. Only one of the four studies was a randomized controlled trial. This was conducted in Argentina and the SMS intervention did not significantly improve adherence to tuberculosis treatment compared to self-administration of tuberculosis treatment (risk ratio [RR] 1.49, 95% confidence intervals [CI] 0.90 to 2.42). One of the non-randomized studies, conducted in South Africa, which compared SMS reminders to directly observed therapy short course (DOTS) reported similar rates of tuberculosis cure (62.35% vs. 66.4%) and treatment success (72.94% vs. 69.4%). A second study from South Africa, utilized SMS reminders when patients delayed in opening their pill bottles and reported increased tuberculosis cure (RR 2.32, 95% CI 1.60 to 3.36) and smear conversion (RR 1.62, 95% CI 1.09 to 2.42) rates compared to DOTS. In the third non-randomized study, conducted in Kenya, use of SMS reminders increased rates of clinic attendance on scheduled days compared to standard care (RR 1.56, 95% CI 1.06 to 2.29). Using the GRADE approach, we rate the quality of the evidence as low, mainly because of the high risk of bias and heterogeneity of effects across studies. Conclusions This systematic review indicates that there is a paucity of high-quality data on the effectiveness of SMS interventions for improving patients’ adherence to tuberculosis treatment. The low quality of the current evidence implies that further studies (in particular randomized trials) on the subject are needed. In the interim, if the intervention is implemented outside research settings an impact evaluation is warranted. PMID:24295439

Impact of community tracer teams on treatment outcomes among tuberculosis patients in South Africa

PubMed Central

2012-01-01

Background Tuberculosis (TB) indicators in South Africa currently remain well below global targets. In 2008, the National Tuberculosis Program (NTP) implemented a community mobilization program in all nine provinces to trace TB patients that had missed a treatment or clinic visit. Implementation sites were selected by TB program managers and teams liaised with health facilities to identify patients for tracing activities. The objective of this analysis was to assess the impact of the TB Tracer Project on treatment outcomes among TB patients. Methods The study population included all smear positive TB patients registered in the Electronic TB Registry from Quarter 1 2007-Quarter 1 2009 in South Africa. Subdistricts were used as the unit of analysis, with each designated as either tracer (standard TB program plus tracer project) or non-tracer (standard TB program only). Mixed linear regression models were utilized to calculate the percent quarterly change in treatment outcomes and to compare changes in treatment outcomes from Quarter 1 2007 to Quarter 1 2009 between tracer and non-tracer subdistricts. Results For all provinces combined, the percent quarterly change decreased significantly for default treatment outcomes among tracer subdistricts (−0.031%; p < 0.001) and increased significantly for successful treatment outcomes among tracer subdistricts (0.003%; p = 0.03). A significant decrease in the proportion of patient default was observed for all provinces combined over the time period comparing tracer and non-tracer subdistricts (p = 0.02). Examination in stratified models revealed the results were not consistent across all provinces; significant differences were observed between tracer and non-tracer subdistricts over time in five of nine provinces for treatment default. Conclusions Community mobilization of teams to trace TB patients that missed a clinic appointment or treatment dose may be an effective strategy to mitigate default rates and improve treatment outcomes. Additional information is necessary to identify best practices and elucidate discrepancies across provinces; these findings will help guide the NTP in optimizing the adoption of tracing activities for TB control. PMID:22871071

Quality DOTS management and empowering tuberculosis patients.

PubMed

Chugh, Satish

2009-03-01

Central Tuberculosis Division (CTD) has covered whole of India under DOTS. IMA is a proud partner of RNTCP which is managed by CTD. International Standards for Tuberculosis Care is expected from all healthcare providers. The basic principles of care is same worldwide. IMA GFATM RNTCP PPM is completing 2 years of its inception. Sensitisation programme and district training programmes has yielded DOTS/DMC centres in the target states. IMA is having 100% commitment for containing tuberculosis in India. There are International Standards for quality management in tuberculosis control, some of the Standards are elaborated in this write-up. In the Indian context, DOTS needs some innovations that is discussed in this article.

Paradoxical results of two automated real-time PCR assays in the diagnosis of pleural tuberculosis.

PubMed

Morales-López, Soraya E; Yepes, Jayr A; Anzola, Irina; Aponte, Hernán; Llerena-Polo, Claudia R

2017-01-01

Tuberculosis (TB) is a major cause of worldwide mortality. We report the case of a non-HIV-infected woman with clinical suspicion of pleural tuberculosis and contradictory results between Xpert ® MTB/RIF and Abbott RealTime MTB assays from pleural fluid specimen. Liquid and solid cultures for tuberculosis were performed with negative results. The patient received treatment, and clinical improvement was observed. Both techniques detect Mycobacterium tuberculosis complex, but they have different targets and limits of detection. Abbott RealTime MTB results correlated well with the clinical findings of the patient. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Simple method for production of internal control DNA for Mycobacterium tuberculosis polymerase chain reaction assays.

PubMed Central

deWit, D; Wootton, M; Allan, B; Steyn, L

1993-01-01

A simple method for the production of internal control DNA for two well-established Mycobacterium tuberculosis polymerase chain reaction assays is described. The internal controls were produced from Mycobacterium kansasii DNA with the same primers but at a lower annealing temperature than that used in the standard assays. In both assays, therefore, the internal control DNA has the same primer-binding sequences at the target DNA. One-microgram quantities of internal control DNA which was not contaminated with target DNA could easily be produced by this method. The inclusion of the internal control in the reaction mixture did not affect the efficiency of amplification of the target DNA. The method is simple and rapid and should be adaptable to most M. tuberculosis polymerase chain reaction assays. Images PMID:8370752

Moving new vaccines for tuberculosis through the regulatory process.

PubMed

Brennan, M J

2000-06-01

The development of novel vaccines for the prevention of tuberculosis is an area of intense interest for scientific researchers, public health agencies, and pharmaceutical manufacturers. Development of effective new vaccines directed against tuberculosis for use in target populations will require close cooperation among several different international organizations, including regulatory agencies responsible for evaluating the safety and effectiveness of new biologics for human use.

Characterisation of Mycobacterium tuberculosis isolates lacking IS6110 in Viet Nam.

PubMed

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

2013-11-01

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 tests. We quantified the frequency of M. tuberculosis strains lacking IS6110 in rural Viet Nam and studied their epidemiological and clinical characteristics. Consecutively diagnosed adult TB patients in rural Southern Viet Nam submitted two sputum samples for culture, IS6110 restriction fragment length polymorphism (RFLP) spoligotyping and 15-loci variable number tandem repeat typing. Polymerase chain reaction (PCR) was performed to confirm the absence of IS6110 elements in strains lacking IS6110 hybridisation in RFLP. Among 2664 TB patient isolates examined, 109 (4.1%) had no IS6110 element. Compared to other strains, these no-copy strains were less often resistant to anti-tuberculosis drugs, particularly to streptomycin (adjusted OR 0.2, 95%CI 0.1-0.5), and showed significant geographic variation. No associations with TB history or demographic factors were found. Strains without the IS6110 target pose a problem in Viet Nam as regards false-negative molecular TB diagnosis in PCR. Compared to other strains circulating in Viet Nam, no-copy strains are more susceptible to anti-tuberculosis drugs.

Role of the horizontal gene exchange in evolution of pathogenic Mycobacteria.

PubMed

Reva, Oleg; Korotetskiy, Ilya; Ilin, Aleksandr

2015-01-01

Mycobacterium tuberculosis is one of the most dangerous human pathogens, the causative agent of tuberculosis. While this pathogen is considered as extremely clonal and resistant to horizontal gene exchange, there are many facts supporting the hypothesis that on the early stages of evolution the development of pathogenicity of ancestral Mtb has started with a horizontal acquisition of virulence factors. Episodes of infections caused by non-tuberculosis Mycobacteria reported worldwide may suggest a potential for new pathogens to appear. If so, what is the role of horizontal gene transfer in this process? Availing of accessibility of complete genomes sequences of multiple pathogenic, conditionally pathogenic and saprophytic Mycobacteria, a genome comparative study was performed to investigate the distribution of genomic islands among bacteria and identify ontological links between these mobile elements. It was shown that the ancient genomic islands from M. tuberculosis still may be rooted to the pool of mobile genetic vectors distributed among Mycobacteria. A frequent exchange of genes was observed between M. marinum and several saprophytic and conditionally pathogenic species. Among them M. avium was the most promiscuous species acquiring genetic materials from diverse origins. Recent activation of genetic vectors circulating among Mycobacteria potentially may lead to emergence of new pathogens from environmental and conditionally pathogenic Mycobacteria. The species which require monitoring are M. marinum and M. avium as they eagerly acquire genes from different sources and may become donors of virulence gene cassettes to other micro-organisms.

Domestic and donor financing for tuberculosis care and control in low-income and middle-income countries: an analysis of trends, 2002-11, and requirements to meet 2015 targets.

PubMed

Floyd, Katherine; Fitzpatrick, Christopher; Pantoja, Andrea; Raviglione, Mario

2013-08-01

Progress in tuberculosis control worldwide, including achievement of 2015 global targets, requires adequate financing sustained for many years. WHO began yearly monitoring of tuberculosis funding in 2002. We used data reported to WHO to analyse tuberculosis funding from governments and international donors (in real terms, constant 2011 US$) and associated progress in tuberculosis control in low-income and middle-income countries between 2002 and 2011. We then assessed funding needed to 2015 and how this funding could be mobilised. We included low-income and middle-income countries that reported data about financing for tuberculosis to WHO and had at least three observations between 2002 and 2011. When data were missing for specific country-year combinations, we imputed the missing data. We aggregated country-specific results for eight country groups defined according to income level, political and economic profile, geography, and tuberculosis burden. We compared absolute changes in total funding with those in the total number of patients successfully treated and did cross-country comparisons of cost per successfully treated patient relative to gross domestic product. We estimated funding needs for tuberculosis care and control for all low-income and middle-income countries to 2015, and compared these needs with domestic funding that could be mobilised. Total funding grew from $1·7 billion in 2002 to $4·4 billion in 2011. It was mostly spent on diagnosis and treatment of drug-susceptible tuberculosis. 43 million patients were successfully treated, usually for $100-500 per person in countries with high burdens of tuberculosis. Domestic funding rose from $1·5 billion to $3·9 billion per year, mostly in Brazil, Russia, India, China, and South Africa (BRICS), which collectively account for 45% of global cases, where national contributions accounted for more than 95% of yearly funding. Donor funding increased from $0·2 billion in 2002 to $0·5 billion in 2011, and accounted for a mean of 39% of funding in the 17 countries with the highest burdens (excluding BRICS) and a mean of 67% in low-income countries by 2011. BRICS and upper middle-income countries could mobilise almost all of their funding needs to 2015 from domestic sources. A full response to the tuberculosis epidemic to 2015, including investments to tackle multidrug-resistant tuberculosis, will require international donor funding of $1·6-2·3 billion each year. Funding for tuberculosis control increased substantially between 2002 and 2011, resulting in impressive and cost-effective gains. The increasing self-sufficiency of many countries, including BRICS, which account for almost half the world's tuberculosis cases, is a success story for control of tuberculosis. Nonetheless, international donor funding remains crucial in many countries and more is needed to achieve 2015 targets. None. Copyright © 2013 World Health Organization; licensee Elsevier. Published by .. All rights reserved.

From the NIH Director: A Global Health System

MedlinePlus

... world, people will continue to contract diseases like malaria. They will also suffer as we do from ... AIDS Relief and the Global Fund for AIDS, Malaria, and Tuberculosis. Billions of dollars have been mobilized ...

The impact of social protection and poverty elimination on global tuberculosis incidence: a statistical modelling analysis of Sustainable Development Goal 1.

PubMed

Carter, Daniel J; Glaziou, Philippe; Lönnroth, Knut; Siroka, Andrew; Floyd, Katherine; Weil, Diana; Raviglione, Mario; Houben, Rein M G J; Boccia, Delia

2018-05-01

The End TB Strategy and the Sustainable Development Goals (SDGs) are intimately linked by their common targets and approaches. SDG 1 aims to end extreme poverty and expand social protection coverage by 2030. Achievement of SDG 1 is likely to affect the tuberculosis epidemic through a range of pathways. We estimate the reduction in global tuberculosis incidence that could be obtained by reaching SDG 1. We developed a conceptual framework linking key indicators of SDG 1 progress to tuberculosis incidence via well described risk factor pathways and populated it with data from the SDG data repository and the WHO tuberculosis database for 192 countries. Correlations and mediation analyses informed the strength of the association between the SDG 1 subtargets and tuberculosis incidence, resulting in a simplified framework for modelling. The simplified framework linked key indicators for SDG 1 directly to tuberculosis incidence. We applied an exponential decay model based on linear associations between SDG 1 indicators and tuberculosis incidence to estimate tuberculosis incidence in 2035. Ending extreme poverty resulted in a reduction in global incidence of tuberculosis of 33·4% (95% credible interval 15·5-44·5) by 2035 and expanding social protection coverage resulted in a reduction in incidence of 76·1% (45·2-89·9) by 2035; both pathways together resulted in a reduction in incidence of 84·3% (54·7-94·9). Full achievement of SDG 1 could have a substantial effect on the global burden of tuberculosis. Cross-sectoral approaches that promote poverty reduction and social protection expansion will be crucial complements to health interventions, accelerating progress towards the End TB targets. World Health Organization. Copyright © 2018 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license. Published by Elsevier Ltd.. All rights reserved.

Pathway-selective sensitization of Mycobacterium tuberculosis for target-based whole-cell screening

PubMed Central

Abrahams, Garth L.; Kumar, Anuradha; Savvi, Suzana; Hung, Alvin W.; Wen, Shijun; Abell, Chris; Barry, Clifton E.; Sherman, David R.; Boshoff, Helena I.M.; Mizrahi, Valerie

2012-01-01

SUMMARY Whole-cell screening of Mycobacterium tuberculosis (Mtb) remains a mainstay of drug discovery but subsequent target elucidation often proves difficult. Conditional mutants that under-express essential genes have been used to identify compounds with known mechanism of action by target-based whole-cell screening (TB-WCS). Here, the feasibility of TB-WCS in Mtb was assessed by generating mutants that conditionally express pantothenate synthetase (panC), diaminopimelate decarboxylase (lysA) and isocitrate lyase (icl1). The essentiality of panC and lysA, and conditional essentiality of icl1 for growth on fatty acids, was confirmed. Depletion of PanC and Icl1 rendered the mutants hypersensitive to target-specific inhibitors. Stable reporter strains were generated for use in high-throughput screening, and their utility demonstrated by identifying compounds that display greater potency against a PanC-depleted strain. These findings illustrate the power of TB-WCS as a tool for tuberculosis drug discovery. PMID:22840772

Oxidative Phosphorylation as a Target Space for Tuberculosis: Success, Caution, and Future Directions.

PubMed

Cook, Gregory M; Hards, Kiel; Dunn, Elyse; Heikal, Adam; Nakatani, Yoshio; Greening, Chris; Crick, Dean C; Fontes, Fabio L; Pethe, Kevin; Hasenoehrl, Erik; Berney, Michael

2017-06-01

The emergence and spread of drug-resistant pathogens, and our inability to develop new antimicrobials to combat resistance, have inspired scientists to seek out new targets for drug development. The Mycobacterium tuberculosis complex is a group of obligately aerobic bacteria that have specialized for inhabiting a wide range of intracellular and extracellular environments. Two fundamental features in this adaptation are the flexible utilization of energy sources and continued metabolism in the absence of growth. M. tuberculosis is an obligately aerobic heterotroph that depends on oxidative phosphorylation for growth and survival. However, several studies are redefining the metabolic breadth of the genus. Alternative electron donors and acceptors may provide the maintenance energy for the pathogen to maintain viability in hypoxic, nonreplicating states relevant to latent infection. This hidden metabolic flexibility may ultimately decrease the efficacy of drugs targeted against primary dehydrogenases and terminal oxidases. However, it may also open up opportunities to develop novel antimycobacterials targeting persister cells. In this review, we discuss the progress in understanding the role of energetic targets in mycobacterial physiology and pathogenesis and the opportunities for drug discovery.

Latent tuberculosis infection: screening and treatment in an urban setting.

PubMed

Morano, Jamie P; Walton, Mary R; Zelenev, Alexei; Bruce, R Douglas; Altice, Frederick L

2013-10-01

Despite its benefit for treating active tuberculosis, directly observed therapy (DOT) for latent tuberculosis infection (LTBI) has been largely understudied among challenging inner city populations. Utilizing questionnaire data from a comprehensive mobile healthcare clinic in New Haven, CT from 2003 to July 2011, a total of 2,523 completed tuberculin skin tests (TSTs) resulted in 356 new LTBIs. Multivariate logistic regression correlated covariates of the two outcomes (a) initiation of isoniazid preventative therapy (IPT) and (b) completion of 9 months of IPT. Of the 357 newly positive TSTs, 86.3 % (n = 308) completed screening chest radiographs (CXRs): 90.3 % (n = 278) were normal, and 0.3 % (n = 1) had active tuberculosis. Of those completing CXR screening, 44.0 % (n = 135) agreed to IPT: 69.6 % (n = 94) selected DOT, and 30.4 % (n = 41) selected self-administered therapy (SAT). Initiating IPT was correlated with undocumented status (AOR = 3.43; p < 0.001) and being born in a country of highest and third highest tuberculosis prevalence (AOR = 14.09; p = 0.017 and AOR = 2.25; p = 0.005, respectively). Those selecting DOT were more likely to be Hispanic (83.0 vs 53.7 %; p < 0.0001), undocumented (57.4 vs 41.5 %; p = 0.012), employed (p < 0.0001), uninsured (p = 0.014), and have stable housing (p = 0.002), no prior cocaine or crack use (p = 0.013) and no recent incarceration (p = 0.001). Completing 9 months of IPT was correlated with no recent incarceration (AOR 5.95; p = 0.036) and younger age (AOR 1.03; p = 0.031). SAT and DOT participants did not significantly differ for IPT duration (6.54 vs 5.68 months; p = 0.216) nor 9-month completion (59.8 vs 46.3 %; p = 0.155). In an urban mobile healthcare sample, screening completion for LTBI was high with nearly half initiating IPT. Undocumented, Hispanic immigrants from high prevalence tuberculosis countries were more likely to self-select DOT at the mobile outreach clinic, potentially because of more culturally, linguistically, and logistically accessible services and self-selection optimization phenomena. Within a diverse, urban environment, DOT and SAT IPT models for LTBI treatment resulted in similar outcomes, yet outcomes were hampered by differential measurement bias between DOT and SAT participants.

Target prioritization and strategy selection for active case-finding of pulmonary tuberculosis: a tool to support country-level project planning.

PubMed

Nishikiori, Nobuyuki; Van Weezenbeek, Catharina

2013-02-02

Despite the progress made in the past decade, tuberculosis (TB) control still faces significant challenges. In many countries with declining TB incidence, the disease tends to concentrate in vulnerable populations that often have limited access to health care. In light of the limitations of the current case-finding approach and the global urgency to improve case detection, active case-finding (ACF) has been suggested as an important complementary strategy to accelerate tuberculosis control especially among high-risk populations. The present exercise aims to develop a model that can be used for county-level project planning. A simple deterministic model was developed to calculate the number of estimated TB cases diagnosed and the associated costs of diagnosis. The model was designed to compare cost-effectiveness parameters, such as the cost per case detected, for different diagnostic algorithms when they are applied to different risk populations. The model was transformed into a web-based tool that can support national TB programmes and civil society partners in designing ACF activities. According to the model output, tuberculosis active case-finding can be a costly endeavor, depending on the target population and the diagnostic strategy. The analysis suggests the following: (1) Active case-finding activities are cost-effective only if the tuberculosis prevalence among the target population is high. (2) Extensive diagnostic methods (e.g. X-ray screening for the entire group, use of sputum culture or molecular diagnostics) can be applied only to very high-risk groups such as TB contacts, prisoners or people living with human immunodeficiency virus (HIV) infection. (3) Basic diagnostic approaches such as TB symptom screening are always applicable although the diagnostic yield is very limited. The cost-effectiveness parameter was sensitive to local diagnostic costs and the tuberculosis prevalence of target populations. The prioritization of appropriate target populations and careful selection of cost-effective diagnostic strategies are critical prerequisites for rational active case-finding activities. A decision to conduct such activities should be based on the setting-specific cost-effectiveness analysis and programmatic assessment. A web-based tool was developed and is available to support national tuberculosis programmes and partners in the formulation of cost-effective active case-finding activities at the national and subnational levels.

[Epidemic of tuberculosis in Meiji and Taisho eras in Japan and excess deaths from tuberculosis in females].

PubMed

Aoki, K

1995-08-01

Acute increase in tuberculosis mortality between 1885 and 1910 could be explained by rapidly increased birth rate, consequently large expansion of noninfected population, and gradual increase in opportunity of contact with infectious patients by changing working environments and living conditions. Prevalence of tuberculosis patients was not so few in the beginning of Meiji era. Vicious spiral of increased young susceptibles, many infectious sources and increased opportunity of infection had been continued for long. Lower nutrition from infant to adult, hard work and poor living conditions had worsen prognosis of the patients. Nation-wide tuberculosis control campaign, mainly avoiding contact with patients and contaminated materials had started around 1910 and then issued Factory act which had been improved working conditions in the factories, although the speed was very slow. Tuberculosis mortality began to decrease in 1910s, but sharp temporary rise of tuberculosis mortality was marked in 1918-19 by epidemic of influenza, then the mortality had been declined again. Excess mortality by influenza caused temporary reduction of infectious sources, which had affected mortality rate of tuberculosis in the younger ages after 1920. Large raise-up of wages for factory workers around 1920 and increase trend in income for other workers by economic growth since 1900 had been improved not only working and living conditions, but also dietary life with increased higher intake of animal foods. Female excess deaths from tuberculosis comparing those of males had continued until 1930, then male mortality exceeded females. Mobilization of young women to spinning and textile industries in Meiji and Taisho eras forced to increase in tuberculosis mortality among them.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of highly conserved hsp65-specific nested PCR primers for diagnosing Mycobacterium tuberculosis.

PubMed

Priyadarshini, P; Tiwari, K; Das, A; Kumar, D; Mishra, M N; Desikan, P; Nath, G

2017-02-01

To evaluate the sensitivity and specificity of a new nested set of primers designed for the detection of Mycobacterium tuberculosis complex targeting a highly conserved heat shock protein gene (hsp65). The nested primers were designed using multiple sequence alignment assuming the nucleotide sequence of the M. tuberculosis H37Rv hsp65 genome as base. Multidrug-resistant Mycobacterium species along with other non-mycobacterial and fungal species were included to evaluate the specificity of M. tuberculosis hsp65 gene-specific primers. The sensitivity of the primers was determined using serial 10-fold dilutions, and was 100% as shown by the bands in the case of M. tuberculosis complex. None of the other non M. tuberculosis complex bacterial and fungal species yielded any band on nested polymerase chain reaction (PCR). The first round of amplification could amplify 0.3 ng of the template DNA, while nested PCR could detect 0.3 pg. The present hsp65-specific primers have been observed to be sensitive, specific and cost-effective, without requiring interpretation of biochemical tests, real-time PCR, sequencing or high-performance liquid chromatography. These primer sets do not have the drawbacks associated with those protocols that target insertion sequence 6110, 16S rDNA, rpoB, recA and MPT 64.

Structural and functional characterization of Mycobacterium tuberculosis triosephosphate isomerase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Connor, Sean E.; Capodagli, Glenn C.; Deaton, Michelle K.

Tuberculosis (TB) is a major infectious disease that accounts for over 1.7 million deaths every year. Mycobacterium tuberculosis, the causative agent of tuberculosis, enters the human host by the inhalation of infectious aerosols. Additionally, one third of the world's population is likely to be infected with latent TB. The incidence of TB is on the rise owing in part to the emergence of multidrug-resistant strains. As a result, there is a growing need to focus on novel M. tuberculosis enzyme targets. M. tuberculosis triosephosphate isomerase (MtTPI) is an essential enzyme for gluconeogenetic pathways, making it a potential target for futuremore » therapeutics. In order to determine its structure, the X-ray crystal structure of MtTPI has been determined, as well as that of MtTPI bound with a reaction-intermediate analog. As a result, two forms of the active site were revealed. In conjunction with the kinetic parameters obtained for the MtTPI-facilitated conversion of dihydroxyacetone phosphate (DHAP) to D-glyceraldehyde-3-phosphate (D-GAP), this provides a greater structural and biochemical understanding of this enzyme. Additionally, isothermal titration calorimetry was used to determine the binding constant for a reaction-intermediate analog bound to the active site of MtTPI.« less

Targeted Intracellular Delivery of Antituberculosis Drugs to Mycobacterium tuberculosis-Infected Macrophages via Functionalized Mesoporous Silica Nanoparticles

PubMed Central

Lee, Bai-Yu; Xue, Min; Thomas, Courtney R.; Meng, Huan; Ferris, Daniel; Nel, Andre E.; Zink, Jeffrey I.

2012-01-01

Delivery of antituberculosis drugs by nanoparticles offers potential advantages over free drug, including the potential to target specifically the tissues and cells that are infected by Mycobacterium tuberculosis, thereby simultaneously increasing therapeutic efficacy and decreasing systemic toxicity, and the capacity for prolonged release of drug, thereby allowing less-frequent dosing. We have employed mesoporous silica nanoparticle (MSNP) drug delivery systems either equipped with a polyethyleneimine (PEI) coating to release rifampin or equipped with cyclodextrin-based pH-operated valves that open only at acidic pH to release isoniazid (INH) into M. tuberculosis-infected macrophages. The MSNP are internalized efficiently by human macrophages, traffic to acidified endosomes, and release high concentrations of antituberculosis drugs intracellularly. PEI-coated MSNP show much greater loading of rifampin than uncoated MSNP and much greater efficacy against M. tuberculosis-infected macrophages. MSNP were devoid of cytotoxicity at the particle doses employed for drug delivery. Similarly, we have demonstrated that the isoniazid delivered by MSNP equipped with pH-operated nanovalves kill M. tuberculosis within macrophages significantly more effectively than an equivalent amount of free drug. These data demonstrate that MSNP provide a versatile platform that can be functionalized to optimize the loading and intracellular release of specific drugs for the treatment of tuberculosis. PMID:22354311

Efficient heterologous expression and one-step purification of fully active c-terminal histidine-tagged uridine monophosphate kinase from Mycobacterium tuberculosis.

PubMed

Penpassakarn, Praweenuch; Chaiyen, Pimchai; Palittapongarnpim, Prasit

2011-11-01

Tuberculosis has long been recognized as one of the most significant public health problems. Finding novel antituberculous drugs is always a necessary approach for controlling the disease. Mycobacterium tuberculosis pyrH gene (Rv2883c) encodes for uridine monophosphate kinase (UMK), which is a key enzyme in the uridine nucleotide interconversion pathway. The enzyme is essential for M. tuberculosis to sustain growth and hence is a potential drug target. In this study, we have developed a rapid protocol for production and purification of M. tuberculosis UMK by cloning pyrH (Rv2883c) of M. tuberculosis H37Rv with the addition of 6-histidine residues to the C-terminus of the protein, and expressing in E. coli BL21-CodonPlus (DE3)-RIPL using an auto-induction medium. The enzyme was efficiently purified by a single-step TALON cobalt affinity chromatography with about 8 fold increase in specific activity, which was determined by a coupled assay with the pyruvate kinase and lactate dehydrogenase. The molecular mass of monomeric UMK was 28.2 kDa and that of the native enzyme was 217 kDa. The enzyme uses UMP as a substrate but not CMP and TMP and activity was enhanced by GTP. Measurements of enzyme kinetics revealed the kcat value of 7.6 +/- 0.4 U mg(-1) or 0.127 +/- 0.006 sec(-1).The protocol reported here can be used for expression of M. tuberculosis UMK in large quantity for formulating a high throughput target-based assay for screening anti-tuberculosis UMK compounds.

Phenotypic and genotypic characteristics of drug resistance in Mycobacterium tuberculosis isolates from pediatric population of Chennai, India.

PubMed

Therese, K Lily; Gayathri, R; Balasubramanian, S; Natrajan, S; Madhavan, H N

2012-01-01

Multidrug-resistant TB (MDR-TB) has been reported in almost all parts of the world. Childhood TB is accorded low priority by national TB control programs. Probable reasons include diagnostic difficulties, limited resources, misplaced faith in BCG and lack of data on treatment. Good data on the burden of all forms of TB among children in India are not available. To study the drug sensitivity pattern of tuberculosis in children aged from 3 months to 18 years and the outcome of drug-resistant tuberculosis by BACTEC culture system and PCR-based DNA sequencing technique. This is a retrospective study. One hundred and fifty-nine clinical specimens were processed for Ziehl-Neelsen stain, Mycobacterial culture by BACTEC method, phenotypic DST for first-line drugs for Mycobacterium tuberculosis (M. tuberculosis) isolates and PCR-based DNA sequencing was performed for the M. tuberculosis isolates targeting rpoB, katG, inhA, oxyR-ahpC, rpsL, rrs and pncA. Out of the 159 Mycobacterial cultures performed during the study period, 17 clinical specimens (10.7%) were culture positive for M. tuberculosis. Among the 17 M. tuberculosis isolates, 2 were multidrug-resistant TB. PCR-based DNA sequencing revealed the presence of many novel mutations targeting katG, inhA, oxyR-ahpC and pncA and the most commonly reported mutation Ser531Leu in the rpoB gene. This study underlines the urgent need to take efforts to develop methods for rapid detection and drug susceptibility of tubercle bacilli in the pediatric population.

Discovery of Imidazo[1,2-a]pyridine Ethers and Squaramides as Selective and Potent Inhibitors of Mycobacterial Adenosine Triphosphate (ATP) Synthesis.

PubMed

Tantry, Subramanyam J; Markad, Shankar D; Shinde, Vikas; Bhat, Jyothi; Balakrishnan, Gayathri; Gupta, Amit K; Ambady, Anisha; Raichurkar, Anandkumar; Kedari, Chaitanyakumar; Sharma, Sreevalli; Mudugal, Naina V; Narayan, Ashwini; Naveen Kumar, C N; Nanduri, Robert; Bharath, Sowmya; Reddy, Jitendar; Panduga, Vijender; Prabhakar, K R; Kandaswamy, Karthikeyan; Saralaya, Ramanatha; Kaur, Parvinder; Dinesh, Neela; Guptha, Supreeth; Rich, Kirsty; Murray, David; Plant, Helen; Preston, Marian; Ashton, Helen; Plant, Darren; Walsh, Jarrod; Alcock, Peter; Naylor, Kathryn; Collier, Matthew; Whiteaker, James; McLaughlin, Robert E; Mallya, Meenakshi; Panda, Manoranjan; Rudrapatna, Suresh; Ramachandran, Vasanthi; Shandil, Radha; Sambandamurthy, Vasan K; Mdluli, Khisi; Cooper, Christopher B; Rubin, Harvey; Yano, Takahiro; Iyer, Pravin; Narayanan, Shridhar; Kavanagh, Stefan; Mukherjee, Kakoli; Balasubramanian, V; Hosagrahara, Vinayak P; Solapure, Suresh; Ravishankar, Sudha; Hameed P, Shahul

2017-02-23

The approval of bedaquiline to treat tuberculosis has validated adenosine triphosphate (ATP) synthase as an attractive target to kill Mycobacterium tuberculosis (Mtb). Herein, we report the discovery of two diverse lead series imidazo[1,2-a]pyridine ethers (IPE) and squaramides (SQA) as inhibitors of mycobacterial ATP synthesis. Through medicinal chemistry exploration, we established a robust structure-activity relationship of these two scaffolds, resulting in nanomolar potencies in an ATP synthesis inhibition assay. A biochemical deconvolution cascade suggested cytochrome c oxidase as the potential target of IPE class of molecules, whereas characterization of spontaneous resistant mutants of SQAs unambiguously identified ATP synthase as its molecular target. Absence of cross resistance against bedaquiline resistant mutants suggested a different binding site for SQAs on ATP synthase. Furthermore, SQAs were found to be noncytotoxic and demonstrated efficacy in a mouse model of tuberculosis infection.

Inhibiting Mycobacterium tuberculosis within and without.

PubMed

Cole, Stewart T

2016-11-05

Tuberculosis remains a scourge of global health with shrinking treatment options due to the spread of drug-resistant strains of Mycobacterium tuberculosis Intensive efforts have been made in the past 15 years to find leads for drug development so that better, more potent drugs inhibiting new targets could be produced and thus shorten treatment duration. Initial attempts focused on repurposing drugs that had been developed for other therapeutic areas but these agents did not meet their goals in clinical trials. Attempts to find new lead compounds employing target-based screens were unsuccessful as the leads were inactive against M. tuberculosis Greater success was achieved using phenotypic screening against live tubercle bacilli and this gave rise to the drugs bedaquiline, pretomanid and delamanid, currently in phase III trials. Subsequent phenotypic screens also uncovered new leads and targets but several of these targets proved to be promiscuous and inhibited by a variety of seemingly unrelated pharmacophores. This setback sparked an interest in alternative screening approaches that mimic the disease state more accurately. Foremost among these were cell-based screens, often involving macrophages, as these should reflect the bacterium's niche in the host more faithfully. A major advantage of this approach is its ability to uncover functions that are central to infection but not necessarily required for growth in vitro For instance, inhibition of virulence functions mediated by the ESX-1 secretion system severely attenuates intracellular M. tuberculosis, preventing intercellular spread and ultimately limiting tissue damage. Cell-based screens have highlighted the druggability of energy production via the electron transport chain and cholesterol metabolism. Here, I review the scientific progress and the pipeline, but warn against over-optimism due to the lack of industrial commitment for tuberculosis drug development and other socio-economic factors.This article is part of the themed issue 'The new bacteriology'. © 2016 The Author(s).

Standardization of a TaqMan-based real-time PCR for the detection of Mycobacterium tuberculosis-complex in human sputum.

PubMed

Barletta, Francesca; Vandelannoote, Koen; Collantes, Jimena; Evans, Carlton A; Arévalo, Jorge; Rigouts, Leen

2014-10-01

Real-time polymerase chain reaction (qPCR) was optimized for detecting Mycobacterium tuberculosis in sputum. Sputum was collected from patients (N = 112) with suspected pulmonary tuberculosis, tested by smear microscopy, decontaminated, and split into equal aliquots that were cultured in Löwenstein-Jensen medium and tested by qPCR for the small mobile genetic element IS6110. The human ERV3 sequence was used as an internal control. 3 of 112 (3%) qPCR failed. For the remaining 109 samples, qPCR diagnosed tuberculosis in 79 of 84 patients with culture-proven tuberculosis, and sensitivity was greater than microscopy (94% versus 76%, respectively, P < 0.05). The qPCR sensitivity was similar (P = 0.9) for smear-positive (94%, 60 of 64) and smear-negative (95%, 19 of 20) samples. The qPCR was negative for 24 of 25 of the sputa with negative microscopy and culture (diagnostic specificity 96%). The qPCR had 99.5% sensitivity and specificity for 211 quality control samples including 84 non-tuberculosis mycobacteria. The qPCR cost ∼5US$ per sample and provided same-day results compared with 2-6 weeks for culture. © The American Society of Tropical Medicine and Hygiene.

Recurrent tuberculosis in an urban area in China: Relapse or exogenous reinfection?

PubMed

Shen, Xin; Yang, Chongguang; Wu, Jie; Lin, Senlin; Gao, Xu; Wu, Zheyuan; Tian, Jiyun; Gan, Mingyu; Luo, Tao; Wang, Lili; Yu, Chenlei; Mei, Jian; Pan, Qichao; DeRiemer, Kathryn; Yuan, ZhengAn; Gao, Qian

2017-03-01

Recurrent tuberculosis is an important indicator of the effectiveness of tuberculosis control and can occur by relapse or exogenous reinfection. We conducted a retrospective cohort study on all bacteriologically confirmed tuberculosis cases that were successfully treated between 2000 and 2012 in Shanghai, an urban area with a high number but a low prevalence rate of tuberculosis cases and a low prevalence of HIV infection. Genotyping the Mycobacterium tuberculosis from clinical isolates was used to distinguish between relapse and reinfection. In total, 5.3% (710/13,417) of successfully treated cases had a recurrence, a rate of 7.55 (95% CI 7.01-8.13) episodes per 1000 person-years, more than 18 times the rate of tuberculosis in the general population. Patients who were male, age 30-59, retreatment cases, had cavitation, diabetes, drug-resistant or multidrug-resistant tuberculosis in their initial episode of tuberculosis, were at high risk for a recurrence. Among 141 recurrent cases that had paired isolates, 59 (41.8%) had different genotypes, indicating reinfection with a different strain. Patients who completed treatment were still at high risk of another episode of tuberculosis and exogenous reinfection contributed a significant proportion of the recurrent tuberculosis cases. Targeted control strategies are needed to prevent new tuberculosis infections in this setting. Copyright © 2017 Elsevier Ltd. All rights reserved.

Recurrent tuberculosis in an urban area in China: relapse or exogenous reinfection?

PubMed Central

Shen, Xin; Yang, Chongguang; Wu, Jie; Lin, Senlin; Gao, Xu; Wu, Zheyuan; Tian, Jiyun; Gan, Mingyu; Luo, Tao; Wang, Lili; Yu, Chenlei; Mei, Jian; Pan, Qichao; DeRiemer, Kathryn; Yuan, ZhengAn; Gao, Qian

2017-01-01

Recurrent tuberculosis is an important indicator of the effectiveness of tuberculosis control and can occur by relapse or exogenous reinfection. We conducted a retrospective cohort study on all bacteriologically confirmed tuberculosis cases that were successfully treated between 2000 and 2012 in Shanghai, an urban area with a high number but a low prevalence rate of tuberculosis cases and a low prevalence of HIV infection. Genotyping the Mycobacterium tuberculosis from clinical isolates was used to distinguish between relapse and reinfection. In total, 5.3% (710/13,417) of successfully treated cases had a recurrence, a rate of 7.55 (95% CI 7.01–8.13) episodes per 1000 person-years, more than 18 times the rate of tuberculosis in the general population. Patients who were male, age 30–59, retreatment cases, had cavitation, diabetes, drug-resistant or multidrug-resistant tuberculosis in their initial episode of tuberculosis, were at high risk for a recurrence. Among 141 recurrent cases that had paired isolates, 59 (41.8%) had different genotypes, indicating reinfection with a different strain. Patients who completed treatment were still at high risk of another episode of tuberculosis and exogenous reinfection contributed a significant proportion of the recurrent tuberculosis cases. Targeted control strategies are needed to prevent new tuberculosis infections in this setting. PMID:28237039

Mycobacterium tuberculosis surface protein Rv0227c contains high activity binding peptides which inhibit cell invasion.

PubMed

Rodríguez, Diana Marcela; Ocampo, Marisol; Curtidor, Hernando; Vanegas, Magnolia; Patarroyo, Manuel Elkin; Patarroyo, Manuel Alfonso

2012-12-01

Mycobacterium tuberculosis surface proteins involved in target cell invasion may be identified as a strategy for developing subunit-based, chemically-synthesized vaccines. The Rv0227c protein was thus selected to assess its role in the invasion and infection of Mycobacterium tuberculosis target cells. Results revealed Rv0227c localization on mycobacterial surface by immunoelectron microscopy and Western blot. Receptor-ligand assays using 20-mer, non-overlapping peptides covering the complete Rv0227c protein sequence revealed three high activity binding peptides for U937 phagocytic cells and seven for A549 cells. Peptide 16944 significantly inhibited mycobacterial entry to both cell lines while 16943 and 16949 only managed to inhibit entrance to U937 cells and 16951 to A549 cells. The Jnet bioinformatics tool predicted secondary structure elements for the complete protein, agreeing with elements determined for such chemically-synthesized peptides. It was thus concluded that high activity binding peptides which were able to inhibit mycobacterial entry to target cells are of great importance when selecting peptide candidates for inclusion in an anti-tuberculosis vaccine. Copyright © 2012 Elsevier Inc. All rights reserved.

Immune activation of the host cell induces drug tolerance in Mycobacterium tuberculosis both in vitro and in vivo

PubMed Central

Liu, Yancheng; Tan, Shumin; Huang, Lu; Abramovitch, Robert B.; Rohde, Kyle H.; Zimmerman, Matthew D.; Chen, Chao; Dartois, Véronique; VanderVen, Brian C.

2016-01-01

Successful chemotherapy against Mycobacterium tuberculosis (Mtb) must eradicate the bacterium within the context of its host cell. However, our understanding of the impact of this environment on antimycobacterial drug action remains incomplete. Intriguingly, we find that Mtb in myeloid cells isolated from the lungs of experimentally infected mice exhibit tolerance to both isoniazid and rifampin to a degree proportional to the activation status of the host cells. These data are confirmed by in vitro infections of resting versus activated macrophages where cytokine-mediated activation renders Mtb tolerant to four frontline drugs. Transcriptional analysis of intracellular Mtb exposed to drugs identified a set of genes common to all four drugs. The data imply a causal linkage between a loss of fitness caused by drug action and Mtb’s sensitivity to host-derived stresses. Interestingly, the environmental context exerts a more dominant impact on Mtb gene expression than the pressure on the drugs’ primary targets. Mtb’s stress responses to drugs resemble those mobilized after cytokine activation of the host cell. Although host-derived stresses are antimicrobial in nature, they negatively affect drug efficacy. Together, our findings demonstrate that the macrophage environment dominates Mtb’s response to drug pressure and suggest novel routes for future drug discovery programs. PMID:27114608

Controlling the Seedbeds of Tuberculosis: Diagnosis and Treatment of Tuberculosis Infection

PubMed Central

Rangaka, Molebogeng X.; Cavalcante, Solange C.; Marais, Ben J.; Thim, Sok; Martinson, Neil A.; Swaminathan, Soumya; Chaisson, Richard E.

2015-01-01

The billions of people with latent tuberculosis infection serve as the seedbeds for future cases of active tuberculosis. Virtually all episodes of tuberculosis disease are preceded by a period of asymptomatic Mycobacterium tuberculosis infection; therefore, identifying infected individuals most likely to progress to disease and treating such subclinical infections to prevent future disease provides a critical opportunity to interrupt tuberculosis transmission and reduce the global burden of tuberculosis disease. Programs focusing on single strategies rather than comprehensive programs that deliver an integrated arsenal for tuberculosis control may continue to struggle. Tuberculosis preventive therapy is a poorly utilized tool that is essential for controlling the reservoirs of disease that drive the current epidemic. Comprehensive control strategies that combine preventive therapy for the most high-risk populations and communities with improved case-finding and treatment, control of transmission and health systems strengthening could ultimately lead to worldwide tuberculosis elimination. This paper outlines challenges to implementation of preventive therapy and provides pragmatic suggestions for overcoming them. It further advocates for tuberculosis preventive therapy as the core of a renewed global focus to implement a comprehensive epidemic control strategy that would reduce new tuberculosis cases to elimination targets. This strategy would be underpinned by accelerated research to further understand the biology of subclinical tuberculosis infections, develop novel diagnostics, and drug regimens specifically for subclinical tuberculosis infection, strengthen health systems, community engagement, and enhance sustainable large scale implementation of preventive therapy programs. PMID:26515679

Evaluation of small molecule tuberculostats for targeting tuberculosis infections of the central nervous system.

PubMed

Bartzatt, Ronald

2012-03-01

Tuberculosis infection of the central nervous system is a serious and frequently fatal disease. Four drugs have been found to very efficiently inhibit the growth of Mycobacterium tuberculosis and are examined for molecular properties that enable penetration of the blood-brain barrier. Drugs 1, 2, and 3 are aromatic compounds having a single bromine atom in ortho, meta, and para-position, respectively, relative to the hydrazide group (-C(O)NHNH2). A paraposition for bromine enabled the strongest inhibition of Mycobacterium tuberculosis. Drug 4 is a hydrazide derivative of ciprofloxacin. All drugs showed molecular properties suitable for targeting tuberculosis infections of the central nervous system. Drugs 1, 2, 3, 4, and isoniazid showed zero violations of the Rule of 5 and potential capability for oral administration. Values of BB (Cbrain/Cblood) suggested that drugs 1, 2, and 3 will be able to penetrate the brain approximately three times greater than isoniazid. Similarly, the calculated value of BB for drug 4 is comparable to that of isoniazid. Calculated values of polar surface area for drugs 1, 2, 3, and isoniazid indicated a potential rate of intestinal absorption of greater than 75% of drug amount present. The intestinal absorption of drug 4 is predicted to be greater than 50% of total amount present. Drug concentrations necessary for achieving MIC50 for 1, 2, 3, 4, and isoniazid are determined to be 65.9 μg/mL, 29.5 μg/mL, 21.5 μg/mL, 36.4 μg/mL, and 16.7 μg/mL, respectively. The position of the bromine atom within drugs 1, 2, and 3 appears to substantially influence the effectiveness of growth inhibition. These compounds show substantial potential for targeting tuberculosis infections within the central nervous system.

Unique Mechanism of Action of the Thiourea Drug Isoxyl on Mycobacterium tuberculosis*

PubMed Central

Phetsuksiri, Benjawan; Jackson, Mary; Scherman, Hataichanok; McNeil, Michael; Besra, Gurdyal S.; Baulard, Alain R.; Slayden, Richard A.; DeBarber, Andrea E.; Barry, Clifton E.; Baird, Mark S.; Crick, Dean C.; Brennan, Patrick J.

2016-01-01

The thiourea isoxyl (thiocarlide; 4,4′-diisoamyloxydiphenylthiourea) is known to be an effective anti-tuberculosis drug, active against a range of multidrug-resistant strains of Mycobacterium tuberculosis and has been used clinically. Little was known of its mode of action. We now demonstrate that isoxyl results in a dose-dependent decrease in the synthesis of oleic and, consequently, tuberculostearic acid in M. tuberculosis with complete inhibition at 3 μg/ml. Synthesis of mycolic acid was also affected. The anti-bacterial effect of isoxyl was partially reversed by supplementing growth medium with oleic acid. The specificity of this inhibition pointed to a Δ9-stearoyl desaturase as the drug target. Development of a cell-free assay for Δ9-desaturase activity allowed direct demonstration of the inhibition of oleic acid synthesis by isoxyl. Interestingly, sterculic acid, a known inhibitor of Δ9-desaturases, emulated the effect of isoxyl on oleic acid synthesis but did not affect mycolic acid synthesis, demonstrating the lack of a relationship between the two effects of the drug. The three putative fatty acid desaturases in the M. tuberculosis genome, desA1, desA2, and desA3, were cloned and expressed in Mycobacterium bovis BCG. Cell-free assays and whole cell labeling demonstrated increased Δ9-desaturase activity and oleic acid synthesis only in the desA3-overexpressing strain and an increase in the minimal inhibitory concentration for isoxyl, indicating that DesA3 is the target of the drug. These results validate membrane-bound Δ9-desaturase, DesA3, as a new therapeutic target, and the thioureas as anti-tuberculosis drugs worthy of further development. PMID:14559907

Computer laboratory notification system via short message service to reduce health care delays in management of tuberculosis in Taiwan.

PubMed

Chen, Tun-Chieh; Lin, Wei-Ru; Lu, Po-Liang; Lin, Chun-Yu; Lin, Shu-Hui; Lin, Chuen-Ju; Feng, Ming-Chu; Chiang, Horn-Che; Chen, Yen-Hsu; Huang, Ming-Shyan

2011-06-01

We investigated the impacts of introducing an expedited acid-fast bacilli (AFB) smear laboratory procedure and an automatic, real-time laboratory notification system by short message with mobile phones on delays in prompt isolation of patients with pulmonary tuberculosis (TB). We analyzed the data for all patients with active pulmonary tuberculosis at a hospital in Kaohsiung, Taiwan, a 1,600-bed medical center, during baseline (January 2004 to February 2005) and intervention (July 2005 to August 2006) phases. A total of 96 and 127 patients with AFB-positive TB was reported during the baseline and intervention phases, respectively. There were significant decreases in health care system delays (ie, laboratory delays: reception of sputum to reporting, P < .001; response delays: reporting to patient isolation, P = .045; and interval from admission to patient isolation, P < .001) during the intervention phase. Significantly fewer nurses were exposed to each patient with active pulmonary TB during the intervention phase (P = .039). Implementation of expedited AFB smear laboratory procedures and an automatic, real-time laboratory mobile notification system significantly decreased delays in the diagnosis and isolation of patients with active TB. Copyright © 2011 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Mosby, Inc. All rights reserved.

Sulfonamide-Based Inhibitors of Aminoglycoside Acetyltransferase Eis Abolish Resistance to Kanamycin in Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garzan, Atefeh; Willby, Melisa J.; Green, Keith D.

A two-drug combination therapy where one drug targets an offending cell and the other targets a resistance mechanism to the first drug is a time-tested, yet underexploited approach to combat or prevent drug resistance. By high-throughput screening, we identified a sulfonamide scaffold that served as a pharmacophore to generate inhibitors of Mycobacterium tuberculosis acetyltransferase Eis, whose upregulation causes resistance to the aminoglycoside (AG) antibiotic kanamycin A (KAN) in Mycobacterium tuberculosis. Rational systematic derivatization of this scaffold to maximize Eis inhibition and abolish the Eis-mediated KAN resistance of M. tuberculosis yielded several highly potent agents. A crystal structure of Eis inmore » complex with one of the most potent inhibitors revealed that the inhibitor bound Eis in the AG-binding pocket held by a conformationally malleable region of Eis (residues 28–37) bearing key hydrophobic residues. These Eis inhibitors are promising leads for preclinical development of innovative AG combination therapies against resistant TB.« less

Colworth prize lecture 2016: exploiting new biological targets from a whole-cell phenotypic screening campaign for TB drug discovery.

PubMed

Moynihan, Patrick Joseph; Besra, Gurdyal S

2017-10-01

Mycobacterium tuberculosis is the aetiological agent of tuberculosis (TB) and is the leading bacterial cause of mortality and morbidity in the world. One third of the world's population is infected with TB, and in conjunction with HIV represents a serious problem that urgently needs addressing. TB is a disease of poverty and mostly affects young adults in their productive years, primarily in the developing world. The most recent report from the World Health Organisation states that 8 million new cases of TB were reported and that ~1.5 million people died from TB. The efficacy of treatment is threatened by the emergence of multi-drug and extensively drug-resistant strains of M. tuberculosis. It can be argued that, globally, M. tuberculosis is the single most important infectious agent affecting mankind. Our research aims to establish an academic-industrial partnership with the goal of discovering new drug targets and hit-to-lead new chemical entities for TB drug discovery.

The World Health Organization and public health research and practice in tuberculosis in India.

PubMed

Banerji, Debabar

2012-01-01

Two major research studies carried out in India fundamentally affected tuberculosis treatment practices worldwide. One study demonstrated that home treatment of the disease is as efficacious as sanatorium treatment. The other showed that BCG vaccination is of little protective value from a public health viewpoint. India had brought together an interdisciplinary team at the National Tuberculosis Institute (NTI) with a mandate to formulate a nationally applicable, socially acceptable, and epidemiologically sound National Tuberculosis Programme (NTP). Work at the NTI laid the foundation for developing an operational research approach to dealing with tuberculosis as a public health problem. The starting point for this was not operational research as enunciated by experts in this field; rather, the NTI achieved operational research by starting from the people. This approach was enthusiastically welcomed by the World Health Organization's Expert Committee on Tuberculosis of 1964. The NTP was designed to "sink or sail with the general health services of the country." The program was dealt a major blow when, starting in 1967, a virtual hysteria was worked up to mobilize most of the health services for imposing birth control on the people. Another blow to the general health services occurred when the WHO joined the rich countries in instituting a number of vertical programs called "Global Initiatives". An ill-conceived, ill-designed, and ill-managed Global Programme for Tuberculosis was one outcome. The WHO has shown rank public health incompetence in taking a very casual approach to operational research and has been downright quixotic in its thinking on controlling tuberculosis worldwide.

Mycobacterium tuberculosis thymidylate synthase gene thyX is essential and potentially bifunctional, while thyA deletion confers resistance to p-aminosalicylic acid

PubMed Central

Houghton, Joanna; Davis, Elaine O.

2012-01-01

Thymidylate synthase (TS) enzymes catalyse the biosynthesis of deoxythymidine monophosphate (dTMP or thymidylate), and so are important for DNA replication and repair. Two different types of TS proteins have been described (ThyA and ThyX), which have different enzymic mechanisms and unrelated structures. Mycobacteria are unusual as they encode both thyA and thyX, and the biological significance of this is not yet understood. Mycobacterium tuberculosis ThyX is thought to be essential and a potential drug target. We therefore analysed M. tuberculosis thyA and thyX expression levels, their essentiality and roles in pathogenesis. We show that both thyA and thyX are expressed in vitro, and that this expression significantly increased within murine macrophages. Under all conditions tested, thyA expression exceeded that of thyX. Mutational studies show that M. tuberculosis thyX is essential, confirming that the enzyme is a plausible drug target. The requirement for M. tuberculosis thyX in the presence of thyA implies that the essential function of ThyX is something other than dTMP synthase. We successfully deleted thyA from the M. tuberculosis genome, and this deletion conferred an in vitro growth defect that was not observed in vivo. Presumably ThyX performs TS activity within M. tuberculosis ΔthyA at a sufficient rate in vivo for normal growth, but the rate in vitro is less than optimal. We also demonstrate that thyA deletion confers M. tuberculosis p-aminosalicylic acid resistance, and show by complementation studies that ThyA T202A and V261G appear to be functional and non-functional, respectively. PMID:22034487

Mycobacterium tuberculosis thymidylate synthase gene thyX is essential and potentially bifunctional, while thyA deletion confers resistance to p-aminosalicylic acid.

PubMed

Fivian-Hughes, Amanda S; Houghton, Joanna; Davis, Elaine O

2012-02-01

Thymidylate synthase (TS) enzymes catalyse the biosynthesis of deoxythymidine monophosphate (dTMP or thymidylate), and so are important for DNA replication and repair. Two different types of TS proteins have been described (ThyA and ThyX), which have different enzymic mechanisms and unrelated structures. Mycobacteria are unusual as they encode both thyA and thyX, and the biological significance of this is not yet understood. Mycobacterium tuberculosis ThyX is thought to be essential and a potential drug target. We therefore analysed M. tuberculosis thyA and thyX expression levels, their essentiality and roles in pathogenesis. We show that both thyA and thyX are expressed in vitro, and that this expression significantly increased within murine macrophages. Under all conditions tested, thyA expression exceeded that of thyX. Mutational studies show that M. tuberculosis thyX is essential, confirming that the enzyme is a plausible drug target. The requirement for M. tuberculosis thyX in the presence of thyA implies that the essential function of ThyX is something other than dTM synthesis [corrected].We successfully deleted thyA from the M. tuberculosis genome, and this deletion conferred an in vitro growth defect that was not observed in vivo. Presumably ThyX performs TS activity within M. tuberculosis ΔthyA at a sufficient rate in vivo for normal growth, but the rate in vitro is less than optimal. We also demonstrate that thyA deletion confers M. tuberculosis p-aminosalicylic acid resistance, and show by complementation studies that ThyA T202A and V261G appear to be functional and non-functional, respectively.

Systematic Survey of Serine Hydrolase Activity in Mycobacterium tuberculosis Defines Changes Associated with Persistence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ortega, Corrie; Anderson, Lindsey N.; Frando, Andrew

The transition between replication and non-replication underlies much of Mycobacterium tuberculosis (Mtb) pathogenicity, as non- or slowly replicating Mtb are responsible for persistence and poor treatment outcomes. Therapeutic targeting of non-replicating, persistent populations is a priority for tuberculosis treatment, but only few drug targets in non-replicating Mtb are currently known. Here, we directly measure the activity of the highly diverse and druggable serine hydrolases (SHs) during active replication and non-replication by activity-based proteomics. We predict serine hydrolase activity for 78 proteins, including 27 proteins with previously unknown function, and identify 37 SHs that remain active even in the absence ofmore » replication, providing a set of candidate persistence targets. Non-replication was associated with large shifts in the activity of the majority of SHs. These activity changes were largely independent of SH abundance, indicating extensive post-translational regulation. By probing a large cross-section of druggable Mtb enzyme space during replication and non-replication, we identify new SHs and suggest new persistence targets.« less

Targeted pulmonary delivery of inducers of host macrophage autophagy as a potential host-directed chemotherapy of tuberculosis.

PubMed

Gupta, Anuradha; Misra, Amit; Deretic, Vojo

2016-07-01

One of the promising host-directed chemotherapeutic interventions in tuberculosis (TB) is based on inducing autophagy as an immune effector. Here we consider the strengths and weaknesses of potential autophagy-based pharmacological intervention. Using the existing drugs that induce autophagy is an option, but it has limitations given the broad role of autophagy in most cells, tissues, and organs. Thus, it may be desirable that the agent being used to modulate autophagy is applied in a targeted manner, e.g. delivered to affected tissues, with infected macrophages being an obvious choice. This review addresses the advantages and disadvantages of delivering drugs to induce autophagy in M. tuberculosis-infected macrophages. One option, already being tested in models, is to design particles for inhalation delivery to lung macrophages. The choice of drugs, drug release kinetics and intracellular residence times, non-target cell exposure and feasibility of use by patients is discussed. We term here this (still experimental) approach, of compartment-targeting, autophagy-based, host-directed therapy as "Track-II antituberculosis chemotherapy." Copyright © 2016. Published by Elsevier B.V.

The South African Tuberculosis Care Cascade: Estimated Losses and Methodological Challenges

PubMed Central

Naidoo, Pren; Theron, Grant; Rangaka, Molebogeng X; Chihota, Violet N; Vaughan, Louise; Brey, Zameer O; Pillay, Yogan

2017-01-01

Abstract Background While tuberculosis incidence and mortality are declining in South Africa, meeting the goals of the End TB Strategy requires an invigorated programmatic response informed by accurate data. Enumerating the losses at each step in the care cascade enables appropriate targeting of interventions and resources. Methods We estimated the tuberculosis burden; the number and proportion of individuals with tuberculosis who accessed tests, had tuberculosis diagnosed, initiated treatment, and successfully completed treatment for all tuberculosis cases, for those with drug-susceptible tuberculosis (including human immunodeficiency virus (HIV)–coinfected cases) and rifampicin-resistant tuberculosis. Estimates were derived from national electronic tuberculosis register data, laboratory data, and published studies. Results The overall tuberculosis burden was estimated to be 532005 cases (range, 333760–764480 cases), with successful completion of treatment in 53% of cases. Losses occurred at multiple steps: 5% at test access, 13% at diagnosis, 12% at treatment initiation, and 17% at successful treatment completion. Overall losses were similar among all drug-susceptible cases and those with HIV coinfection (54% and 52%, respectively, successfully completed treatment). Losses were substantially higher among rifampicin- resistant cases, with only 22% successfully completing treatment. Conclusion Although the vast majority of individuals with tuberculosis engaged the public health system, just over half were successfully treated. Urgent efforts are required to improve implementation of existing policies and protocols to close gaps in tuberculosis diagnosis, treatment initiation, and successful treatment completion. PMID:29117342

The Cording Phenotype of Mycobacterium tuberculosis Induces the Formation of Extracellular Traps in Human Macrophages.

PubMed

Kalsum, Sadaf; Braian, Clara; Koeken, Valerie A C M; Raffetseder, Johanna; Lindroth, Margaretha; van Crevel, Reinout; Lerm, Maria

2017-01-01

The causative agent of tuberculosis, Mycobacterium tuberculosis , shares several characteristics with organisms that produce biofilms during infections. One of these is the ability to form tight bundles also known as cords. However, little is known of the physiological relevance of the cording phenotype. In this study, we investigated whether cord-forming M. tuberculosis induce the formation of macrophage extracellular traps (METs) in human monocyte-derived macrophages. Macrophages have previously been shown to produce extracellular traps in response to various stimuli. We optimized bacterial culturing conditions that favored the formation of the cord-forming phenotype as verified by scanning electron microscopy. Microscopy analysis of METs formation during experimental infection of macrophages with M. tuberculosis revealed that cord-forming M. tuberculosis induced significantly more METs compared to the non-cording phenotype. Deletion of early secreted antigenic target-6 which is an important virulence factor of M. tuberculosis , abrogated the ability of the bacteria to induce METs. The release of extracellular DNA from host cells during infection may represent a defense mechanism against pathogens that are difficult to internalize, including cord-forming M. tuberculosis .

Trend in case detection rate for all tuberculosis cases notified in Ebonyi, Southeastern Nigeria during 1999-2009.

PubMed

Ukwaja, Kingsley Nnanna; Alobu, Isaac; Ifebunandu, Ngozi Appolonia; Osakwe, Chijioke; Igwenyi, Chika

2013-01-01

Unlike previous annual WHO tuberculosis reports that reported case detection rate for only smear-positive tuberculosis cases, the 2010 report presented case detection rate for all tuberculosis cases notified in line with the current Stop TB strategy. To help us understand how tuberculosis control programmes performed in terms of detecting tuberculosis, there is need to document the trend in case detection rate for all tuberculosis cases notified in high burden countries. This evidence is currently lacking from Nigeria. Therefore, this study aimed to assess the trend in case detection rate for all tuberculosis cases notified from Ebonyi state compared to Nigeria national figures. Reports of tuberculosis cases notified between 1999 and 2009 were reviewed from the Ebonyi State Ministry of Health tuberculosis quarterly reports. Tuberculosis case detection rates were computed according to WHO guidelines. 22, 508 patients with all forms of tuberculosis were notified during the study. Case detection rate for all tuberculosis rose from 27% in 1999 to gradually reach a peak of 40% during 2007 to 2008 before a slight decline in 2009 to 38%. However, the national case detection rate for all tuberculosis cases in Nigeria rose from 7% in 1999 and progressively increased to reach a peak of 19% during 2008 and 2009. Since the introduction of DOTS in Ebonyi, the programme has achieved 40% case detection rate for all tuberculosis cases - about 20% better than national figures. However, with the current low case detection rates, alternative mechanisms are needed to achieve the current global stop- TB targets in Nigeria.

Analysis of uracil phosphoribosyltransferase expression in Mycobacterium tuberculosis and evaluation of upp knockout strain in infected mice.

PubMed

Villela, Anne Drumond; Pham, Ha; Jones, Victoria; Grzegorzewicz, Anna E; Rodrigues-Junior, Valnês da Silva; Campos, Maria Martha; Basso, Luiz Augusto; Jackson, Mary; Santos, Diógenes Santiago

2017-02-01

The upp (Rv3309c)-encoded uracil phosphoribosyltransferase from Mycobacterium tuberculosis (MtUPRT) converts uracil and 5-phosphoribosyl-α-1-pyrophosphate into pyrophosphate and uridine 5΄-monophosphate, the precursor of all pyrimidine nucleotides. A M. tuberculosis knockout strain for upp gene was generated by allelic replacement. Knockout and complemented strains were validated by a functional assay of uracil incorporation. A basal level of MtUPRT expression is shown to be independent of either growth medium used, addition of bases, or oxygen presence/absence. The upp disruption does not affect M. tuberculosis growth in Middlebrook 7H9 medium, and it is not required for M. tuberculosis virulence in a mouse model of infection. Thus, MtUPRT is unlikely to be a good target for drugs against M. tuberculosis. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

New drug candidates and therapeutic targets for tuberculosis therapy.

PubMed

Zhang, Ying; Post-Martens, Katrin; Denkin, Steven

2006-01-01

Despite advances in chemotherapy and the BCG (Bacillus Calmette-Guérin) vaccine, tuberculosis remains a significant infectious disease. Although it can be cured, the therapy takes at least 6-9 months, and the laborious and lengthy treatment brings with it dangers of noncompliance, significant toxicity and drug resistance. The increasing emergence of drug resistance and the problem of mycobacterial persistence highlight the need to develop novel TB drugs that are active against drug resistant bacteria but, more importantly, kill persistent bacteria and shorten the length of treatment. Recent new and exciting developments in tuberculosis drug discovery show good promise of a possible revolution in the chemotherapy of tuberculosis.

A Mycobacterium tuberculosis Cytochrome bd Oxidase Mutant Is Hypersensitive to Bedaquiline

PubMed Central

Hartman, Travis E.

2014-01-01

ABSTRACT The new medicinal compound bedaquiline (BDQ) kills Mycobacterium tuberculosis by inhibiting F1Fo-ATP synthase. BDQ is bacteriostatic for 4 to 7 days and kills relatively slowly compared to other frontline tuberculosis (TB) drugs. Here we show that killing with BDQ can be improved significantly by inhibiting cytochrome bd oxidase, a non-proton-pumping terminal oxidase. BDQ was instantly bactericidal against a cytochrome bd oxidase null mutant of M. tuberculosis, and the rate of killing was increased by more than 50%. We propose that this exclusively bacterial enzyme should be a high-priority target for new drug discovery. PMID:25028424

The characteristic profiles of PD-1 and PD-L1 expressions and dynamic changes during treatment in active tuberculosis.

PubMed

Shen, Lei; Shi, Hong; Gao, Yan; Ou, Qinfang; Liu, Qianqian; Liu, Yuanyuan; Wu, Jing; Zhang, Wenhong; Fan, Lin; Shao, Lingyun

2016-12-01

PD-1 is a cell surface receptor of activated T and B lymphocytes and it's role in tuberculosis is controversial because of lack of congruence between clinical study and animal model. To investigate the immunological pathogenesis mechanisms of tuberculosis and to develop the immune therapy target essential for controlling tuberculosis, here we explored the expression characteristics and dynamic changes of PD-1/PD-L1 pathway in different CD4+T cell subsets. We enrolled 24 human subjects including 15 active tuberculosis (ATB) patients and 9 healthy donors (HD). The expressions of PD-1 and PD-L1 on CD4+T cells increased significantly in ATB patients than HD. ATB patients had a higher proportion of regulatory T cells (Treg, CD4 + CD25 + Foxp3+) than HD. The expressions of PD-1 and PD-L1 increased remarkably on CD4+T cell subsets, including Treg cells, Tresp (CD4 + CD25 - ) cells and Teff (CD4 + CD25 + Foxp3-) cells. Finally, clinical improvement following effective anti-TB therapy is correlated with significantly decreased expression of PD-1 in Tresp and Teff cells, but not in Treg cells. Thus, expression profiles of PD-1 in T cell subpopulations may be used as a candidate to predict the clinical efficacy of anti-tuberculosis therapy. Modulation of PD-1/PD-L1 pathway in CD4 subsets may offer an immunotherapy target for the control of tuberculosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Smear Conversion, Treatment Outcomes and the Time of Default in Registered Tuberculosis Patients on RNTCP DOTS in Puducherry, Southern India

PubMed Central

Jayakumar, Niranjana; Gnanasekaran, Dhivyalakshmi

2014-01-01

Background: Revised National Tuberculosis Control Programme (RNTCP) in India has achieved improved cure rates. Objectives: This study describes the achievements under RNTCP in terms of conversion rates, treatment outcomes and pattern of time of default in patients on directly observed short-course treatment for Tuberculosis in Puducherry, Southern India. Settings: Retrospective cohort study; Tuberculosis Unit in District Tuberculosis Centre, Puducherry, India. Materials and Methods: Cohort analysis of patients of registered at the Tuberculosis Unit during 1st and 2nd quarter of the year 2011. Details about sputum conversion, treatment outcome and time of default were obtained from the tuberculosis register. Statistical Analysis: Kaplan-Meier plots & log rank tests. Results: RNTCP targets with respect to success rate (85.7%), death rate (2.7%) and failure rate (2.1%) in new cases have been achieved but the sputum conversion rate (88%) and default rate (5.9%) targets have not been achieved. The overall default rate for all registered TB patients was 7.4%; significantly higher in category II. In retreatment cases registered as treatment after default, the default rate was high (9%). The cumulative default rate; though similar in the initial two months of treatment; was consistently higher in category II as compared to that in category I. Nearly 40% of all defaulters interrupted treatment between the second and fourth month after treatment initiation. Conclusion: Defaulting from treatment is more common among the retreatment cases and usually occurs during the transition phase from intensive phase to continuation phase. PMID:25478371

Knowledge, Risk Perception and Practice Regarding Tuberculosis Transmission among Long Distance Bus Drivers in Addis Ababa, Ethiopia: A Cross Sectional Study.

PubMed

Gebrehiwot, Tsegaye Tewelde; Tesfamichael, Fessahaye Alemseged

2017-11-01

Window opening during bus transportation is recommended as a tuberculosis prevention strategy.Yet, drivers are affected by lack knowledge and risk perception of passengers and assistants. Boosting knowledge of and notifying the high risk of tuberculosis transmission for every passenger could be too costly. However, strategies targeting bus drivers as key agents unlike targeting all passengers might be less costly for window opening. Data were collected from November 18/2014 to December 21/2014 in inter-region bus stations of Addis Ababa using cross sectional study design. Samples of 306 participants were selected using simple random sampling, and data were collected through face-to-face interview. Data were entered into Epi-data version 3.1 andanalyzed using IBM SPSS version 21. From a sample of 306 bus drivers, 303 were interviewed. Nine in ten and nearly half of participants believed in the need for opening all windows and avoiding overcrowding of passengers as TB preventive measures respectively. Few bus drivers (7.3%) believed that bus drivers and their assistants could be at risk of tuberculosis. The majority (85.7%) of bus drivers opened side window the whole day without precondition. Hearing tuberculosis related information from radio was a promoting factor for tuberculosis preventive measures among bus drivers. Tuberculosis preventive practices and knowledge of bus drivers seempositive (opportunities), despite their low risk perception (challenge). Using the opportunity, further empowering bus drivers to persuade passengers and assistants to open all the rest of the windows is needed.

Crystal structures of the transpeptidase domain of the Mycobacterium tuberculosis penicillin-binding protein PonA1 reveal potential mechanisms of antibiotic resistance.

PubMed

Filippova, Ekaterina V; Kieser, Karen J; Luan, Chi-Hao; Wawrzak, Zdzislaw; Kiryukhina, Olga; Rubin, Eric J; Anderson, Wayne F

2016-06-01

Mycobacterium tuberculosis is a human respiratory pathogen that causes the deadly disease tuberculosis. The rapid global spread of antibiotic-resistant M. tuberculosis makes tuberculosis infections difficult to treat. To overcome this problem new effective antimicrobial strategies are urgently needed. One promising target for new therapeutic approaches is PonA1, a class A penicillin-binding protein, which is required for maintaining physiological cell wall synthesis and cell shape during growth in mycobacteria. Here, crystal structures of the transpeptidase domain, the enzymatic domain responsible for penicillin binding, of PonA1 from M. tuberculosis in the inhibitor-free form and in complex with penicillin V are reported. We used site-directed mutagenesis, antibiotic profiling experiments, and fluorescence thermal shift assays to measure PonA1's sensitivity to different classes of β-lactams. Structural comparison of the PonA1 apo-form and the antibiotic-bound form shows that binding of penicillin V induces conformational changes in the position of the loop β4'-α3 surrounding the penicillin-binding site. We have also found that binding of different antibiotics including penicillin V positively impacts protein stability, while other tested β-lactams such as clavulanate or meropenem resulted in destabilization of PonA1. Our antibiotic profiling experiments indicate that the transpeptidase activity of PonA1 in both M. tuberculosis and M. smegmatis mediates tolerance to specific cell wall-targeting antibiotics, particularly to penicillin V and meropenem. Because M. tuberculosis is an important human pathogen, these structural data provide a template to design novel transpeptidase inhibitors to treat tuberculosis infections. Structural data are available in the PDB database under the accession numbers 5CRF and 5CXW. © 2016 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

Use of Microsphere Technology for Targeted Delivery of Rifampin to Mycobacterium tuberculosis-Infected Macrophages

PubMed Central

Barrow, Esther L. W.; Winchester, Gary A.; Staas, Jay K.; Quenelle, Debra C.; Barrow, William W.

1998-01-01

Microsphere technology was used to develop formulations of rifampin for targeted delivery to host macrophages. These formulations were prepared by using biocompatible polymeric excipients of lactide and glycolide copolymers. Release characteristics were examined in vitro and also in two monocytic cell lines, the murine J774 and the human Mono Mac 6 cell lines. Bioassay assessment of cell culture supernatants from monocyte cell lines showed release of bioactive rifampin during a 7-day experimental period. Treatment of Mycobacterium tuberculosis H37Rv-infected monocyte cell lines with rifampin-loaded microspheres resulted in a significant decrease in numbers of CFU at 7 days following initial infection, even though only 8% of the microsphere-loaded rifampin was released. The levels of rifampin released from microsphere formulations within monocytes were more effective at reducing M. tuberculosis intracellular growth than equivalent doses of rifampin given as a free drug. These results demonstrate that rifampin-loaded microspheres can be formulated for effective sustained and targeted delivery to host macrophages. PMID:9756777

Mycolic acids: deciphering and targeting the Achilles' heel of the tubercle bacillus

PubMed Central

Nataraj, Vijayashankar; Varela, Cristian; Javid, Asma; Singh, Albel; Besra, Gurdyal S.

2015-01-01

Summary Mycolic acids are unique long chain fatty acids found in the lipid‐rich cell walls of mycobacteria including the tubercle bacillus M ycobacterium tuberculosis. Essential for viability and virulence, enzymes involved in the biosynthesis of mycolic acids represent novel targets for drug development. This is particularly relevant to the impact on global health given the rise of multidrug resistant and extensively drug resistant strains of M . tuberculosis. In this review, we discuss recent advances in our understanding of how mycolic acid are synthesised, especially the potential role of specialised fatty acid synthase complexes. Also, we examine the role of a recently reported mycolic acid transporter MmpL3 with reference to several reports of the targeting of this transporter by diverse compounds with anti‐M . tuberculosis activity. Additionally, we consider recent findings that place mycolic acid biosynthesis in the context of the cell biology of the bacterium, viz its localisation and co‐ordination with the bacterial cytoskeleton, and its role beyond maintaining cell envelope integrity. PMID:26135034

Reagent Precoated Targets for Rapid In-Tissue Derivatization of the Anti-Tuberculosis Drug Isoniazid Followed by MALDI Imaging Mass Spectrometry

NASA Astrophysics Data System (ADS)

Manier, M. Lisa; Reyzer, Michelle L.; Goh, Anne; Dartois, Veronique; Via, Laura E.; Barry, Clifton E.; Caprioli, Richard M.

2011-08-01

Isoniazid (INH) is an important component of front-line anti-tuberculosis therapy with good serum pharmacokinetics but unknown ability to penetrate tuberculous lesions. However, endogenous background interferences hinder our ability to directly analyze INH in tissues. Chemical derivatization has been successfully used to measure isoniazid directly from tissue samples using matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS). MALDI targets were pretreated with trans-cinnamaldehyde (CA) prior to mounting tissue slices. Isoniazid present in the tissues was efficiently derivatized and the INH-CA product measured by MS/MS. Precoating of MALDI targets allows the tissues to be directly thaw-mounted and derivatized, thus simplifying the preparation. A time-course series of tissues from tuberculosis infected/INH dosed animals were assayed and the MALDI MS/MS response correlates well with the amount of INH determined to be in the tissues by high-performance liquid chromatography (HPLC)-MS/MS.

The outcome of tuberculosis treatment in subjects with chronic kidney disease in Brazil: a multinomial analysis*

PubMed Central

Reis-Santos, Barbara; Gomes, Teresa; Horta, Bernardo Lessa; Maciel, Ethel Leonor Noia

2013-01-01

OBJECTIVE: To analyze the association between clinical/epidemiological characteristics and outcomes of tuberculosis treatment in patients with concomitant tuberculosis and chronic kidney disease (CKD) in Brazil. METHODS: We used the Brazilian Ministry of Health National Case Registry Database to identify patients with tuberculosis and CKD, treated between 2007 and 2011. The tuberculosis treatment outcomes were compared with epidemiological and clinical characteristics of the subjects using a hierarchical multinomial logistic regression model, in which cure was the reference outcome. RESULTS: The prevalence of CKD among patients with tuberculosis was 0.4% (95% CI: 0.37-0.42%). The sample comprised 1,077 subjects. The outcomes were cure, in 58%; treatment abandonment, in 7%; death from tuberculosis, in 13%; and death from other causes, in 22%. The characteristics that differentiated the ORs for treatment abandonment or death were age; alcoholism; AIDS; previous noncompliance with treatment; transfer to another facility; suspected tuberculosis on chest X-ray; positive results in the first smear microscopy; and indications for/use of directly observed treatment, short-course strategy. CONCLUSIONS: Our data indicate the importance of sociodemographic characteristics for the diagnosis of tuberculosis in patients with CKD and underscore the need for tuberculosis control strategies targeting patients with chronic noncommunicable diseases, such as CKD. PMID:24310632

Understanding Latent Tuberculosis: A Moving Target

PubMed Central

Lin, Philana Ling; Flynn, JoAnne L.

2012-01-01

Tuberculosis (TB) remains a threat to the health of people worldwide. Infection with Mycobacterium tuberculosis can result in active TB or, more commonly, latent infection. Latently infected persons, of which there are estimated to be ~2 billion in the world, represent an enormous reservoir of potential reactivation TB, which can spread to other people. The immunology of TB is complex and multifaceted. Identifying the immune mechanisms that lead to control of initial infection and prevent reactivation of latent infection is crucial to combating this disease. PMID:20562268

Total synthesis of cyclomarins A, C and D, marine cyclic peptides with interesting anti-tuberculosis and anti-malaria activities.

PubMed

Barbie, Philipp; Kazmaier, Uli

2016-07-07

Cyclomarins are cyclic heptapeptides containing four unusual amino acids. New synthetic protocols toward their synthesis have been developed, leading to the synthesis and biological evaluation of three natural occurring cyclomarins. Interestingly, cyclomarins address two completely different targets: Clp C1, a subunit of the caseinolytic protease of Mycobacterium tuberculosis (MTB), as well as PfAp3Ase of Plasmodium falciparum. Therefore, cyclomarins are interesting lead structures for the development of drugs against tuberculosis and malaria.

Global Fund-supported programmes contribution to international targets and the Millennium Development Goals: an initial analysis.

PubMed

Komatsu, Ryuichi; Low-Beer, Daniel; Schwartländer, Bernhard

2007-10-01

The Global Fund to Fight AIDS, Tuberculosis and Malaria is one of the largest funders to fight these diseases. This paper discusses the programmatic contribution of Global Fund-supported programmes towards achieving international targets and Millennium Development Goals, using data from Global Fund grants. Results until June 2006 of 333 grants supported by the Global Fund in 127 countries were aggregated and compared against international targets for HIV/AIDS, tuberculosis and malaria. Progress reports to the Global Fund secretariat were used as a basis to calculate results. Service delivery indicators for antiretrovirals (ARV) for HIV/AIDS, case detection under the DOTS strategy for tuberculosis (DOTS) and insecticide-treated nets (ITNs) for malaria prevention were selected to estimate programmatic contributions to international targets for the three diseases. Targets of Global Fund-supported programmes were projected based on proposals for Rounds 1 to 4 and compared to international targets for 2009. Results for Global Fund-supported programmes total 544,000 people on ARV, 1.4 million on DOTS and 11.3 million for ITNs by June 2006. Global Fund-supported programmes contributed 18% of international ARV targets, 29% of DOTS targets and 9% of ITNs in sub-Saharan Africa by mid-2006. Existing Global Fund-supported programmes have agreed targets that are projected to account for 19% of the international target for ARV delivery expected for 2009, 28% of the international target for DOTS and 84% of ITN targets in sub-Saharan Africa. Global Fund-supported programmes have already contributed substantially to international targets by mid-2006, but there is a still significant gap. Considerably greater financial support is needed, particularly for HIV, in order to achieve international targets for 2009.

Management of children exposed to Mycobacterium tuberculosis: a public health evaluation in West Java, Indonesia.

PubMed

Rutherford, Merrin E; Ruslami, Rovina; Anselmo, Melissa; Alisjahbana, Bachti; Yulianti, Neti; Sampurno, Hedy; van Crevel, Reinout; Hill, Philip C

2013-12-01

To investigate qualitatively and quantitatively the performance of a programme for managing the child contacts of adult tuberculosis patients in Indonesia. A public health evaluation framework was used to assess gaps in a child contact management programme at a lung clinic. Targets for programme performance indicators were derived from established programme indicator targets, the scientific literature and expert opinion. Compliance with tuberculosis screening, the initiation of isoniazid preventive therapy in children younger than 5 years, the accuracy of tuberculosis diagnosis and adherence to preventive therapy were assessed in 755 child contacts in two cohorts. In addition, 22 primary caregivers and 34 clinic staff were interviewed to evaluate knowledge and acceptance of child contact management. The cost to caregivers was recorded. Gaps between observed and target indicator values were quantified. THE GAPS BETWEEN OBSERVED AND TARGET PERFORMANCE INDICATORS WERE: 82% for screening compliance; 64 to 100% for diagnostic accuracy, 50% for the initiation of preventive therapy, 54% for adherence to therapy and 50% for costs. Many staff did not have adequate knowledge of, or an appropriate attitude towards, child contact management, especially regarding isoniazid preventive therapy. Caregivers had good knowledge of screening but not of preventive therapy and had difficulty travelling to the clinic and paying costs. The study identified widespread gaps in the performance of a child contact management system in Indonesia, all of which appear amenable to intervention. The public health evaluation framework used could be applied in other settings where child contact management is failing.

Evidence for the role of Mycobacterium tuberculosis RecG helicase in DNA repair and recombination.

PubMed

Thakur, Roshan S; Basavaraju, Shivakumar; Somyajit, Kumar; Jain, Akshatha; Subramanya, Shreelakshmi; Muniyappa, Kalappa; Nagaraju, Ganesh

2013-04-01

In order to survive and replicate in a variety of stressful conditions during its life cycle, Mycobacterium tuberculosis must possess mechanisms to safeguard the integrity of the genome. Although DNA repair and recombination related genes are thought to play key roles in the repair of damaged DNA in all organisms, so far only a few of them have been functionally characterized in the tubercle bacillus. In this study, we show that M. tuberculosis RecG (MtRecG) expression was induced in response to different genotoxic agents. Strikingly, expression of MtRecG in Escherichia coli ∆recG mutant strain provided protection against mitomycin C, methyl methane sulfonate and UV induced cell death. Purified MtRecG exhibited higher binding affinity for the Holliday junction (HJ) compared with a number of canonical recombinational DNA repair intermediates. Notably, although MtRecG binds at the core of the mobile and immobile HJs, and with higher binding affinity for the immobile HJ, branch migration was evident only in the case of the mobile HJ. Furthermore, immobile HJs stimulate MtRecG ATPase activity less efficiently than mobile HJs. In addition to HJ substrates, MtRecG exhibited binding affinity for a variety of branched DNA structures including three-way junctions, replication forks, flap structures, forked duplex and a D-loop structure, but demonstrated strong unwinding activity on replication fork and flap DNA structures. Together, these results support that MtRecG plays an important role in processes related to DNA metabolism under normal as well as stress conditions. © 2013 The Authors Journal compilation © 2013 FEBS.

Human CD8 T lymphocytes recognize Mycobacterium tuberculosis antigens presented by HLA-E during active tuberculosis and express type 2 cytokines.

PubMed

Caccamo, Nadia; Pietra, Gabriella; Sullivan, Lucy C; Brooks, Andrew G; Prezzemolo, Teresa; La Manna, Marco P; Di Liberto, Diana; Joosten, Simone A; van Meijgaarden, Krista E; Di Carlo, Paola; Titone, Lucina; Moretta, Lorenzo; Mingari, Maria C; Ottenhoff, Tom H M; Dieli, Francesco

2015-04-01

CD8 T cells contribute to protective immunity against Mycobacterium tuberculosis. In humans, M. tuberculosis reactive CD8 T cells typically recognize peptides associated to classical MHC class Ia molecules, but little information is available on CD8 T cells recognizing M. tuberculosis Ags presented by nonclassical MHC class Ib molecules. We show here that CD8 T cells from tuberculosis (TB) patients recognize HLA-E-binding M. tuberculosis peptides in a CD3/TCR αβ mediated and CD8-dependent manner, and represent an additional type of effector cells playing a role in immune response to M. tuberculosis during active infection. HLA-E-restricted recognition of M. tuberculosis peptides is detectable by a significant enhanced ex vivo frequency of tetramer-specific circulating CD8 T cells during active TB. These CD8 T cells produce type 2 cytokines upon antigenic in vitro stimulation, help B cells for Ab production, and mediate limited TRAIL-dependent cytolytic and microbicidal activity toward M. tuberculosis infected target cells. Our results, together with the finding that HLA-E/M. tuberculosis peptide specific CD8 T cells are detected in TB patients with or without HIV coinfection, suggest that this is a new human T-cell population that participates in immune response in TB. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Genotype heterogeneity of Mycobacterium tuberculosis within geospatial hotspots suggests foci of imported infection in Sydney, Australia.

PubMed

Gurjav, Ulziijargal; Jelfs, Peter; Hill-Cawthorne, Grant A; Marais, Ben J; Sintchenko, Vitali

2016-06-01

In recent years the State of New South Wales (NSW), Australia, has maintained a low tuberculosis incidence rate with little evidence of local transmission. Nearly 90% of notified tuberculosis cases occurred in people born in tuberculosis-endemic countries. We analyzed geographic, epidemiological and genotypic data of all culture-confirmed tuberculosis cases to identify the bacterial and demographic determinants of tuberculosis hotspot areas in NSW. Standard 24-loci mycobacterium interspersed repetitive unit-variable number tandem repeat (MIRU-24) typing was performed on all isolates recovered between 2009 and 2013. In total 1692/1841 (91.9%) cases with confirmed Mycobacterium tuberculosis infection had complete MIRU-24 and demographic data and were included in the study. Despite some year-to-year variability, spatio-temporal analysis identified four tuberculosis hotspots. The incidence rate and the relative risk of tuberculosis in these hotspots were 2- to 10-fold and 4- to 8-fold higher than the state average, respectively. MIRU-24 profiles of M. tuberculosis isolates associated with these hotspots revealed high levels of heterogeneity. This suggests that these spatio-temporal hotspots, within this low incidence setting, can represent areas of predominantly imported infection rather than clusters of cases due to local transmission. These findings provide important epidemiological insight and demonstrate the value of combining tuberculosis genotyping and spatiotemporal data to guide better-targeted public health interventions. Copyright © 2015 Elsevier B.V. All rights reserved.

Identifying Hotspots of Multidrug-Resistant Tuberculosis Transmission Using Spatial and Molecular Genetic Data

PubMed Central

Zelner, Jonathan L.; Murray, Megan B.; Becerra, Mercedes C.; Galea, Jerome; Lecca, Leonid; Calderon, Roger; Yataco, Rosa; Contreras, Carmen; Zhang, Zibiao; Manjourides, Justin; Grenfell, Bryan T.; Cohen, Ted

2016-01-01

Background. We aimed to identify and determine the etiology of “hotspots” of concentrated multidrug-resistant tuberculosis (MDR-tuberculosis) risk in Lima, Peru. Methods. From 2009 to 2012, we conducted a prospective cohort study among households of tuberculosis cases from 106 health center (HC) areas in Lima, Peru. All notified tuberculosis cases and their household contacts were followed for 1 year. Symptomatic individuals were screened by microscopy and culture; positive cultures were tested for drug susceptibility (DST) and genotyped by 24-loci mycobacterial interspersed repetitive units-variable-number tandem repeats (MIRU-VNTR). Results. 3286 individuals with culture-confirmed disease, DST, and 24-loci MIRU-VNTR were included in our analysis. Our analysis reveals: (1) heterogeneity in annual per-capita incidence of tuberculosis and MDR-tuberculosis by HC, with a rate of MDR-tuberculosis 89 times greater (95% confidence interval [CI], 54,185) in the most-affected versus the least-affected HC; (2) high risk for MDR-tuberculosis in a region spanning several HCs (odds ratio = 3.19, 95% CI, 2.33, 4.36); and (3) spatial aggregation of MDR-tuberculosis genotypes, suggesting localized transmission. Conclusions. These findings reveal that localized transmission is an important driver of the epidemic of MDR-tuberculosis in Lima. Efforts to interrupt transmission may be most effective if targeted to this area of the city. PMID:26175455

Exploiting the synthetic lethality between terminal respiratory oxidases to kill Mycobacterium tuberculosis and clear host infection

PubMed Central

Kalia, Nitin P.; Hasenoehrl, Erik J.; Ab Rahman, Nurlilah B.; Koh, Vanessa H.; Ang, Michelle L. T.; Sajorda, Dannah R.; Hards, Kiel; Grüber, Gerhard; Alonso, Sylvie; Cook, Gregory M.; Berney, Michael; Pethe, Kevin

2017-01-01

The recent discovery of small molecules targeting the cytochrome bc1:aa3 in Mycobacterium tuberculosis triggered interest in the terminal respiratory oxidases for antituberculosis drug development. The mycobacterial cytochrome bc1:aa3 consists of a menaquinone:cytochrome c reductase (bc1) and a cytochrome aa3-type oxidase. The clinical-stage drug candidate Q203 interferes with the function of the subunit b of the menaquinone:cytochrome c reductase. Despite the affinity of Q203 for the bc1:aa3 complex, the drug is only bacteriostatic and does not kill drug-tolerant persisters. This raises the possibility that the alternate terminal bd-type oxidase (cytochrome bd oxidase) is capable of maintaining a membrane potential and menaquinol oxidation in the presence of Q203. Here, we show that the electron flow through the cytochrome bd oxidase is sufficient to maintain respiration and ATP synthesis at a level high enough to protect M. tuberculosis from Q203-induced bacterial death. Upon genetic deletion of the cytochrome bd oxidase-encoding genes cydAB, Q203 inhibited mycobacterial respiration completely, became bactericidal, killed drug-tolerant mycobacterial persisters, and rapidly cleared M. tuberculosis infection in vivo. These results indicate a synthetic lethal interaction between the two terminal respiratory oxidases that can be exploited for anti-TB drug development. Our findings should be considered in the clinical development of drugs targeting the cytochrome bc1:aa3, as well as for the development of a drug combination targeting oxidative phosphorylation in M. tuberculosis. PMID:28652330

The South African Tuberculosis Care Cascade: Estimated Losses and Methodological Challenges.

PubMed

Naidoo, Pren; Theron, Grant; Rangaka, Molebogeng X; Chihota, Violet N; Vaughan, Louise; Brey, Zameer O; Pillay, Yogan

2017-11-06

While tuberculosis incidence and mortality are declining in South Africa, meeting the goals of the End TB Strategy requires an invigorated programmatic response informed by accurate data. Enumerating the losses at each step in the care cascade enables appropriate targeting of interventions and resources. We estimated the tuberculosis burden; the number and proportion of individuals with tuberculosis who accessed tests, had tuberculosis diagnosed, initiated treatment, and successfully completed treatment for all tuberculosis cases, for those with drug-susceptible tuberculosis (including human immunodeficiency virus (HIV)-coinfected cases) and rifampicin-resistant tuberculosis. Estimates were derived from national electronic tuberculosis register data, laboratory data, and published studies. The overall tuberculosis burden was estimated to be 532005 cases (range, 333760-764480 cases), with successful completion of treatment in 53% of cases. Losses occurred at multiple steps: 5% at test access, 13% at diagnosis, 12% at treatment initiation, and 17% at successful treatment completion. Overall losses were similar among all drug-susceptible cases and those with HIV coinfection (54% and 52%, respectively, successfully completed treatment). Losses were substantially higher among rifampicin- resistant cases, with only 22% successfully completing treatment. Although the vast majority of individuals with tuberculosis engaged the public health system, just over half were successfully treated. Urgent efforts are required to improve implementation of existing policies and protocols to close gaps in tuberculosis diagnosis, treatment initiation, and successful treatment completion. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.

Mycobacterium tuberculosis exploits the formation of new blood vessels for its dissemination

PubMed Central

Polena, Helena; Boudou, Frédéric; Tilleul, Sylvain; Dubois-Colas, Nicolas; Lecointe, Cécile; Rakotosamimanana, Niaina; Pelizzola, Mattia; Andriamandimby, Soa Fy; Raharimanga, Vaomalala; Charles, Patricia; Herrmann, Jean-Louis; Ricciardi-Castagnoli, Paola; Rasolofo, Voahangy; Gicquel, Brigitte; Tailleux, Ludovic

2016-01-01

The mechanisms by which the airborne pathogen Mycobacterium tuberculosis spreads within the lung and leaves its primary niche to colonize other organs, thus inducing extrapulmonary forms of tuberculosis (TB) in humans, remains poorly understood. Herein, we used a transcriptomic approach to investigate the host cell gene expression profile in M. tuberculosis–infected human macrophages (ΜΦ). We identified 33 genes, encoding proteins involved in angiogenesis, for which the expression was significantly modified during infection, and we show that the potent angiogenic factor VEGF is secreted by M. tuberculosis-infected ΜΦ, in an RD1-dependent manner. In vivo these factors promote the formation of blood vessels in murine models of the disease. Inhibiting angiogenesis, via VEGF inactivation, abolished mycobacterial spread from the infection site. In accordance with our in vitro and in vivo results, we show that the level of VEGF in TB patients is elevated and that endothelial progenitor cells are mobilized from the bone marrow. These results strongly strengthen the most recent data suggesting that mycobacteria take advantage of the formation of new blood vessels to disseminate. PMID:27616470

Considerations for biomarker-targeted intervention strategies for tuberculosis disease prevention.

PubMed

Fiore-Gartland, Andrew; Carpp, Lindsay N; Naidoo, Kogieleum; Thompson, Ethan; Zak, Daniel E; Self, Steve; Churchyard, Gavin; Walzl, Gerhard; Penn-Nicholson, Adam; Scriba, Thomas J; Hatherill, Mark

2018-03-01

Current diagnostic tests for Mycobacterium tuberculosis (MTB) infection have low prognostic specificity for identifying individuals who will develop tuberculosis (TB) disease, making mass preventive therapy strategies targeting all MTB-infected individuals impractical in high-burden TB countries. Here we discuss general considerations for a risk-targeted test-and-treat strategy based on a highly specific transcriptomic biomarker that can identify individuals who are most likely to progress to active TB disease as well as individuals with TB disease who have not yet presented for medical care. Such risk-targeted strategies may offer a rapid, ethical and cost-effective path towards decreasing the burden of TB disease and interrupting transmission and would also be critical to achieving TB elimination in countries nearing elimination. We also discuss design considerations for a Correlate of Risk Targeted Intervention Study (CORTIS), which could provide proof-of-concept for the strategy. One such study in South Africa is currently enrolling 1500 high-risk and 1700 low-risk individuals, as defined by biomarker status, and is randomizing high-risk participants to TB preventive therapy or standard of care treatment. All participants are monitored for progression to active TB with primary objectives to assess efficacy of the treatment and performance of the biomarker. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Cigarette smoking and health-promoting behaviours among tuberculosis patients in rural areas.

PubMed

Tsai, Shu-Lan; Lai, Chun-Liang; Chi, Miao-Ching; Chen, Mei-Yen

2016-09-01

To explore cigarette smoking and health-promoting behaviours among disadvantaged adults before their tuberculosis diagnosis and after their tuberculosis treatment. Although tuberculosis infection is associated with impaired immune function, healthy lifestyle habits can play a role in improving the immune system. However, limited research has explored the health-promoting behaviours and cigarette smoking habits among tuberculosis patients in Taiwan. A cross-sectional retrospective study with a convenience sample. This study was conducted between May 2013-June 2014 with 123 patients at a rural district hospital in Chiayi County, Taiwan. Statistical analyses included descriptive statistics, univariate analysis and stepwise regression analysis. Tuberculosis tended to be associated with less education, male sex, malnutrition, cigarette smoking and unhealthy lifestyle habits before the tuberculosis diagnosis. The percentage of smoking decreased from 46·9% before to 30·2% after the tuberculosis diagnosis. Body mass index and health-promoting behaviours also significantly improved after tuberculosis treatment. After controlling for potential confounding factors, multivariate analysis identified chronic disease and completed treatment as significant factors that were associated with current health-promoting behaviours. A high prevalence of cigarette smoking and low levels of health-promoting behaviours were observed before the diagnosis and during or after completing tuberculosis treatment. This study's findings indicate the importance of promoting healthy lifestyle changes among tuberculosis patients; aggressive measures should be implemented immediately after the first diagnosis of tuberculosis. Furthermore, health promotion and smoking cessation programmes should be initiated in the general population to prevent activation of latent tuberculosis infection, and these programmes should specifically target men and rural residents. © 2016 John Wiley & Sons Ltd.

Microarray analysis of Mycobacterium tuberculosis-infected monocytes reveals IL26 as a new candidate gene for tuberculosis susceptibility.

PubMed

Guerra-Laso, José M; Raposo-García, Sara; García-García, Silvia; Diez-Tascón, Cristina; Rivero-Lezcano, Octavio M

2015-02-01

Differences in the activity of monocytes/macrophages, important target cells of Mycobacterium tuberculosis, might influence tuberculosis progression. With the purpose of identifying candidate genes for tuberculosis susceptibility we infected monocytes from both healthy elderly individuals (a tuberculosis susceptibility group) and elderly tuberculosis patients with M. tuberculosis, and performed a microarray experiment. We detected 78 differentially expressed transcripts and confirmed these results by quantitative PCR of selected genes. We found that monocytes from tuberculosis patients showed similar expression patterns for these genes, regardless of whether they were obtained from younger or older patients. Only one of the detected genes corresponded to a cytokine: IL26, a member of the interleukin-10 (IL-10) cytokine family which we found to be down-regulated in infected monocytes from tuberculosis patients. Non-infected monocytes secreted IL-26 constitutively but they reacted strongly to M. tuberculosis infection by decreasing IL-26 production. Furthermore, IL-26 serum concentrations appeared to be lower in the tuberculosis patients. When whole blood was infected, IL-26 inhibited the observed pathogen-killing capability. Although lymphocytes expressed IL26R, the receptor mRNA was not detected in either monocytes or neutrophils, suggesting that the inhibition of anti-mycobacterial activity may be mediated by lymphocytes. Additionally, IL-2 concentrations in infected blood were lower in the presence of IL-26. The negative influence of IL-26 on the anti-mycobacterial activity and its constitutive presence in both serum and monocyte supernatants prompt us to propose IL26 as a candidate gene for tuberculosis susceptibility. © 2014 John Wiley & Sons Ltd.

Consensus Document on Prevention and Treatment of Tuberculosis in Patients for Biological Treatment.

PubMed

Mir Viladrich, Isabel; Daudén Tello, Esteban; Solano-López, Guillermo; López Longo, Francisco Javier; Taxonera Samso, Carlos; Sánchez Martínez, Paquita; Martínez Lacasa, Xavier; García Gasalla, Mercedes; Dorca Sargatal, Jordi; Arias-Guillén, Miguel; García García, José Maria

2016-01-01

Tuberculosis risk is increased in patients with chronic inflammatory diseases receiving any immunosuppressive treatment, notably tumor necrosis factor (TNF) antagonists therapy. Screening for the presence of latent infection with Mycobacterium tuberculosis and targeted preventive treatment to reduce the risk of progression to TB is mandatory in these patients. This Consensus Document summarizes the current knowledge and expert opinion of biologic therapies including TNF-blocking treatments. It provides recommendations for the use of interferon-gamma release assays (IGRA) and tuberculin skin test (TST) for the diagnosis of latent tuberculosis infection in these patients, and for the type and duration of preventive therapy. Copyright © 2015 SEPAR. Published by Elsevier Espana. All rights reserved.

Propelling novel vaccines directed against tuberculosis through the regulatory process.

PubMed

Brennan, M J; Collins, F M; Morris, S L

1999-01-01

The development of novel vaccines for use in the prevention and immunotherapy of tuberculosis is an area of intense interest for scientific researchers, public health agencies and pharmaceutical manufacturers. Development of effective anti-tuberculosis vaccines for use in specific target populations will require close cooperation among several different international organizations including agencies responsible for evaluating the safety and effectiveness of new biologics for human use. In this review, the major issues that are addressed by regulatory agencies to ensure that vaccines are pure, potent, safe, and effective are discussed. It is hoped that the comments provided here will help accelerate the development of new effective vaccines for the prevention and treatment of tuberculosis.

Signal Regulatory Protein alpha (SIRPalpha)+ Cells in the Adaptive Response to ESAT-6/CFP-10 Protein of Tuberculous Mycobacteria

USDA-ARS?s Scientific Manuscript database

Early secretory antigenic target-6 (ESAT-6) and culture filtrate protein-10(CFP-10) are co-secreted proteins of Mycobacterium tuberculosis complex mycobacteria (includes M. bovis, the zoonotic agent of bovine tuberculosis) involved in phagolysosome escape of the bacillus and, potentially, in the eff...

FadA5 a thiolase from Mycobacterium tuberculosis - a unique steroid-binding pocket reveals the potential for drug development against tuberculosis

PubMed Central

Schaefer, Christin M.; Lu, Rui; Nesbitt, Natasha M.; Schiebel, Johannes; Sampson, Nicole S.; Kisker, Caroline

2014-01-01

Summary With the exception of HIV, tuberculosis (TB) is the leading cause of mortality among infectious diseases. The urgent need to develop new anti-tubercular drugs is apparent due to the increasing number of drug resistant Mycobacterium tuberculosis (Mtb) strains. Proteins involved in cholesterol import and metabolism have recently been discovered as potent targets against TB. FadA5, a thiolase from Mtb, is catalyzing the last step of the β-oxidation reaction of the cholesterol side-chain degradation under release of critical metabolites and was shown to be of importance during the chronic stage of TB infections. To gain structural and mechanistic insight on FadA5 we characterized the enzyme in different stages of the cleavage reaction and with a steroid bound to the binding pocket. Structural comparisons to human thiolases revealed that it should be possible to target FadA5 specifically and the steroid-bound structure provides a solid basis for the development of inhibitors against FadA5. PMID:25482540

Hacking into the granuloma: could antibody antibiotic conjugates be developed for TB?

PubMed

Ekins, Sean

2014-12-01

Alternatives to small molecule or vaccine approaches to treating tuberculosis are rarely discussed. Attacking Mycobacterium tuberculosis in the granuloma represents a challenge. It is proposed that the conjugation of small molecules onto a monoclonal antibody that recognizes macrophage or lymphocytes cell surface receptors, might be a way to target the bacteria in the granuloma. This antibody drug conjugate approach is currently being used in 2 FDA approved targeted cancer therapies. The pros and cons of this proposal for further research are discussed. Copyright © 2014 Elsevier Ltd. All rights reserved.

The naphthoquinone diospyrin is an inhibitor of DNA gyrase with a novel mechanism of action.

PubMed

Karkare, Shantanu; Chung, Terence T H; Collin, Frederic; Mitchenall, Lesley A; McKay, Adam R; Greive, Sandra J; Meyer, Jacobus J M; Lall, Namrita; Maxwell, Anthony

2013-02-15

Tuberculosis and other bacterial diseases represent a significant threat to human health. The DNA topoisomerases are excellent targets for chemotherapy, and DNA gyrase in particular is a well-validated target for antibacterial agents. Naphthoquinones (e.g. diospyrin and 7-methyljuglone) have been shown to have therapeutic potential, particularly against Mycobacterium tuberculosis. We have found that these compounds are inhibitors of the supercoiling reaction catalyzed by M. tuberculosis gyrase and other gyrases. Our evidence strongly suggests that the compounds bind to the N-terminal domain of GyrB, which contains the ATPase active site, but are not competitive inhibitors of the ATPase reaction. We propose that naphthoquinones bind to GyrB at a novel site close to the ATPase site. This novel mode of action could be exploited to develop new antibacterial agents.

The Naphthoquinone Diospyrin Is an Inhibitor of DNA Gyrase with a Novel Mechanism of Action*

PubMed Central

Karkare, Shantanu; Chung, Terence T. H.; Collin, Frederic; Mitchenall, Lesley A.; McKay, Adam R.; Greive, Sandra J.; Meyer, Jacobus J. M.; Lall, Namrita; Maxwell, Anthony

2013-01-01

Tuberculosis and other bacterial diseases represent a significant threat to human health. The DNA topoisomerases are excellent targets for chemotherapy, and DNA gyrase in particular is a well-validated target for antibacterial agents. Naphthoquinones (e.g. diospyrin and 7-methyljuglone) have been shown to have therapeutic potential, particularly against Mycobacterium tuberculosis. We have found that these compounds are inhibitors of the supercoiling reaction catalyzed by M. tuberculosis gyrase and other gyrases. Our evidence strongly suggests that the compounds bind to the N-terminal domain of GyrB, which contains the ATPase active site, but are not competitive inhibitors of the ATPase reaction. We propose that naphthoquinones bind to GyrB at a novel site close to the ATPase site. This novel mode of action could be exploited to develop new antibacterial agents. PMID:23275348

Locked Nucleic Acid Probe-Based Real-Time PCR Assay for the Rapid Detection of Rifampin-Resistant Mycobacterium tuberculosis

PubMed Central

Sun, Chongyun; Li, Chao; Wang, Xiaochen; Liu, Haican; Zhang, Pingping; Zhao, Xiuqin; Wang, Xinrui; Jiang, Yi; Yang, Ruifu; Wan, Kanglin; Zhou, Lei

2015-01-01

Drug-resistant Mycobacterium tuberculosis can be rapidly diagnosed through nucleic acid amplification techniques by analyzing the variations in the associated gene sequences. In the present study, a locked nucleic acid (LNA) probe-based real-time PCR assay was developed to identify the mutations in the rpoB gene associated with rifampin (RFP) resistance in M. tuberculosis. Six LNA probes with the discrimination capability of one-base mismatch were designed to monitor the 23 most frequent rpoB mutations. The target mutations were identified using the probes in a “probe dropout” manner (quantification cycle = 0); thus, the proposed technique exhibited superiority in mutation detection. The LNA probe-based real-time PCR assay was developed in a two-tube format with three LNA probes and one internal amplification control probe in each tube. The assay showed excellent specificity to M. tuberculosis with or without RFP resistance by evaluating 12 strains of common non-tuberculosis mycobacteria. The limit of detection of M. tuberculosis was 10 genomic equivalents (GE)/reaction by further introducing a nested PCR method. In a blind validation of 154 clinical mycobacterium isolates, 142/142 (100%) were correctly detected through the assay. Of these isolates, 88/88 (100%) were determined as RFP susceptible and 52/54 (96.3%) were characterized as RFP resistant. Two unrecognized RFP-resistant strains were sequenced and were found to contain mutations outside the range of the 23 mutation targets. In conclusion, this study established a sensitive, accurate, and low-cost LNA probe-based assay suitable for a four-multiplexing real-time PCR instrument. The proposed method can be used to diagnose RFP-resistant tuberculosis in clinical laboratories. PMID:26599667

Sliding Clamp of DNA Polymerase III as a Drug Target for TB Therapy: Comprehensive Conformational and Binding Analysis from Molecular Dynamic Simulations.

PubMed

Machaba, Kgothatso E; Cele, Favorite N; Mhlongo, Ndumiso N; Soliman, Mahmoud E S

2016-12-01

Tuberculosis, caused by Mycobacterium tuberculosis, is one of the most common causes of death in the world. Mycobacterium tuberculosis -sliding clamp is a protein essential for many important DNA transactions including replication and DNA repair proteins, thus, a potential drug target for tuberculosis. Further investigation is needed in understanding DNA polymerase sliding clamp structure, especially from a computational perspective. In this study, we employ a wide-range of comparative molecular dynamic analyses on two systems: Mycobacterium tuberculosis - sliding clamp enzyme in its apo and bound form. The results reported in this study shows apo conformation to be less stable, as compared to bound conformation with an average radius of gyration of 25.812 and 25.459 Å, respectively. This was further supported by root mean square fluctuation, where an apo enzyme showed a higher degree of flexibility. However, the presence of the ligand lowers radius of gyration and root mean square fluctuation and also leads to an existence of negative correlated motions. Principal component analysis further justifies the same findings, whereby the apo enzyme exhibits a higher fluctuation compared to the bound complex. In addition, a stable 3 10 helix located at the binding site appears to be unstable in the presence of the ligand. Hence, it is possible that the binding of the ligand may have caused a rearrangement of the structure, leading to a change in the unwinding of 3 10 helix. Findings reported in this study further enhance the understanding of Mycobacterium tuberculosis -DnaN and also give a lead to the development of potent tuberculosis drugs.

Lipid transport in Mycobacterium tuberculosis and its implications in virulence and drug development.

PubMed

Bailo, Rebeca; Bhatt, Apoorva; Aínsa, José A

2015-08-01

Tuberculosis is still a major health problem worldwide and one of the main causes of death by a single infectious agent. Only few drugs are really effective to treat tuberculosis, hence, the emergence of multiple, extensively, and totally drug resistant bacilli compromises the already difficult antituberculosis treatments. Given the persistent global burden of tuberculosis, it is crucial to understand the underlying mechanisms required for the pathogenicity of Mycobacterium tuberculosis (Mtb), the causal agent of tuberculosis, in order to pave the way for developing better drugs and strategies to treat and prevent tuberculosis. The exclusive mycobacterial cell wall lipids such as trehalose monomycolate and dimycolate (TMM, TDM), phthiocerol dimycocerosate (PDIM), sulpholipid-1 (SL-1), diacyl trehalose (DAT), and pentacyl trehalose (PAT), among others, are known to play an important role in pathogenesis; thus, proteins responsible for their transport are potential virulence factors. MmpL and MmpS proteins mediate transport of important cell wall lipids across the mycobacterial membrane. In Mtb, MmpL3, MmpL7 and MmpL8 transport TMM, PDIM and SL-1 respectively. The translocation of DAT and biosynthesis of PAT is likely due to MmpL10. MmpL and MmpS proteins are involved in other processes such as drug efflux (MmpL5 and MmpL7), siderophore export (MmpL4/MmpS4 and MmpL5/MmpS5), and heme uptake (MmpL3 and MmpL11). Altogether, these proteins can be regarded as new potential targets for antituberculosis drug development. We will review recent advances in developing inhibitors of MmpL proteins, in the challenging context of targeting membrane proteins and the future prospects for potential antituberculosis drug candidates. Copyright © 2015 Elsevier Inc. All rights reserved.

Macrophage defense mechanisms against intracellular bacteria

PubMed Central

Weiss, Günter; Schaible, Ulrich E

2015-01-01

Macrophages and neutrophils play a decisive role in host responses to intracellular bacteria including the agent of tuberculosis (TB), Mycobacterium tuberculosis as they represent the forefront of innate immune defense against bacterial invaders. At the same time, these phagocytes are also primary targets of intracellular bacteria to be abused as host cells. Their efficacy to contain and eliminate intracellular M. tuberculosis decides whether a patient initially becomes infected or not. However, when the infection becomes chronic or even latent (as in the case of TB) despite development of specific immune activation, phagocytes have also important effector functions. Macrophages have evolved a myriad of defense strategies to combat infection with intracellular bacteria such as M. tuberculosis. These include induction of toxic anti-microbial effectors such as nitric oxide and reactive oxygen intermediates, the stimulation of microbe intoxication mechanisms via acidification or metal accumulation in the phagolysosome, the restriction of the microbe's access to essential nutrients such as iron, fatty acids, or amino acids, the production of anti-microbial peptides and cytokines, along with induction of autophagy and efferocytosis to eliminate the pathogen. On the other hand, M. tuberculosis, as a prime example of a well-adapted facultative intracellular bacterium, has learned during evolution to counter-balance the host's immune defense strategies to secure survival or multiplication within this otherwise hostile environment. This review provides an overview of innate immune defense of macrophages directed against intracellular bacteria with a focus on M. tuberculosis. Gaining more insights and knowledge into this complex network of host-pathogen interaction will identify novel target sites of intervention to successfully clear infection at a time of rapidly emerging multi-resistance of M. tuberculosis against conventional antibiotics. PMID:25703560

Differential inhibition of adenylylated and deadenylylated forms of M. tuberculosis glutamine synthetase as a drug discovery platform

PubMed Central

Theron, A.; Roth, R. L.; Hoppe, H.; Parkinson, C.; van der Westhuyzen, C. W.; Stoychev, S.; Wiid, I.; Pietersen, R. D.; Baker, B.

2017-01-01

Glutamine synthetase is a ubiquitous central enzyme in nitrogen metabolism that is controlled by up to four regulatory mechanisms, including adenylylation of some or all of the twelve subunits by adenylyl transferase. It is considered a potential therapeutic target for the treatment of tuberculosis, being essential for the growth of Mycobacterium tuberculosis, and is found extracellularly only in the pathogenic Mycobacterium strains. Human glutamine synthetase is not regulated by the adenylylation mechanism, so the adenylylated form of bacterial glutamine synthetase is of particular interest. Previously published reports show that, when M. tuberculosis glutamine synthetase is expressed in Escherichia coli, the E. coli adenylyl transferase does not optimally adenylylate the M. tuberculosis glutamine synthetase. Here, we demonstrate the production of soluble adenylylated M. tuberulosis glutamine synthetase in E. coli by the co-expression of M. tuberculosis glutamine synthetase and M. tuberculosis adenylyl transferase. The differential inhibition of adenylylated M. tuberulosis glutamine synthetase and deadenylylated M. tuberulosis glutamine synthetase by ATP based scaffold inhibitors are reported. Compounds selected on the basis of their enzyme inhibition were also shown to inhibit M. tuberculosis in the BACTEC 460TB™ assay as well as the intracellular inhibition of M. tuberculosis in a mouse bone-marrow derived macrophage assay. PMID:28972974

Nitazoxanide stimulates autophagy and inhibits mTORC1 signaling and intracellular proliferation of Mycobacterium tuberculosis.

PubMed

Lam, Karen K Y; Zheng, Xingji; Forestieri, Roberto; Balgi, Aruna D; Nodwell, Matt; Vollett, Sarah; Anderson, Hilary J; Andersen, Raymond J; Av-Gay, Yossef; Roberge, Michel

2012-01-01

Tuberculosis, caused by Mycobacterium tuberculosis infection, is a major cause of morbidity and mortality in the world today. M. tuberculosis hijacks the phagosome-lysosome trafficking pathway to escape clearance from infected macrophages. There is increasing evidence that manipulation of autophagy, a regulated catabolic trafficking pathway, can enhance killing of M. tuberculosis. Therefore, pharmacological agents that induce autophagy could be important in combating tuberculosis. We report that the antiprotozoal drug nitazoxanide and its active metabolite tizoxanide strongly stimulate autophagy and inhibit signaling by mTORC1, a major negative regulator of autophagy. Analysis of 16 nitazoxanide analogues reveals similar strict structural requirements for activity in autophagosome induction, EGFP-LC3 processing and mTORC1 inhibition. Nitazoxanide can inhibit M. tuberculosis proliferation in vitro. Here we show that it inhibits M. tuberculosis proliferation more potently in infected human THP-1 cells and peripheral monocytes. We identify the human quinone oxidoreductase NQO1 as a nitazoxanide target and propose, based on experiments with cells expressing NQO1 or not, that NQO1 inhibition is partly responsible for mTORC1 inhibition and enhanced autophagy. The dual action of nitazoxanide on both the bacterium and the host cell response to infection may lead to improved tuberculosis treatment.

Hepatotoxicity during Treatment for Tuberculosis in People Living with HIV/AIDS.

PubMed

Araújo-Mariz, Carolline; Lopes, Edmundo Pessoa; Acioli-Santos, Bartolomeu; Maruza, Magda; Montarroyos, Ulisses Ramos; Ximenes, Ricardo Arraes de Alencar; Lacerda, Heloísa Ramos; Miranda-Filho, Demócrito de Barros; Albuquerque, Maria de Fátima P Militão de

2016-01-01

Hepatotoxicity is frequently reported as an adverse reaction during the treatment of tuberculosis. The aim of this study was to determine the incidence of hepatotoxicity and to identify predictive factors for developing hepatotoxicity after people living with HIV/AIDS (PLWHA) start treatment for tuberculosis. This was a prospective cohort study with PLWHA who were monitored during the first 60 days of tuberculosis treatment in Pernambuco, Brazil. Hepatotoxicity was considered increased levels of aminotransferase, namely those that rose to three times higher than the level before initiating tuberculosis treatment, these levels being associated with symptoms of hepatitis. We conducted a multivariate logistic regression analysis and the magnitude of the associations was expressed by the odds ratio with a confidence interval of 95%. Hepatotoxicity was observed in 53 (30.6%) of the 173 patients who started tuberculosis treatment. The final multivariate logistic regression model demonstrated that the use of fluconazole, malnutrition and the subject being classified as a phenotypically slow acetylator increased the risk of hepatotoxicity significantly. The incidence of hepatotoxicity during treatment for tuberculosis in PLWHA was high. Those classified as phenotypically slow acetylators and as malnourished should be targeted for specific care to reduce the risk of hepatotoxicity during treatment for tuberculosis. The use of fluconazole should be avoided during tuberculosis treatment in PLWHA.

Influence of diabetes mellitus and risk factors in activating latent tuberculosis infection: a case for targeted screening in malaysia.

PubMed

Swarna Nantha, Y

2012-10-01

A review of the epidemiology of tuberculosis, its contributing risk factors (excluding HIV) and the role of screening latent tuberculosis infection in Malaysia was done. Despite the global and domestic decrease in prevalence rates of tuberculosis in the past decade, there is an alarming increase in the trend of non communicable diseases in the country. High prevalence rates of major risk factors leading to reactivation of tuberculosis were seen within the population, with diabetes mellitus being in the forefront. The rising numbers in the ageing population of Malaysia poses a further threat of re-emergence of tuberculosis in the years to come. Economically, screening of diabetic patients with comorbidities for latent tuberculosis infection (LTBI) using two major techniques, namely tuberculin sensitivity (TST) and Interferon gamma release assay tests (IGRA) could be a viable option. The role of future research in the detection of LTBI in the Malaysian setting might be necessary to gauge the disease reservoir before implementing prophylactic measures for high risk groups involved.

Identification of Host-Targeted Small Molecules That Restrict Intracellular Mycobacterium tuberculosis Growth

PubMed Central

Silvis, Melanie R.; Luo, Samantha S.; Sogi, Kimberly; Vokes, Martha; Bray, Mark-Anthony; Carpenter, Anne E.; Moore, Christopher B.; Siddiqi, Noman; Rubin, Eric J.; Hung, Deborah T.

2014-01-01

Mycobacterium tuberculosis remains a significant threat to global health. Macrophages are the host cell for M. tuberculosis infection, and although bacteria are able to replicate intracellularly under certain conditions, it is also clear that macrophages are capable of killing M. tuberculosis if appropriately activated. The outcome of infection is determined at least in part by the host-pathogen interaction within the macrophage; however, we lack a complete understanding of which host pathways are critical for bacterial survival and replication. To add to our understanding of the molecular processes involved in intracellular infection, we performed a chemical screen using a high-content microscopic assay to identify small molecules that restrict mycobacterial growth in macrophages by targeting host functions and pathways. The identified host-targeted inhibitors restrict bacterial growth exclusively in the context of macrophage infection and predominantly fall into five categories: G-protein coupled receptor modulators, ion channel inhibitors, membrane transport proteins, anti-inflammatories, and kinase modulators. We found that fluoxetine, a selective serotonin reuptake inhibitor, enhances secretion of pro-inflammatory cytokine TNF-α and induces autophagy in infected macrophages, and gefitinib, an inhibitor of the Epidermal Growth Factor Receptor (EGFR), also activates autophagy and restricts growth. We demonstrate that during infection signaling through EGFR activates a p38 MAPK signaling pathway that prevents macrophages from effectively responding to infection. Inhibition of this pathway using gefitinib during in vivo infection reduces growth of M. tuberculosis in the lungs of infected mice. Our results support the concept that screening for inhibitors using intracellular models results in the identification of tool compounds for probing pathways during in vivo infection and may also result in the identification of new anti-tuberculosis agents that work by modulating host pathways. Given the existing experience with some of our identified compounds for other therapeutic indications, further clinically-directed study of these compounds is merited. PMID:24586159

Mycobacterium tuberculosis infection in cattle from the Eastern Cape Province of South Africa.

PubMed

Hlokwe, Tiny Motlatso; Said, Halima; Gcebe, Nomakorinte

2017-10-10

Mycobacterium tuberculosis is the main causative agent of tuberculosis (TB) in human and Mycobacterium bovis commonly causes tuberculosis in animals. Transmission of tuberculosis caused by both pathogens can occur from human to animals and vice versa. In the current study, M. tuberculosis, as confirmed by polymerase chain reaction (PCR) using primers targeting 3 regions of difference (RD4, RD9 and RD12) on the genomes, was isolated from cattle originating from two epidemiologically unrelated farms in the Eastern Cape (E.C) Province of South Africa. Although the isolates were genotyped with variable number of tandem repeat (VNTR) typing, no detailed epidemiological investigation was carried out on the respective farms to unequivocally confirm or link humans as sources of TB transmission to cattle, a move that would have embraced the 'One Health' concept. In addition, strain comparison with human M. tuberculosis in the database from the E.C Province and other provinces in the country did not reveal any match. This is the first report of cases of M. tuberculosis infection in cattle in South Africa. The VNTR profiles of the M. tuberculosis strains identified in the current study will form the basis for creating M. tuberculosis VNTR database for animals including cattle for future epidemiological studies. Our findings however, call for urgent reinforcement of collaborative efforts between the veterinary and the public health services of the country.

A Review of Moxifloxacin for the Treatment of Drug-Susceptible Tuberculosis.

PubMed

Naidoo, Anushka; Naidoo, Kogieleum; McIlleron, Helen; Essack, Sabiha; Padayatchi, Nesri

2017-11-01

Moxifloxacin, an 8-methoxy quinolone, is an important drug in the treatment of multidrug-resistant tuberculosis and is being investigated in novel drug regimens with pretomanid, bedaquiline, and pyrazinamide, or rifapentine, for the treatment of drug-susceptible tuberculosis. Early results of these studies are promising. Although current evidence does not support the use of moxifloxacin in treatment-shortening regimens for drug-susceptible tuberculosis, it may be recommended in patients unable to tolerate standard first-line drug regimens or for isoniazid monoresistance. Evidence suggests that the standard 400-mg dose of moxifloxacin used in the treatment of tuberculosis may be suboptimal in some patients, leading to worse tuberculosis treatment outcomes and emergence of drug resistance. Furthermore, a drug interaction with the rifamycins results in up to 31% reduced plasma concentrations of moxifloxacin when these are combined for treatment of drug-susceptible tuberculosis, although the clinical relevance of this interaction is unclear. Moxifloxacin exhibits extensive interindividual pharmacokinetic variability. Higher doses of moxifloxacin may be needed to achieve drug exposures required for improved clinical outcomes. Further study is, however, needed to determine the safety of proposed higher doses and clinically validated targets for drug exposure to moxifloxacin associated with improved tuberculosis treatment outcomes. We discuss in this review the evidence for the use of moxifloxacin in drug-susceptible tuberculosis and explore the role of moxifloxacin pharmacokinetics, pharmacodynamics, and drug interactions with rifamycins, on tuberculosis treatment outcomes when used in first-line tuberculosis drug regimens. © 2017, The American College of Clinical Pharmacology.

Molecular epidemiology of tuberculosis in foreign-born persons living in San Francisco.

PubMed

Suwanpimolkul, Gompol; Jarlsberg, Leah G; Grinsdale, Jennifer A; Osmond, Dennis; Kawamura, L Masae; Hopewell, Philip C; Kato-Maeda, Midori

2013-05-01

In San Francisco, 70% of the tuberculosis cases occur among foreign-born persons, mainly from China, the Philippines, and Mexico. We postulate that there are differences in the characteristics and risk factors for tuberculosis among these populations. To determine the clinical, epidemiological and microbiological characteristics of tuberculosis caused by recent infection and rapid evolution in the major groups of foreign-born and the U.S.-born populations. We analyzed data from a 20-year prospective community-based study of the molecular epidemiology of tuberculosis in San Francisco. We included all culture-positive tuberculosis cases in the City during the study period. We calculated and compared incidence rates, clinical and microbiological characteristics, and risk factors for being a secondary case between the various foreign-born and U.S.-born tuberculosis populations. Between 1991 and 2010, there were 4,058 new cases of tuberculosis, of which 1,226 (30%) were U.S.-born and 2,832 (70%) were foreign-born. A total of 3,278 (81%) were culture positive, of which 2,419 (74%) had complete data for analysis. The incidence rate, including the incidence rate of tuberculosis due to recent infection and rapid evolution, decreased significantly in the U.S.-born and the major foreign-born populations. The clinical and microbiological characteristics and the risk factors for tuberculosis due to recent infection differed among the groups. There are differences in the characteristics and the risk factors for tuberculosis due to recent transmission among the major foreign-born and U.S.-born populations in San Francisco. These differences should be considered for the design of targeted tuberculosis control interventions.

Pulmonary Delivery of Anti-Tubercular Drugs Using Ligand Anchored pH Sensitive Liposomes for the Treatment of Pulmonary Tuberculosis.

PubMed

Bhardwaj, Ankur; Grobler, Anne; Rath, Goutam; Goyal, Amit Kumar; Jain, Amit Kumar; Mehta, Abhinav

2016-01-01

Mycobacterium tuberculosis (M. TB) remains the prime cause of bacterial mortality and morbidity world-wide. Therefore, effective delivery and targeting of drug to the cellular tropics is essentially required to generate significant results for tuberculosis treatment. The aim of the present study was to develop and characterize ligand anchored pH sensitive liposomes (TPSL) as dry powder inhaler for the targeted delivery of drugs in the target site i.e. lungs. Ligand anchored PSL (TPSL) was prepared by thin film hydration for the combined delivery of Isoniazid (INH) and Ciprofloxacin HCl (CIP HCl) using 4-aminophenyl-α-D mannopyranoside (Man) as surface functionalized ligand and characterized using different parameters. It was observed that size of the ligand anchored liposomes (TPSL) was slightly more than the non-ligand anchored liposomes (PSL). Drug release was studied at different pH for 24 hrs and it was observed that liposomes exhibited slow release at alkaline pH (58-64%) as compared to macrophage pH (81-87%) where it increased dramatically due to the destabilization of pH sensitive liposome (PSL). In vitro cellular uptake study showed that much higher concentration was achieved in the alveolar macrophage using ligand anchored liposomes as compared to its counterpart. In vivo study showed that maximum drug accumulation was achieved in the lung by delivering drug using ligand anchored PSL as compared to conventional PSL. It was concluded that ligand anchored pH sensitive liposome is one of the promising systems for the targeted drug therapy in pulmonary tuberculosis.

Management of children exposed to Mycobacterium tuberculosis: a public health evaluation in West Java, Indonesia

PubMed Central

Ruslami, Rovina; Anselmo, Melissa; Alisjahbana, Bachti; Yulianti, Neti; Sampurno, Hedy; van Crevel, Reinout; Hill, Philip C

2013-01-01

Abstract Objective To investigate qualitatively and quantitatively the performance of a programme for managing the child contacts of adult tuberculosis patients in Indonesia. Methods A public health evaluation framework was used to assess gaps in a child contact management programme at a lung clinic. Targets for programme performance indicators were derived from established programme indicator targets, the scientific literature and expert opinion. Compliance with tuberculosis screening, the initiation of isoniazid preventive therapy in children younger than 5 years, the accuracy of tuberculosis diagnosis and adherence to preventive therapy were assessed in 755 child contacts in two cohorts. In addition, 22 primary caregivers and 34 clinic staff were interviewed to evaluate knowledge and acceptance of child contact management. The cost to caregivers was recorded. Gaps between observed and target indicator values were quantified. Findings The gaps between observed and target performance indicators were: 82% for screening compliance; 64 to 100% for diagnostic accuracy, 50% for the initiation of preventive therapy, 54% for adherence to therapy and 50% for costs. Many staff did not have adequate knowledge of, or an appropriate attitude towards, child contact management, especially regarding isoniazid preventive therapy. Caregivers had good knowledge of screening but not of preventive therapy and had difficulty travelling to the clinic and paying costs. Conclusion The study identified widespread gaps in the performance of a child contact management system in Indonesia, all of which appear amenable to intervention. The public health evaluation framework used could be applied in other settings where child contact management is failing. PMID:24347732

Trehalose-6-Phosphate-Mediated Toxicity Determines Essentiality of OtsB2 in Mycobacterium tuberculosis In Vitro and in Mice.

PubMed

Korte, Jan; Alber, Marina; Trujillo, Carolina M; Syson, Karl; Koliwer-Brandl, Hendrik; Deenen, René; Köhrer, Karl; DeJesus, Michael A; Hartman, Travis; Jacobs, William R; Bornemann, Stephen; Ioerger, Thomas R; Ehrt, Sabine; Kalscheuer, Rainer

2016-12-01

Trehalose biosynthesis is considered an attractive target for the development of antimicrobials against fungal, helminthic and bacterial pathogens including Mycobacterium tuberculosis. The most common biosynthetic route involves trehalose-6-phosphate (T6P) synthase OtsA and T6P phosphatase OtsB that generate trehalose from ADP/UDP-glucose and glucose-6-phosphate. In order to assess the drug target potential of T6P phosphatase, we generated a conditional mutant of M. tuberculosis allowing the regulated gene silencing of the T6P phosphatase gene otsB2. We found that otsB2 is essential for growth of M. tuberculosis in vitro as well as for the acute infection phase in mice following aerosol infection. By contrast, otsB2 is not essential for the chronic infection phase in mice, highlighting the substantial remodelling of trehalose metabolism during infection by M. tuberculosis. Blocking OtsB2 resulted in the accumulation of its substrate T6P, which appears to be toxic, leading to the self-poisoning of cells. Accordingly, blocking T6P production in a ΔotsA mutant abrogated otsB2 essentiality. T6P accumulation elicited a global upregulation of more than 800 genes, which might result from an increase in RNA stability implied by the enhanced neutralization of toxins exhibiting ribonuclease activity. Surprisingly, overlap with the stress response caused by the accumulation of another toxic sugar phosphate molecule, maltose-1-phosphate, was minimal. A genome-wide screen for synthetic lethal interactions with otsA identified numerous genes, revealing additional potential drug targets synergistic with OtsB2 suitable for combination therapies that would minimize the emergence of resistance to OtsB2 inhibitors.

Trehalose-6-Phosphate-Mediated Toxicity Determines Essentiality of OtsB2 in Mycobacterium tuberculosis In Vitro and in Mice

PubMed Central

Koliwer-Brandl, Hendrik; Hartman, Travis; Jacobs, William R.; Ioerger, Thomas R.; Ehrt, Sabine

2016-01-01

Trehalose biosynthesis is considered an attractive target for the development of antimicrobials against fungal, helminthic and bacterial pathogens including Mycobacterium tuberculosis. The most common biosynthetic route involves trehalose-6-phosphate (T6P) synthase OtsA and T6P phosphatase OtsB that generate trehalose from ADP/UDP-glucose and glucose-6-phosphate. In order to assess the drug target potential of T6P phosphatase, we generated a conditional mutant of M. tuberculosis allowing the regulated gene silencing of the T6P phosphatase gene otsB2. We found that otsB2 is essential for growth of M. tuberculosis in vitro as well as for the acute infection phase in mice following aerosol infection. By contrast, otsB2 is not essential for the chronic infection phase in mice, highlighting the substantial remodelling of trehalose metabolism during infection by M. tuberculosis. Blocking OtsB2 resulted in the accumulation of its substrate T6P, which appears to be toxic, leading to the self-poisoning of cells. Accordingly, blocking T6P production in a ΔotsA mutant abrogated otsB2 essentiality. T6P accumulation elicited a global upregulation of more than 800 genes, which might result from an increase in RNA stability implied by the enhanced neutralization of toxins exhibiting ribonuclease activity. Surprisingly, overlap with the stress response caused by the accumulation of another toxic sugar phosphate molecule, maltose-1-phosphate, was minimal. A genome-wide screen for synthetic lethal interactions with otsA identified numerous genes, revealing additional potential drug targets synergistic with OtsB2 suitable for combination therapies that would minimize the emergence of resistance to OtsB2 inhibitors. PMID:27936238

Mycobacterium tuberculosis Rv1152 is a Novel GntR Family Transcriptional Regulator Involved in Intrinsic Vancomycin Resistance and is a Potential Vancomycin Adjuvant Target

PubMed Central

Zeng, Jie; Deng, Wanyan; Yang, Wenmin; Luo, Hongping; Duan, Xiangke; Xie, Longxiang; Li, Ping; Wang, Rui; Fu, Tiwei; Abdalla, Abualgasim Elgaili; Xie, Jianping

2016-01-01

Novel factors involved in Mycobacteria antibiotics resistance are crucial for better targets to combat the ever-increasing drug resistant strains. Mycobacterium tuberculosis Rv1152, a novel GntR family transcriptional regulator and a promising vancomycin adjuvant target, was firstly characterized in our study. Overexpression of Rv1152 in Mycobacterium smegmatis decreased bacterial susceptibility to vancomycin. Moreover, a deficiency in MSMEG_5174, an Rv1152 homolog made M. smegmatis more sensitive to vancomycin, which was reverted by complementing the MSMEG_5174 deficiency with Rv1152 of M. tuberculosis. Rv1152 negatively regulated four vancomycin responsive genes, namely genes encoding the ribosome binding protein Hsp, small unit of sulfate adenylyltransferase CysD, L-lysine-epsilon aminotransferase Lat, and protease HtpX. Taken together, Rv1152 controls the expression of genes required for the susceptibility to vancomycin. This is the first report that links the GntR family transcriptional factor with vancomycin susceptibility. Inhibitors of Rv1152 might be ideal vancomycin adjuvants for controlling multi-drug resistant Mycobacterial infections. PMID:27349953

Addressing Institutional Amplifiers in the Dynamics and Control of Tuberculosis Epidemics

PubMed Central

Basu, Sanjay; Stuckler, David; McKee, Martin

2011-01-01

Tuberculosis outbreaks originating in prisons, mines, or hospital wards can spread to the larger community. Recent proposals have targeted these high-transmission institutional amplifiers by improving case detection, treatment, or reducing the size of the exposed population. However, what effects these alternative proposals may have is unclear. We mathematically modeled these control strategies and found case detection and treatment methods insufficient in addressing epidemics involving common types of institutional amplifiers. Movement of persons in and out of amplifiers fundamentally altered the transmission dynamics of tuberculosis in a manner not effectively mitigated by detection or treatment alone. Policies increasing the population size exposed to amplifiers or the per-person duration of exposure within amplifiers potentially worsened incidence, even in settings with high rates of detection and treatment success. However, reducing the total population size entering institutional amplifiers significantly lowered tuberculosis incidence and the risk of propagating new drug-resistant tuberculosis strains. PMID:21212197

DNA Replication Fidelity in the Mycobacterium tuberculosis Complex.

PubMed

Warner, Digby F; Rock, Jeremy M; Fortune, Sarah M; Mizrahi, Valerie

2017-01-01

Mycobacterium tuberculosis is genetically isolated, with no evidence for horizontal gene transfer or the acquisition of episomal genetic information in the modern evolution of strains of the Mycobacterium tuberculosis complex. When considered in the context of the specific features of the disease M. tuberculosis causes (e.g., transmission via cough aerosol, replication within professional phagocytes, subclinical persistence, and stimulation of a destructive immune pathology), this implies that to understand the mechanisms ensuring preservation of genomic integrity in infecting mycobacterial populations is to understand the source of genetic variation, including the emergence of microdiverse sub-populations that may be linked to the acquisition of drug resistance. In this chapter, we focus on mechanisms involved in maintaining DNA replication fidelity in M. tuberculosis, and consider the potential to target components of the DNA replication machinery as part of novel therapeutic regimens designed to curb the emerging threat of drug-resistance.

Prioritizing multiple therapeutic targets in parallel using automated DNA-encoded library screening

NASA Astrophysics Data System (ADS)

Machutta, Carl A.; Kollmann, Christopher S.; Lind, Kenneth E.; Bai, Xiaopeng; Chan, Pan F.; Huang, Jianzhong; Ballell, Lluis; Belyanskaya, Svetlana; Besra, Gurdyal S.; Barros-Aguirre, David; Bates, Robert H.; Centrella, Paolo A.; Chang, Sandy S.; Chai, Jing; Choudhry, Anthony E.; Coffin, Aaron; Davie, Christopher P.; Deng, Hongfeng; Deng, Jianghe; Ding, Yun; Dodson, Jason W.; Fosbenner, David T.; Gao, Enoch N.; Graham, Taylor L.; Graybill, Todd L.; Ingraham, Karen; Johnson, Walter P.; King, Bryan W.; Kwiatkowski, Christopher R.; Lelièvre, Joël; Li, Yue; Liu, Xiaorong; Lu, Quinn; Lehr, Ruth; Mendoza-Losana, Alfonso; Martin, John; McCloskey, Lynn; McCormick, Patti; O'Keefe, Heather P.; O'Keeffe, Thomas; Pao, Christina; Phelps, Christopher B.; Qi, Hongwei; Rafferty, Keith; Scavello, Genaro S.; Steiginga, Matt S.; Sundersingh, Flora S.; Sweitzer, Sharon M.; Szewczuk, Lawrence M.; Taylor, Amy; Toh, May Fern; Wang, Juan; Wang, Minghui; Wilkins, Devan J.; Xia, Bing; Yao, Gang; Zhang, Jean; Zhou, Jingye; Donahue, Christine P.; Messer, Jeffrey A.; Holmes, David; Arico-Muendel, Christopher C.; Pope, Andrew J.; Gross, Jeffrey W.; Evindar, Ghotas

2017-07-01

The identification and prioritization of chemically tractable therapeutic targets is a significant challenge in the discovery of new medicines. We have developed a novel method that rapidly screens multiple proteins in parallel using DNA-encoded library technology (ELT). Initial efforts were focused on the efficient discovery of antibacterial leads against 119 targets from Acinetobacter baumannii and Staphylococcus aureus. The success of this effort led to the hypothesis that the relative number of ELT binders alone could be used to assess the ligandability of large sets of proteins. This concept was further explored by screening 42 targets from Mycobacterium tuberculosis. Active chemical series for six targets from our initial effort as well as three chemotypes for DHFR from M. tuberculosis are reported. The findings demonstrate that parallel ELT selections can be used to assess ligandability and highlight opportunities for successful lead and tool discovery.

Encoded library technology as a source of hits for the discovery and lead optimization of a potent and selective class of bactericidal direct inhibitors of Mycobacterium tuberculosis InhA.

PubMed

Encinas, Lourdes; O'Keefe, Heather; Neu, Margarete; Remuiñán, Modesto J; Patel, Amish M; Guardia, Ana; Davie, Christopher P; Pérez-Macías, Natalia; Yang, Hongfang; Convery, Maire A; Messer, Jeff A; Pérez-Herrán, Esther; Centrella, Paolo A; Alvarez-Gómez, Daniel; Clark, Matthew A; Huss, Sophie; O'Donovan, Gary K; Ortega-Muro, Fátima; McDowell, William; Castañeda, Pablo; Arico-Muendel, Christopher C; Pajk, Stane; Rullás, Joaquín; Angulo-Barturen, Iñigo; Alvarez-Ruíz, Emilio; Mendoza-Losana, Alfonso; Ballell Pages, Lluís; Castro-Pichel, Julia; Evindar, Ghotas

2014-02-27

Tuberculosis (TB) is one of the world's oldest and deadliest diseases, killing a person every 20 s. InhA, the enoyl-ACP reductase from Mycobacterium tuberculosis, is the target of the frontline antitubercular drug isoniazid (INH). Compounds that directly target InhA and do not require activation by mycobacterial catalase peroxidase KatG are promising candidates for treating infections caused by INH resistant strains. The application of the encoded library technology (ELT) to the discovery of direct InhA inhibitors yielded compound 7 endowed with good enzymatic potency but with low antitubercular potency. This work reports the hit identification, the selected strategy for potency optimization, the structure-activity relationships of a hundred analogues synthesized, and the results of the in vivo efficacy studies performed with the lead compound 65.

Proteomic profile of Mycobacterium tuberculosis after eupomatenoid-5 induction reveals potential drug targets.

PubMed

Ghiraldi-Lopes, Luciana D; Campanerut-Sá, Paula Az; Meneguello, Jean E; Seixas, Flávio Av; Lopes-Ortiz, Mariana A; Scodro, Regiane Bl; Pires, Claudia Ta; da Silva, Rosi Z; Siqueira, Vera Ld; Nakamura, Celso V; Cardoso, Rosilene F

2017-08-01

We investigated a proteome profile, protein-protein interaction and morphological changes of Mycobacterium tuberculosis after different times of eupomatenoid-5 (EUP-5) induction to evaluate the cellular response to the drug-induced damages. The bacillus was induced to sub-minimal inhibitory concentration of EUP-5 at 12 h, 24 h and 48 h. The proteins were separated by 2D gel electrophoresis, identified by LC/MS-MS. Scanning electron microscopy and Search Tool for the Retrieval of Interacting Genes/Proteins analyses were performed. EUP-5 impacts mainly in M. tuberculosis proteins of intermediary metabolism and interactome suggests a multisite disturbance that contributes to bacilli death. Scanning electron microscopy revealed the loss of bacillary form. Some of the differentially expressed proteins have the potential to be drug targets such as citrate synthase (Rv0896), phosphoglycerate kinase (Rv1437), ketol-acid reductoisomerase (Rv3001c) and ATP synthase alpha chain (Rv1308).

Infection of the Invisible: Impressions of a Tuberculosis Intervention Program for Migrants in Istanbul.

PubMed

Yasin, Yesim; Biehl, Kristen; Erol, Maral

2015-10-01

This paper reviews the experience of the Istanbul Tuberculosis Aid Program, which targeted tuberculosis (TB) disease in the growing irregular migrant populations of Istanbul. This experience illustrated the importance of community-based public health interventions when dealing with an infectious disease like TB among vulnerable groups. Our data is derived primarily from a qualitative study carried out with program stakeholders. We summarize lessons for success of ITAP as: (1) Strengthening impact and outreach of TB intervention among irregular migrant communities through involvement of multiple stakeholders (2) Increasing TB awareness through a community targeted approach (3) Increasing TB contact tracing and treatment success among infected irregular migrants, and, (4) Improving overall health seeking behavior of irregular migrants through empowerment and trust. Given these particularities we list our policy suggestions for revision of regulations regarding TB control and healthcare needs of irregular migrant populations.

Targeting the human macrophage with combinations of drugs and inhibitors of Ca2+ and K+ transport to enhance the killing of intracellular multi-drug resistant Mycobacterium tuberculosis (MDR-TB)--a novel, patentable approach to limit the emergence of XDR-TB.

PubMed

Martins, Marta

2011-05-01

The emergence of resistance in tuberculosis has become a serious problem for the control of this disease. For that reason, new therapeutic strategies that can be implemented in the clinical setting are urgently needed. The design of new compounds active against mycobacteria must take into account that tuberculosis is mainly an intracellular infection of the alveolar macrophage and therefore must maintain activity within the host cells. An alternative therapeutic approach will be described in this review, focusing on the activation of the phagocytic cell and the subsequent killing of the internalized bacteria. This approach explores the combined use of antibiotics and phenothiazines, or Ca(2+) and K(+) flux inhibitors, in the infected macrophage. Targeting the infected macrophage and not the internalized bacteria could overcome the problem of bacterial multi-drug resistance. This will potentially eliminate the appearance of new multi-drug resistant tuberculosis (MDR-TB) cases and subsequently prevent the emergence of extensively-drug resistant tuberculosis (XDR-TB). Patents resulting from this novel and innovative approach could be extremely valuable if they can be implemented in the clinical setting. Other patents will also be discussed such as the treatment of TB using immunomodulator compounds (for example: betaglycans).

Fragment-based approaches to TB drugs.

PubMed

Marchetti, Chiara; Chan, Daniel S H; Coyne, Anthony G; Abell, Chris

2018-02-01

Tuberculosis is an infectious disease associated with significant mortality and morbidity worldwide, particularly in developing countries. The rise of antibiotic resistance in Mycobacterium tuberculosis (Mtb) urgently demands the development of new drug leads to tackle resistant strains. Fragment-based methods have recently emerged at the forefront of pharmaceutical development as a means to generate more effective lead structures, via the identification of fragment molecules that form weak but high quality interactions with the target biomolecule and subsequent fragment optimization. This review highlights a number of novel inhibitors of Mtb targets that have been developed through fragment-based approaches in recent years.

Individual and social vulnerabilities upon acquiring tuberculosis: a literature systematic review

PubMed Central

2014-01-01

Tuberculosis is a contagious infectious disease mainly caused by the bacteria Mycobacterium tuberculosis that still meets the priority criteria - high magnitude, transcendence and vulnerability - due to the threat it poses to public health. When taking into consideration the vulnerability conditions that favor the onset of the disease, this article aimed to investigate the implications originated from individual and social vulnerability conditions in which tuberculosis patients are inserted. Databases like MEDLINE, LILACS and SciELO were searched in Portuguese, Spanish and English using the descriptors tuberculosis and vulnerability, and 183 articles were found. After the selection criterion was applied, there were 22 publications left to be discussed. Some of the aspects that characterize the vulnerability to tuberculosis are: low-income and low-education families, age, poor living conditions, chemical dependency, pre-existing conditions/aggravations like diabetes mellitus and malnutrition, indigenous communities, variables related to health professionals, intense border crossings and migration, difficulty in accessing information and health services and lack of knowledge on tuberculosis. Much as such aspects are present and favor the onset of the disease, several reports show high incidence rates of tuberculosis in low vulnerability places, suggesting that some factors related to the disease are still unclear. In conclusion, health promotion is important in order to disfavor such conditions or factors of vulnerability to tuberculosis, making them a primary target in the public health planning process and disease control. PMID:25067955

Individual and social vulnerabilities upon acquiring tuberculosis: a literature systematic review.

PubMed

Nadjane Batista Lacerda, Sheylla; Cristina de Abreu Temoteo, Rayrla; Maria Ribeiro Monteiro de Figueiredo, Tânia; Darliane Tavares de Luna, Fernanda; Alves Nunes de Sousa, Milena; Carlos de Abreu, Luiz; Luiz Affonso Fonseca, Fernando

2014-01-01

Tuberculosis is a contagious infectious disease mainly caused by the bacteria Mycobacterium tuberculosis that still meets the priority criteria - high magnitude, transcendence and vulnerability - due to the threat it poses to public health. When taking into consideration the vulnerability conditions that favor the onset of the disease, this article aimed to investigate the implications originated from individual and social vulnerability conditions in which tuberculosis patients are inserted. Databases like MEDLINE, LILACS and SciELO were searched in Portuguese, Spanish and English using the descriptors tuberculosis and vulnerability, and 183 articles were found. After the selection criterion was applied, there were 22 publications left to be discussed. Some of the aspects that characterize the vulnerability to tuberculosis are: low-income and low-education families, age, poor living conditions, chemical dependency, pre-existing conditions/aggravations like diabetes mellitus and malnutrition, indigenous communities, variables related to health professionals, intense border crossings and migration, difficulty in accessing information and health services and lack of knowledge on tuberculosis. Much as such aspects are present and favor the onset of the disease, several reports show high incidence rates of tuberculosis in low vulnerability places, suggesting that some factors related to the disease are still unclear. In conclusion, health promotion is important in order to disfavor such conditions or factors of vulnerability to tuberculosis, making them a primary target in the public health planning process and disease control.

Stopping tuberculosis: a biosocial model for sustainable development.

PubMed

Ortblad, Katrina F; Salomon, Joshua A; Bärnighausen, Till; Atun, Rifat

2015-12-05

Tuberculosis transmission and progression are largely driven by social factors such as poor living conditions and poor nutrition. Increased standards of living and social approaches helped to decrease the burden of tuberculosis before the introduction of chemotherapy in the 1940s. Since then, management of tuberculosis has been largely biomedical. More funding for tuberculosis since 2000, coinciding with the Millennium Development Goals, has yielded progress in tuberculosis mortality but smaller reductions in incidence, which continues to pose a risk to sustainable development, especially in poor and susceptible populations. These at-risk populations need accelerated progress to end tuberculosis as resolved by the World Health Assembly in 2015. Effectively addressing the worldwide tuberculosis burden will need not only enhancement of biomedical approaches but also rebuilding of the social approaches of the past. To combine a biosocial approach, underpinned by social, economic, and environmental actions, with new treatments, new diagnostics, and universal health coverage, will need multisectoral coordination and action involving the health and other governmental sectors, as well as participation of the civil society, and especially the poor and susceptible populations. A biosocial approach to stopping tuberculosis will not only target morbidity and mortality from disease but would also contribute substantially to poverty alleviation and sustainable development that promises to meet the needs of the present, especially the poor, and provide them and subsequent generations an opportunity for a better future. Copyright © 2015 Elsevier Ltd. All rights reserved.

The mimic epitopes of Mycobacterium tuberculosis screened by phage display peptide library have serodiagnostic potential for tuberculosis.

PubMed

Wang, Li; Deng, Xiangying; Liu, Haican; Zhao, Lanhua; You, Xiaolong; Dai, Pei; Wan, Kanglin; Zeng, Yanhua

2016-11-01

Mycobacterium tuberculosis is an obligate pathogenic bacterial species in the family of Mycobacteriaceae and attracts excessive immune responses which cause pathology of the lungs in active tuberculosis. The lack of more sensitive and effective diagnosis reagents advocates a further recognition for the fast diagnostic and immunological measures for tuberculosis. Here, two 12-mer peptides with core sequences of SVSVGMKPSPRP (CS1) and TMGFTAPRFPHY (CS2) were screened from a phage display random peptide library using the purified mixed tuberculosis-positive serum as a target. Enzyme-linked immunosorbent assay (ELISA) and dot immunobinding assay verified that positive phages exhibited strong binding affinity to mixed tuberculosis-positive serum. BLAST analysis showed that the two sequences may be mimotopes of the Mycobacterium tuberculosis The diagnostic potential for two synthetic mimotope peptides CS1 and CS2 was evaluated using different panels of serum samples (n = 181) by ELISA, and the diagnostic parameters were calculated. CS1 and CS2 achieved sensitivity of 89.41% and 85.88%, and specificities were 90.63% and 87.50%, respectively. We hypothesized that the diagnostic based on CS1 and CS2 may become a promising strategy to enhance the detection of Mycobacterium tuberculosis infection due to higher specificity and sensitivity. Therefore, CS1 and CS2 may possess potentials to provide an experimental basis for the diagnosis of tuberculosis. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The management of infection with Mycobacterium tuberculosis in young children post-2015: an opportunity to close the policy-practice gap.

PubMed

Graham, Stephen M

2017-01-01

The treatment of infection with Mycobacterium tuberculosis in young children is supported by universal policy based on strong rationale and evidence of effectiveness, but has rarely been implemented in tuberculosis endemic countries. Areas covered: This review highlights a number of important recent developments that provide an unprecedented opportunity to close the policy-practice gap, as well as ongoing needs to facilitate implementation under programmatic conditions and scale-up. Expert commentary: The WHO's End TB Strategy and Stop TB Partnership's Plan to End TB provide ambitious targets for prevention at a time when National Tuberculosis Programs in tuberculosis endemic countries are increasing attention to the challenges of management and prevention of tuberculosis disease in children. This opportunity is greatly enhanced by recent evidence of the effectiveness of shorter, simpler and safer regimens to treat tuberculosis infection. The scale of the challenge for implementation will require a decentralized, integrated, community-based approach. An accurate and low-cost point-of-care test for tuberculous infection would be a major advance to support such implementation. Specific guidance for the treatment of infection in young child contacts of multidrug-resistant tuberculosis cases is a major current need while awaiting further evidence.

Phage display of functional αβ single-chain T-cell receptor molecules specific for CD1b:Ac₂SGL complexes from Mycobacterium tuberculosis-infected cells.

PubMed

Camacho, Frank; Huggett, Jim; Kim, Louise; Infante, Juan F; Lepore, Marco; Perez, Viviana; Sarmiento, María E; Rook, Graham; Acosta, Armando

2013-01-01

The development of molecules specific for M. tuberculosis-infected cells has important implications, as these tools may facilitate understanding of the mechanisms regulating host pathogen interactions in vivo. In addition, development of new tools capable to targeting M. tuberculosis-infected cells may have potential applications to diagnosis, treatment, and prevention of tuberculosis (TB). Due to the lack of CD1b polymorphism, M. tuberculosis lipid-CD1b complexes could be considered as universal tuberculosis infection markers. The aim of the present study was to display on the PIII surface protein of m13 phage, a human αβ single-chain T-cell receptor molecule specific for CD1b:2-stearoyl-3-hydroxyphthioceranoyl-2´-sulfate-α-α´-D-trehalose (Ac₂SGL) which is a complex presented by human cells infected with M. tuberculosis. The results showed the pIII fusion particle was successfully displayed on the phage surface. The study of the recognition of the recombinant phage in ELISA and immunohistochemistry showed the recognition of CD1b:Ac₂SGL complexes and cells in human lung tissue from a tuberculosis patient respectively, suggesting the specific recognition of the lipid-CD1b complex.

The Granuloma in Tuberculosis: Dynamics of a Host–Pathogen Collusion

PubMed Central

Ehlers, Stefan; Schaible, Ulrich E.

2012-01-01

A granuloma is defined as an inflammatory mononuclear cell infiltrate that, while capable of limiting growth of Mycobacterium tuberculosis, also provides a survival niche from which the bacteria may disseminate. The tuberculosis lesion is highly dynamic and shaped by both, immune response elements and the pathogen. In the granuloma, M. tuberculosis switches to a non-replicating but energy-generating life style whose detailed molecular characterization can identify novel targets for chemotherapy. To secure transmission to a new host, M. tuberculosis has evolved to drive T cell immunity to the point that necrotizing granulomas leak into bronchial cavities to facilitate expectoration of bacilli. From an evolutionary perspective it is therefore questionable whether vaccination and immunity enhancing strategies that merely mimic the natural immune response directed against M. tuberculosis infection can overcome pulmonary tuberculosis in the adult population. Juxtaposition of molecular pathology and immunology with microbial physiology and the use of novel imaging approaches afford an integrative view of the granuloma’s contribution to the life cycle of M. tuberculosis. This review revisits the different input of innate and adaptive immunity in granuloma biogenesis, with a focus on the co-evolutionary forces that redirect immune responses also to the benefit of the pathogen, i.e., its survival, propagation, and transmission. PMID:23308075

Evolution of Mycolic Acid Biosynthesis Genes and Their Regulation during Starvation in Mycobacterium tuberculosis

PubMed Central

Jamet, Stevie; Quentin, Yves; Coudray, Coralie; Texier, Pauline; Laval, Françoise; Daffé, Mamadou

2015-01-01

ABSTRACT Mycobacterium tuberculosis, the etiological agent of tuberculosis, is a Gram-positive bacterium with a unique cell envelope composed of an essential outer membrane. Mycolic acids, which are very-long-chain (up to C100) fatty acids, are the major components of this mycomembrane. The enzymatic pathways involved in the biosynthesis and transport of mycolates are fairly well documented and are the targets of the major antituberculous drugs. In contrast, only fragmented information is available on the expression and regulation of the biosynthesis genes. In this study, we report that the hadA, hadB, and hadC genes, which code for the mycolate biosynthesis dehydratase enzymes, are coexpressed with three genes that encode proteins of the translational apparatus. Consistent with the well-established control of the translation potential by nutrient availability, starvation leads to downregulation of the hadABC genes along with most of the genes required for the synthesis, modification, and transport of mycolates. The downregulation of a subset of the biosynthesis genes is partially dependent on RelMtb, the key enzyme of the stringent response. We also report the phylogenetic evolution scenario that has shaped the current genetic organization, characterized by the coregulation of the hadABC operon with genes of the translational apparatus and with genes required for the modification of the mycolates. IMPORTANCE Mycobacterium tuberculosis infects one-third of the human population worldwide, and despite the available therapeutic arsenal, it continues to kill millions of people each year. There is therefore an urgent need to identify new targets and develop a better understanding of how the bacterium is adapting itself to host defenses during infection. A prerequisite of this understanding is knowledge of how this adaptive skill has been implanted by evolution. Nutrient scarcity is an environmental condition the bacterium has to cope with during infection. In many bacteria, adaptation to starvation relies partly on the stringent response. M. tuberculosis's unique outer membrane layer, the mycomembrane, is crucial for its viability and virulence. Despite its being the target of the major antituberculosis drugs, only scattered information exists on how the genes required for biosynthesis of the mycomembrane are expressed and regulated during starvation. This work has addressed this issue as a step toward the identification of new targets in the fight against M. tuberculosis. PMID:26416833

Mycobacterium indicus pranii (MIP) mediated host protective intracellular mechanisms against tuberculosis infection: Involvement of TLR-4 mediated signaling.

PubMed

Das, Shibali; Chowdhury, Bidisha Paul; Goswami, Avranil; Parveen, Shabina; Jawed, Junaid; Pal, Nishith; Majumdar, Subrata

2016-12-01

Mycobacterium tuberculosis infection inflicts the disease Tuberculosis (TB), which is fatal if left untreated. During M. tuberculosis infection, the pathogen modulates TLR-4 receptor down-stream signaling, indicating the possible involvement of TLR-4 in the regulation of the host immune response. Mycobacterium indicus pranii (MIP) possesses immuno-modulatory properties which induces the pro-inflammatory responses via induction of TLR-4-mediated signaling. Here, we observed the immunomodulatory properties of MIP against tuberculosis infection. We have studied the detailed signaling mechanisms employed by MIP in order to restore the host immune response against the in vitro tuberculosis infection. We observed that in infected macrophages MIP treatment significantly increased the TLR-4 expression as well as activation of its downstream signaling, facilitating the activation of P38 MAP kinase. MIP treatment was able to activate NF-κB via involvement of TLR-4 signaling leading to the enhanced pro-inflammatory cytokine and NO generation in the infected macrophages and generation of protective immune response. Therefore, we may suggest that, TLR4 may represent a novel therapeutic target for the activation of the innate immune response during Tuberculosis infection. Copyright © 2016. Published by Elsevier Ltd.

Molecular Epidemiology of Tuberculosis in Foreign-Born Persons Living in San Francisco

PubMed Central

Suwanpimolkul, Gompol; Jarlsberg, Leah G.; Grinsdale, Jennifer A.; Osmond, Dennis; Kawamura, L. Masae; Hopewell, Philip C.

2013-01-01

Rationale: In San Francisco, 70% of the tuberculosis cases occur among foreign-born persons, mainly from China, the Philippines, and Mexico. We postulate that there are differences in the characteristics and risk factors for tuberculosis among these populations. Objectives: To determine the clinical, epidemiological and microbiological characteristics of tuberculosis caused by recent infection and rapid evolution in the major groups of foreign-born and the U.S.-born populations. Methods: We analyzed data from a 20-year prospective community-based study of the molecular epidemiology of tuberculosis in San Francisco. We included all culture-positive tuberculosis cases in the City during the study period. Measurements and Main Results: We calculated and compared incidence rates, clinical and microbiological characteristics, and risk factors for being a secondary case between the various foreign-born and U.S.-born tuberculosis populations. Between 1991 and 2010, there were 4,058 new cases of tuberculosis, of which 1,226 (30%) were U.S.-born and 2,832 (70%) were foreign-born. A total of 3,278 (81%) were culture positive, of which 2,419 (74%) had complete data for analysis. The incidence rate, including the incidence rate of tuberculosis due to recent infection and rapid evolution, decreased significantly in the U.S.-born and the major foreign-born populations. The clinical and microbiological characteristics and the risk factors for tuberculosis due to recent infection differed among the groups. Conclusions: There are differences in the characteristics and the risk factors for tuberculosis due to recent transmission among the major foreign-born and U.S.-born populations in San Francisco. These differences should be considered for the design of targeted tuberculosis control interventions. PMID:23471470

Multiscale Model of Mycobacterium tuberculosis Infection Maps Metabolite and Gene Perturbations to Granuloma Sterilization Predictions

PubMed Central

Pienaar, Elsje; Matern, William M.; Linderman, Jennifer J.

2016-01-01

Granulomas are a hallmark of tuberculosis. Inside granulomas, the pathogen Mycobacterium tuberculosis may enter a metabolically inactive state that is less susceptible to antibiotics. Understanding M. tuberculosis metabolism within granulomas could contribute to reducing the lengthy treatment required for tuberculosis and provide additional targets for new drugs. Two key adaptations of M. tuberculosis are a nonreplicating phenotype and accumulation of lipid inclusions in response to hypoxic conditions. To explore how these adaptations influence granuloma-scale outcomes in vivo, we present a multiscale in silico model of granuloma formation in tuberculosis. The model comprises host immunity, M. tuberculosis metabolism, M. tuberculosis growth adaptation to hypoxia, and nutrient diffusion. We calibrated our model to in vivo data from nonhuman primates and rabbits and apply the model to predict M. tuberculosis population dynamics and heterogeneity within granulomas. We found that bacterial populations are highly dynamic throughout infection in response to changing oxygen levels and host immunity pressures. Our results indicate that a nonreplicating phenotype, but not lipid inclusion formation, is important for long-term M. tuberculosis survival in granulomas. We used virtual M. tuberculosis knockouts to predict the impact of both metabolic enzyme inhibitors and metabolic pathways exploited to overcome inhibition. Results indicate that knockouts whose growth rates are below ∼66% of the wild-type growth rate in a culture medium featuring lipid as the only carbon source are unable to sustain infections in granulomas. By mapping metabolite- and gene-scale perturbations to granuloma-scale outcomes and predicting mechanisms of sterilization, our method provides a powerful tool for hypothesis testing and guiding experimental searches for novel antituberculosis interventions. PMID:26975995

Animal Models of Mycobacteria Infection

PubMed Central

Ordway, Diane J.; Orme, Ian M.

2011-01-01

This unit describes the infection of mice and guinea pigs with mycobacteria via various routes, as well as necropsy methods for the determination of mycobacterial loads within target organs. Additionally, methods for cultivating mycobacteria and preparing stocks are described. The protocols outlined are primarily used for M. tuberculosis, but can also be used for the study of other non-tuberculosis mycobacterial species. PMID:18432756

Targeting Mycobacterium tuberculosis Topoisomerase I by Small-Molecule Inhibitors

PubMed Central

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

2014-01-01

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. PMID:25534741

Smartphone Applications to Support Tuberculosis Prevention and Treatment: Review and Evaluation.

PubMed

Iribarren, Sarah J; Schnall, Rebecca; Stone, Patricia W; Carballo-Diéguez, Alex

2016-05-13

Tuberculosis (TB) remains a major global health problem and is the leading killer due to a single infectious disease. Mobile health (mHealth)-based tools such as smartphone apps have been suggested as tools to support TB control efforts (eg, identification, contact tracing, case management including patient support). The purpose of this review was to identify and assess the functionalities of mobile apps focused on prevention and treatment of TB. We searched 3 online mobile app stores. Apps were included if they were focused on TB and were in English, Spanish, or Portuguese. For each included app, 11 functionalities were assessed (eg, inform, instruct, record), and searches were conducted to identify peer-review publications of rigorous testing of the available apps. A total of 1332 potentially relevant apps were identified, with 24 meeting our inclusion criteria. All of the apps were free to download, but 7 required login and password and were developed for specific clinics, regional sites, or research studies. Targeted users were mainly clinicians (n=17); few (n=4) apps were patient focused. Most apps (n=17) had 4 or fewer functions out of 11 (range 1-6). The most common functionalities were inform and record (n=15). Although a number of apps were identified with various functionalities to support TB efforts, some had issues such as incorrect spelling and grammar, inconsistent responses to data entry, problems with crashing, or links to features that had no data. Of more concern, some apps provided potentially harmful information to patients, such as links to natural remedies for TB and natural healers. One-third of the apps (8/24) had not been updated for more than a year and may no longer be supported. Peer-reviewed publications were identified for only two of the included apps. In the gray literature (not found in the app stores), three TB-related apps were identified as in progress, being launched, or tested. Apps identified for TB prevention and treatment had minimal functionality, primarily targeted frontline health care workers, and focused on TB information (eg, general information, guidelines, and news) or data collection (eg, replace paper-based notification or tracking). Few apps were developed for use by patients and none were developed to support TB patient involvement and management in their care (eg, follow-up alerts/reminders, side effects monitoring) or improve interaction with their health care providers, limiting the potential of these apps to facilitate patient-centered care. Our evaluation shows that more refined work is needed to be done in the area of apps to support patients with active TB. Involving TB patients in treatment in the design of these apps is recommended.

Mycobacterium tuberculosis effectors interfering host apoptosis signaling.

PubMed

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.

A Phenotypic Based Target Screening Approach Delivers New Antitubercular CTP Synthetase Inhibitors.

PubMed

Esposito, Marta; Szadocka, Sára; Degiacomi, Giulia; Orena, Beatrice S; Mori, Giorgia; Piano, Valentina; Boldrin, Francesca; Zemanová, Júlia; Huszár, Stanislav; Barros, David; Ekins, Sean; Lelièvre, Joel; Manganelli, Riccardo; Mattevi, Andrea; Pasca, Maria Rosalia; Riccardi, Giovanna; Ballell, Lluis; Mikušová, Katarína; Chiarelli, Laurent R

2017-06-09

Despite its great potential, the target-based approach has been mostly unsuccessful in tuberculosis drug discovery, while whole cell phenotypic screening has delivered several active compounds. However, for many of these hits, the cellular target has not yet been identified, thus preventing further target-based optimization of the compounds. In this context, the newly validated drug target CTP synthetase PyrG was exploited to assess a target-based approach of already known, but untargeted, antimycobacterial compounds. To this purpose the publically available GlaxoSmithKline antimycobacterial compound set was assayed, uncovering a series of 4-(pyridin-2-yl)thiazole derivatives which efficiently inhibit the Mycobacterium tuberculosis PyrG enzyme activity, one of them showing low activity against the human CTP synthetase. The three best compounds were ATP binding site competitive inhibitors, with K i values ranging from 3 to 20 μM, but did not show any activity against a small panel of different prokaryotic and eukaryotic kinases, thus demonstrating specificity for the CTP synthetases. Metabolic labeling experiments demonstrated that the compounds directly interfere not only with CTP biosynthesis, but also with other CTP dependent biochemical pathways, such as lipid biosynthesis. Moreover, using a M. tuberculosis pyrG conditional knock-down strain, it was shown that the activity of two compounds is dependent on the intracellular concentration of the CTP synthetase. All these results strongly suggest a role of PyrG as a target of these compounds, thus strengthening the value of this kind of approach for the identification of new scaffolds for drug development.

A Data-Driven Evaluation of the Stop TB Global Partnership Strategy of Targeting Key Populations at Greater Risk for Tuberculosis

PubMed Central

Schnippel, Kathryn; Sharp, Alana

2016-01-01

Objective Identifying those infected with tuberculosis (TB) is an important component of any strategy for reducing TB transmission and population prevalence. The Stop TB Global Partnership recently launched an initiative with a focus on key populations at greater risk for TB infection or poor clinical outcomes, due to housing and working conditions, incarceration, low household income, malnutrition, co-morbidities, exposure to tobacco and silica dust, or barriers to accessing medical care. To achieve operational targets, the global health community needs effective, low cost, and large-scale strategies for identifying key populations. Using South Africa as a test case, we assess the feasibility and effectiveness of targeting active case finding to populations with TB risk factors identified from regularly collected sources of data. Our approach is applicable to all countries with TB testing and census data. It allows countries to tailor their outreach activities to the particular risk factors of greatest significance in their national context. Methods We use a national database of TB test results to estimate municipality-level TB infection prevalence, and link it to Census data to measure population risk factors for TB including rates of urban households, informal settlements, household income, unemployment, and mobile phone ownership. To examine the relationship between TB prevalence and risk factors, we perform linear regression analysis and plot the set of population characteristics against TB prevalence and TB testing rate by municipality. We overlay lines of best fit and smoothed curves of best fit from locally weighted scatter plot smoothing. Findings Higher TB prevalence is statistically significantly associated with more urban municipalities (slope coefficient β1 = 0.129, p < 0.0001, R2 = 0.133), lower mobile phone access (β1 = -0.053, p < 0.001, R2 = 0.089), lower unemployment rates (β1 = -0.020, p = 0.003, R2 = 0.048), and a lower proportion of low-income households (β1 = -0.048, p < 0.0001, R2 = 0.084). Municipalities with more low-income households also have marginally higher TB testing rates, however, this association is not statistically significant (β1 = -0.025, p = 0.676, R2 = 0.001). There is no relationship between TB prevalence and the proportion of informal settlement households (β1 = 0.021, p = 0.136, R2 = 0.014). Conclusions These analyses reveal that the set of characteristics identified by the Global Plan as defining key populations do not adequately predict populations with high TB burden. For example, we find that higher TB prevalence is correlated with more urbanized municipalities but not with informal settlements. We highlight several factors that are counter-intuitively those most associated with high TB burdens and which should therefore play a large role in any effective targeting strategy. Targeting active case finding to key populations at higher risk of infection or poor clinical outcomes may prove more cost effective than broad efforts. However, these results should increase caution in current targeting of active case finding interventions. PMID:27732606

A Data-Driven Evaluation of the Stop TB Global Partnership Strategy of Targeting Key Populations at Greater Risk for Tuberculosis.

PubMed

McLaren, Zoë M; Schnippel, Kathryn; Sharp, Alana

2016-01-01

Identifying those infected with tuberculosis (TB) is an important component of any strategy for reducing TB transmission and population prevalence. The Stop TB Global Partnership recently launched an initiative with a focus on key populations at greater risk for TB infection or poor clinical outcomes, due to housing and working conditions, incarceration, low household income, malnutrition, co-morbidities, exposure to tobacco and silica dust, or barriers to accessing medical care. To achieve operational targets, the global health community needs effective, low cost, and large-scale strategies for identifying key populations. Using South Africa as a test case, we assess the feasibility and effectiveness of targeting active case finding to populations with TB risk factors identified from regularly collected sources of data. Our approach is applicable to all countries with TB testing and census data. It allows countries to tailor their outreach activities to the particular risk factors of greatest significance in their national context. We use a national database of TB test results to estimate municipality-level TB infection prevalence, and link it to Census data to measure population risk factors for TB including rates of urban households, informal settlements, household income, unemployment, and mobile phone ownership. To examine the relationship between TB prevalence and risk factors, we perform linear regression analysis and plot the set of population characteristics against TB prevalence and TB testing rate by municipality. We overlay lines of best fit and smoothed curves of best fit from locally weighted scatter plot smoothing. Higher TB prevalence is statistically significantly associated with more urban municipalities (slope coefficient β1 = 0.129, p < 0.0001, R2 = 0.133), lower mobile phone access (β1 = -0.053, p < 0.001, R2 = 0.089), lower unemployment rates (β1 = -0.020, p = 0.003, R2 = 0.048), and a lower proportion of low-income households (β1 = -0.048, p < 0.0001, R2 = 0.084). Municipalities with more low-income households also have marginally higher TB testing rates, however, this association is not statistically significant (β1 = -0.025, p = 0.676, R2 = 0.001). There is no relationship between TB prevalence and the proportion of informal settlement households (β1 = 0.021, p = 0.136, R2 = 0.014). These analyses reveal that the set of characteristics identified by the Global Plan as defining key populations do not adequately predict populations with high TB burden. For example, we find that higher TB prevalence is correlated with more urbanized municipalities but not with informal settlements. We highlight several factors that are counter-intuitively those most associated with high TB burdens and which should therefore play a large role in any effective targeting strategy. Targeting active case finding to key populations at higher risk of infection or poor clinical outcomes may prove more cost effective than broad efforts. However, these results should increase caution in current targeting of active case finding interventions.

Pharmacokinetics and Dosing of Levofloxacin in Children Treated for Active or Latent Multidrug-resistant Tuberculosis, Federated States of Micronesia and Republic of the Marshall Islands.

PubMed

Mase, Sundari R; Jereb, John A; Gonzalez, Daniel; Martin, Fatma; Daley, Charles L; Fred, Dorina; Loeffler, Ann M; Menon, Lakshmy R; Bamrah Morris, Sapna; Brostrom, Richard; Chorba, Terence; Peloquin, Charles A

2016-04-01

In the Federated States of Micronesia and then the Republic of the Marshall Islands (RMI), levofloxacin pharmacokinetics were studied in children receiving directly observed once-daily regimens (10 mg/kg, age >5 years; 15-20 mg/kg, age ≤5 years) for either multidrug-resistant tuberculosis disease or latent infection after multidrug-resistant tuberculosis exposure, to inform future dosing strategies. Blood samples were collected at 0 (RMI only), 1, 2 and 6 hours (50 children, aged 6 months to 15 years) after oral levofloxacin at >6 weeks of treatment. Clinical characteristics and maximal drug concentration (Cmax) of levofloxacin, elimination half-life and area under the curve from 0 to 24 hours (AUC0-24 hours × μg/mL) were correlated to determine the optimal dosage and to examine associations. Population pharmacokinetics and target attainment were modeled. With results from the Federated States of Micronesia, dosages were increased in RMI toward the target Cmax for Mycobacterium tuberculosis, 8-12 µg/mL. Cmax correlated linearly with per-weight dosage. Neither Cmax nor half-life was associated with gender, age, body mass index, concurrent medications or predose meals. At levofloxacin dosage of 15-20 mg/kg, Cmax ≥8 µg/mL was observed, and modeling corroborated a high target attainment across the ratio of the area under the free concentration versus time curve to minimum inhibitory concentration (fAUCss,0-24/MIC) values. Levofloxacin dosage should be 15-20 mg/kg for Cmax ≥8 µg/mL and a high target attainment across fAUCss,0-24/MIC values in children ≥2 years of age.

Feasibility study of strengthening the public-private partnership for tuberculosis case detection in Bandung City, Indonesia.

PubMed

Lestari, Bony Wiem; Arisanti, Nita; Siregar, Adiatma Y M; Sihaloho, Estro Dariatno; Budiman, Gelar; Hill, Philip C; Alisjahbana, Bachti; McAllister, Susan

2017-08-14

Private practitioner's (PPs) collaboration for detection, diagnosis and treatment of tuberculosis (TB) is recommended by the World Health Organization and encouraged by the Indonesian National TB control programme. TB case management by PPs, however, are mostly not in line with current guidelines. Therefore, we developed an intervention package for PPs comprising of TB training, implementation of a mobile phone application for notification of TB cases and a 6-month regular follow-up with PPs. This study aimed to evaluate the feasibility of the intervention package to increase TB case detection and notification rates among PPs in five community health centre areas in Bandung City, Indonesia. A total of 87 PPs were registered within the study area of whom 17 attended the training and 12 had the mobile phone application successfully installed. The remaining five PPs had phones that did not support the application. During the follow-up period, five PPs registered patients with TB symptoms and cases into the application. A total of 36 patients with TB symptoms were identified and 17 were confirmed TB positive.

Universal immunization in urban areas: Calcutta's success story.

PubMed

Chaudhuri, E R

1990-01-01

The Central Government of Calcutta, India aimed to immunize 85% (85,262) of the city's 12 month old infants against polio, diphtheria, measles, tuberculosis, pertussis and tetanus. The Universal Immunization Program (UIP) achieved this target 3 months earlier than intended. In fact, at the end of December 1990, it achieved 110.6% for DPT3, 142.16% for OPV3, 151.96% for BCG, and 97% for measles. UIP was able to surpass its targets by emphasizing team work. Government, the private sector, UNICEF, and the voluntary sector made up the Apex Coordination Committee on Immunization headed up by the mayor. The committee drafted an action plan which included routine immunization sessions on a fixed day and intensive immunization drives. Further the involved organizations pooled together cold chain equipment. In addition, the District Family Welfare Bureau was the distribution center for vaccines, syringes, immunization cards, report formats, vaccine carriers, and ice packs. Health workers administered immunizations from about 300 centers generally on Wednesday, National Immunization Day. Intensive immunization drives focused on measles immunizations. UIP leaders encouraged all center to routinely record coverage and submit monthly progress reports to the District Family Welfare Bureau. The Calcutta Municipal Corporation coordinated promotion activities and social mobilization efforts. Promotion included radio and TV announcements, newspaper advertisements, cinema slides, billboards, and posters. The original UIP plan to use professional communicators to mobilize communities was ineffective, so nongovernmental organizations entered the slums to encourage people to encourage their neighbors to immunize their children. Further Islamic, Protestant, and Catholic leaders encouraged the faithful to immunize their children. A UNICEF officer noted that this success must be sustained, however.

Fragment-Based Whole Cell Screen Delivers Hits against M. tuberculosis and Non-tuberculous Mycobacteria.

PubMed

Moreira, Wilfried; Lim, Jia Jie; Yeo, Si Ying; Ramanujulu, Pondy M; Dymock, Brian W; Dick, Thomas

2016-01-01

Reactive multi-target 'fragment drugs' represent critical components of current tuberculosis regimens. These compounds, such as pyrazinamide, are old synthetic antimycobacterials that are activated inside Mycobacterium tuberculosis bacilli and are smaller than the usual drug-like, single-target molecules. Based on the success of small 'dirty' drugs in the chemotherapy of tuberculosis, we suggested previously that fragment-based whole cell screens should be introduced in our current antimycobacterial drug discovery efforts. Here, we carried out such a screen and characterized bactericidal activity, selectivity and spectrum of hits we obtained. A library of 1725 fragments was tested at a single concentration for growth inhibitory activity against M. bovis BCG as screening strain and 38 of 116 primary hits were confirmed in dose response analyses to be active against virulent M. tuberculosis. Bacterial kill experiments showed that most hits displayed bactericidal activity at their minimal inhibitory concentration. Cytotoxicity assays established that a large proportion of hits displayed a favorable selectivity index for mammalian cells. Importantly, one third of M. tuberculosis active fragments were also active against M. abscessus and M. avium, two emerging non-tuberculous mycobacterial (NTM) pathogens, opening the opportunity to develop broad spectrum antimycobacterials. Activity determination against Gram positive (Staphylococcus aureus) and Gram negative (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa) bacteria, as well as fungi (Candida albicans, Cryptococcus neoformans) showed only a small overlap indicating a generally narrow spectrum of these novel antimicrobial hits for mycobacteria. In conclusion, we carried out the first fragment-based whole cell screen against bacteria and identified a substantial number of hits with excellent physicochemical properties and dual activity against M. tuberculosis and NTM pathogens. These hits will now be evaluated in animal models of mycobacterial infection to determine whether any of them can be moved forward as a new antimycobacterial fragment drug candidate.

Proteome Analysis of the Plasma Membrane of Mycobacterium Tuberculosis

PubMed Central

Arora, Shalini; Kosalai, K.; Namane, Abdelkader; Pym, Alex S.; Cole, Stewart T.

2002-01-01

The plasma membrane of Mycobacterium tuberculosis is likely to contain proteins that could serve as novel drug targets, diagnostic probes or even components of a vaccine against tuberculosis. With this in mind, we have undertaken proteome analysis of the membrane of M. tuberculosis H37Rv. Isolated membrane vesicles were extracted with either a detergent (Triton X114) or an alkaline buffer (carbonate) following two of the protocols recommended for membrane protein enrichment. Proteins were resolved by 2D-GE using immobilized pH gradient (IPG) strips, and identified by peptide mass mapping utilizing the M. tuberculosis genome database. The two extraction procedures yielded patterns with minimal overlap. Only two proteins, both HSPs, showed a common presence. MALDI–MS analysis of 61 spots led to the identification of 32 proteins, 17 of which were new to the M. tuberculosis proteome database. We classified 19 of the identified proteins as ‘membrane-associated’; 14 of these were further classified as ‘membrane-bound’, three of which were lipoproteins. The remaining proteins included four heat-shock proteins and several enzymes involved in energy or lipid metabolism. Extraction with Triton X114 was found to be more effective than carbonate for detecting ‘putative’ M. tuberculosis membrane proteins. The protocol was also found to be suitable for comparing BCG and M. tuberculosis membranes, identifying ESAT-6 as being expressed selectively in M. tuberculosis. While this study demonstrates for the first time some of the membrane proteins of M. tuberculosis, it also underscores the problems associated with proteomic analysis of a complex membrane such as that of a mycobacterium. PMID:18629250

Development of Diazaquinomycin Class Antibiotics for the Treatment of Drug-Resistant TB Infections

DTIC Science & Technology

2016-10-01

exhibited weak antibacterial activity by targeting thymidylate synthase, though no reports of their anti-TB activity existed and our studies have suggested...Murphy, B. T. Diaza-anthracene antibiotics from a freshwater-derived actinomycete with selective antibacterial activity toward M. tuberculosis. ACS Inf...freshwater-derived actinomycete with selective antibacterial activity toward M. tuberculosis. ACS Infectious Diseases, 2015. 1: p. 168-174. (17) Mullowney

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.

PubMed

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.

IL-32 is a molecular marker of a host defense network in human tuberculosis

PubMed Central

Montoya, Dennis; Inkeles, Megan S.; Liu, Phillip T.; Realegeno, Susan; Teles, Rosane M. B.; Vaidya, Poorva; Munoz, Marcos A.; Schenk, Mirjam; Swindell, William R.; Chun, Rene; Zavala, Kathryn; Hewison, Martin; Adams, John S.; Horvath, Steve; Pellegrini, Matteo; Bloom, Barry R.; Modlin, Robert L.

2014-01-01

Tuberculosis is a leading cause of infectious disease–related death worldwide; however, only 10% of people infected with Mycobacterium tuberculosis develop disease. Factors that contribute to protection could prove to be promising targets for M. tuberculosis therapies. Analysis of peripheral blood gene expression profiles of active tuberculosis patients has identified correlates of risk for disease or pathogenesis. We sought to identify potential human candidate markers of host defense by studying gene expression profiles of macrophages, cells that, upon infection by M. tuberculosis, can mount an antimicrobial response. Weighted gene coexpression network analysis revealed an association between the cytokine interleukin-32 (IL-32) and the vitamin D antimicrobial pathway in a network of interferon-γ– and IL-15–induced “defense response” genes. IL-32 induced the vitamin D–dependent antimicrobial peptides cathelicidin and DEFB4 and to generate antimicrobial activity in vitro, dependent on the presence of adequate 25-hydroxyvitamin D. In addition, the IL-15–induced defense response macrophage gene network was integrated with ranked pairwise comparisons of gene expression from five different clinical data sets of latent compared with active tuberculosis or healthy controls and a coexpression network derived from gene expression in patients with tuberculosis undergoing chemotherapy. Together, these analyses identified eight common genes, including IL-32, as molecular markers of latent tuberculosis and the IL-15–induced gene network. As maintaining M. tuberculosis in a latent state and preventing transition to active disease may represent a form of host resistance, these results identify IL-32 as one functional marker and potential correlate of protection against active tuberculosis. PMID:25143364

IL-32 is a molecular marker of a host defense network in human tuberculosis.

PubMed

Montoya, Dennis; Inkeles, Megan S; Liu, Phillip T; Realegeno, Susan; Teles, Rosane M B; Vaidya, Poorva; Munoz, Marcos A; Schenk, Mirjam; Swindell, William R; Chun, Rene; Zavala, Kathryn; Hewison, Martin; Adams, John S; Horvath, Steve; Pellegrini, Matteo; Bloom, Barry R; Modlin, Robert L

2014-08-20

Tuberculosis is a leading cause of infectious disease-related death worldwide; however, only 10% of people infected with Mycobacterium tuberculosis develop disease. Factors that contribute to protection could prove to be promising targets for M. tuberculosis therapies. Analysis of peripheral blood gene expression profiles of active tuberculosis patients has identified correlates of risk for disease or pathogenesis. We sought to identify potential human candidate markers of host defense by studying gene expression profiles of macrophages, cells that, upon infection by M. tuberculosis, can mount an antimicrobial response. Weighted gene coexpression network analysis revealed an association between the cytokine interleukin-32 (IL-32) and the vitamin D antimicrobial pathway in a network of interferon-γ- and IL-15-induced "defense response" genes. IL-32 induced the vitamin D-dependent antimicrobial peptides cathelicidin and DEFB4 and to generate antimicrobial activity in vitro, dependent on the presence of adequate 25-hydroxyvitamin D. In addition, the IL-15-induced defense response macrophage gene network was integrated with ranked pairwise comparisons of gene expression from five different clinical data sets of latent compared with active tuberculosis or healthy controls and a coexpression network derived from gene expression in patients with tuberculosis undergoing chemotherapy. Together, these analyses identified eight common genes, including IL-32, as molecular markers of latent tuberculosis and the IL-15-induced gene network. As maintaining M. tuberculosis in a latent state and preventing transition to active disease may represent a form of host resistance, these results identify IL-32 as one functional marker and potential correlate of protection against active tuberculosis. Copyright © 2014, American Association for the Advancement of Science.

Mycobacterium Tuberculosis Infection within a Warthin Tumor: A Case Report and Literature Review.

PubMed

Ulusan, Murat; Abul, Yasin; Bakır, Sule

2013-10-01

The co-existence of tuberculosis and a Warthin tumor in the parotid gland is extremely rare. A 46-year-old male presented with a mass in the left parotid region of 6-month duration. The patient's history was only remarkable for a facial swelling, night sweats and a 38.5 C° fever. A 2 × 3-cm mobile, non-tender, mass with a smooth surface was palpated on left parotid tail. CT examination showed a well-defined 30 mm in diameter tumor mass in the left superficial lobe of the parotid gland. A superficial parotidectomy was performed. The final pathological diagnosis of the parotidectomy specimen was reported as a Warthin tumor and epitheloid granulomas with caseification necrosis. Purified protein derivative (PPD) was 30 mm in enduration. Two weeks after the antituberculosis treatment fever declined to normal values and night sweats decreased. Tuberculosis can also be seen in parotid tumors which can coexist or mimic pleomorphic adenoma, Warthin tumor.

Tuberculosis Incidence in Elderly in Serbia: Key Trends in Socioeconomic Transition

PubMed Central

Pešut, Dragica P.; Gledović, Zorana B.; Grgurević, Anita D.; Nagorni-Obradović, Ljudmila M.; Adžić, Tatjana N.

2008-01-01

Aim To examine tuberculosis incidence rates among the elderly in Central Serbia in 1992-2006 period, which was characterized by socioeconomic crisis and migration of population. Methods We analyzed all reported active tuberculosis cases in a 15-year period, especially among patients aged ≥65, according to the Annual Reports of the Institute of Lung Diseases and Tuberculosis in Belgrade and Central Tuberculosis Register. Population estimates with extrapolations were based on 1991 and 2002 census data. Results Total tuberculosis incidence rates showed a slight but non-significant decreasing trend (P = 0.535), and no significant increase was found in patients aged ≥65 years (P = 0.064), with an average age-specific incidence rate for the elderly of 64.0 (95% confidence interval, 60.7-67.4). The increase was significant in patients aged ≥70 years (y = 49.3549 + 2.1186x; P = 0.001), both in men (y = 62.8666 + 2.3977x; P = 0.005) and even more prominently in women (y = 39.8240 + 1.9150x; P < 0.001). The proportion of tuberculosis cases in the elderly peaked in 2005, with 35% of all tuberculosis cases. Conclusion High incidence rates and increasing time trend of tuberculosis in the elderly in Central Serbia is a serious problem, especially among those aged 70 years and over, who might present a target group for active case-finding of the disease. PMID:19090606

Peptide Deformylase Inhibitors as Potent Antimycobacterial Agents▿ †

PubMed Central

Teo, Jeanette W. P.; Thayalan, Pamela; Beer, David; Yap, Amelia S. L.; Nanjundappa, Mahesh; Ngew, Xinyi; Duraiswamy, Jeyaraj; Liung, Sarah; Dartois, Veronique; Schreiber, Mark; Hasan, Samiul ; Cynamon, Michael; Ryder, Neil S.; Yang, Xia; Weidmann, Beat; Bracken, Kathryn ; Dick, Thomas; Mukherjee, Kakoli

2006-01-01

Peptide deformylase (PDF) catalyzes the hydrolytic removal of the N-terminal formyl group from nascent proteins. This is an essential step in bacterial protein synthesis, making PDF an attractive target for antibacterial drug development. Essentiality of the def gene, encoding PDF from Mycobacterium tuberculosis, was demonstrated through genetic knockout experiments with Mycobacterium bovis BCG. PDF from M. tuberculosis strain H37Rv was cloned, expressed, and purified as an N-terminal histidine-tagged recombinant protein in Escherichia coli. A novel class of PDF inhibitors (PDF-I), the N-alkyl urea hydroxamic acids, were synthesized and evaluated for their activities against the M. tuberculosis PDF enzyme as well as their antimycobacterial effects. Several compounds from the new class had 50% inhibitory concentration (IC50) values of <100 nM. Some of the PDF-I displayed antibacterial activity against M. tuberculosis, including MDR strains with MIC90 values of <1 μM. Pharmacokinetic studies of potential leads showed that the compounds were orally bioavailable. Spontaneous resistance towards these inhibitors arose at a frequency of ≤5 × 10−7 in M. bovis BCG. DNA sequence analysis of several spontaneous PDF-I-resistant mutants revealed that half of the mutants had acquired point mutations in their formyl methyltransferase gene (fmt), which formylated Met-tRNA. The results from this study validate M. tuberculosis PDF as a drug target and suggest that this class of compounds have the potential to be developed as novel antimycobacterial agents. PMID:16966397

New Targets and Inhibitors of Mycobacterial Sulfur Metabolism§

PubMed Central

Paritala, Hanumantharao; Carroll, Kate S.

2015-01-01

The identification of new antibacterial targets is urgently needed to address multidrug resistant and latent tuberculosis infection. Sulfur metabolic pathways are essential for survival and the expression of virulence in many pathogenic bacteria, including Mycobacterium tuberculosis. In addition, microbial sulfur metabolic pathways are largely absent in humans and therefore, represent unique targets for therapeutic intervention. In this review, we summarize our current understanding of the enzymes associated with the production of sulfated and reduced sulfur-containing metabolites in Mycobacteria. Small molecule inhibitors of these catalysts represent valuable chemical tools that can be used to investigate the role of sulfur metabolism throughout the Mycobacterial lifecycle and may also represent new leads for drug development. In this light, we also summarize recent progress made in the development of inhibitors of sulfur metabolism enzymes. PMID:23808874

The Activity of a Hexameric M17 Metallo-Aminopeptidase Is Associated With Survival of Mycobacterium tuberculosis

PubMed Central

Correa, Andre F.; Bastos, Izabela M. D.; Neves, David; Kipnis, Andre; Junqueira-Kipnis, Ana P.; de Santana, Jaime M.

2017-01-01

Mycobacterium tuberculosis is one of the most prevalent human pathogens causing millions of deaths in the last years. Moreover, tuberculosis (TB) treatment has become increasingly challenging owing to the emergence of multidrug resistant M. tuberculosis strains. Thus, there is an immediate need for the development of new anti-TB drugs. Proteases appear to be a promising approach and may lead to shortened and effective treatments for drug-resistant TB. Although the M. tuberculosis genome predicts more than 100 genes encoding proteases, only a few of them have been studied. Aminopeptidases constitute a set of proteases that selectively remove amino acids from the N-terminus of proteins and peptides and may act as virulence factors, essential for survival and maintenance of many microbial pathogens. Here, we characterized a leucine aminopeptidase of M. tuberculosis (MtLAP) as a cytosolic oligomeric metallo-aminopeptidase. Molecular and enzymatic properties lead us to classify MtLAP as a typical member of the peptidase family M17. Furthermore, the aminopeptidase inhibitor bestatin strongly inhibited MtLAP activity, in vitro M. tuberculosis growth and macrophage infection. In murine model of TB, bestatin treatment reduced bacterial burden and lesion in the lungs of infected mice. Thus, our data suggest that MtLAP participates in important metabolic pathways of M. tuberculosis necessary for its survival and virulence and consequently may be a promising target for new anti-TB drugs. PMID:28396657

Molecular Characteristics of Mycobacterium tuberculosis Strains Isolated from Cutaneous Tuberculosis Patients in China.

PubMed

Jiang, Haiqin; Jin, Yali; Vissa, Varalakshmi; Zhang, Liangfen; Liu, Weijun; Qin, Lianhua; Wan, Kanglin; Wu, Xiaocui; Wang, Hongsheng; Liu, Weida; Wang, Baoxi

2017-04-06

Cutaneous tuberculosis (CTB) is probably underreported due to difficulties in detection and diagnosis. To address this issue, genotypes of Mycobacterium tuberculosis strains isolated from 30 patients with CTB were mapped at multiple loci, namely, RD105 deletions, spacer oligonucleotides, and Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeats (MIRU-VNTRs). Fifty-eight strains of pulmonary tuberculosis (PTB) were mapped as experimental controls. Drug resistance-associated gene mutations were determined by amplicon sequencing of target regions within 7 genes. Beijing family isolates were the most prevalent strains in CTB and PTB. MIRU-VNTR typing separated the Beijing strains from the non-Beijing strains, and the majority of CTB could be separated from PTB counterparts. Drug resistance determining regions showed only one CTB strain expressing isomazid resistance. Thus, while the CTB strains belonged to the same phylogenetic lineages and sub-lineages as the PTB strains, they differed at the level of several MIRU-VNTRs and in the proportion of drug resistance.

Spatial and space-time clustering of tuberculosis in Gurage Zone, Southern Ethiopia.

PubMed

Tadesse, Sebsibe; Enqueselassie, Fikre; Hagos, Seifu

2018-01-01

Spatial targeting is advocated as an effective method that contributes for achieving tuberculosis control in high-burden countries. However, there is a paucity of studies clarifying the spatial nature of the disease in these countries. This study aims to identify the location, size and risk of purely spatial and space-time clusters for high occurrence of tuberculosis in Gurage Zone, Southern Ethiopia during 2007 to 2016. A total of 15,805 patient data that were retrieved from unit TB registers were included in the final analyses. The spatial and space-time cluster analyses were performed using the global Moran's I, Getis-Ord [Formula: see text] and Kulldorff's scan statistics. Eleven purely spatial and three space-time clusters were detected (P <0.001).The clusters were concentrated in border areas of the Gurage Zone. There were considerable spatial variations in the risk of tuberculosis by year during the study period. This study showed that tuberculosis clusters were mainly concentrated at border areas of the Gurage Zone during the study period, suggesting that there has been sustained transmission of the disease within these locations. The findings may help intensify the implementation of tuberculosis control activities in these locations. Further study is warranted to explore the roles of various ecological factors on the observed spatial distribution of tuberculosis.

Phenotypic assays for Mycobacterium tuberculosis infection.

PubMed

Song, Ok-Ryul; Deboosere, Nathalie; Delorme, Vincent; Queval, Christophe J; Deloison, Gaspard; Werkmeister, Elisabeth; Lafont, Frank; Baulard, Alain; Iantomasi, Raffaella; Brodin, Priscille

2017-10-01

Tuberculosis (TB) is still a major global threat, killing more than one million persons each year. With the constant increase of Mycobacterium tuberculosis strains resistant to first- and second-line drugs, there is an urgent need for the development of new drugs to control the propagation of TB. Although screenings of small molecules on axenic M. tuberculosis cultures were successful for the identification of novel putative anti-TB drugs, new drugs in the development pipeline remains scarce. Host-directed therapy may represent an alternative for drug development against TB. Indeed, M. tuberculosis has multiple specific interactions within host phagocytes, which may be targeted by small molecules. In order to enable drug discovery strategies against microbes residing within host macrophages, we developed multiple fluorescence-based HT/CS phenotypic assays monitoring the intracellular replication of M. tuberculosis as well as its intracellular trafficking. What we propose here is a population-based, multi-parametric analysis pipeline that can be used to monitor the intracellular fate of M. tuberculosis and the dynamics of cellular events such as phagosomal maturation (acidification and permeabilization), zinc poisoning system or lipid body accumulation. Such analysis allows the quantification of biological events considering the host-pathogen interplay and may thus be derived to other intracellular pathogens. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

Mycobacterium tuberculosis Transcriptome Profiling in Mice with Genetically Different Susceptibility to Tuberculosis.

PubMed

Skvortsov, T A; Ignatov, D V; Majorov, K B; Apt, A S; Azhikina, T L

2013-04-01

Whole transcriptome profiling is now almost routinely used in various fields of biology, including microbiology. In vivo transcriptome studies usually provide relevant information about the biological processes in the organism and thus are indispensable for the formulation of hypotheses, testing, and correcting. In this study, we describe the results of genome-wide transcriptional profiling of the major human bacterial pathogen M. tuberculosis during its persistence in lungs. Two mouse strains differing in their susceptibility to tuberculosis were used for experimental infection with M. tuberculosis. Mycobacterial transcriptomes obtained from the infected tissues of the mice at two different time points were analyzed by deep sequencing and compared. It was hypothesized that the changes in the M. tuberculosis transcriptome may attest to the activation of the metabolism of lipids and amino acids, transition to anaerobic respiration, and increased expression of the factors modulating the immune response. A total of 209 genes were determined whose expression increased with disease progression in both host strains (commonly upregulated genes, CUG). Among them, the genes related to the functional categories of lipid metabolism, cell wall, and cell processes are of great interest. It was assumed that the products of these genes are involved in M. tuberculosis adaptation to the host immune system defense, thus being potential targets for drug development.

A whole genome bioinformatic approach to determine potential latent phase specific targets in Mycobacterium tuberculosis.

PubMed

Defelipe, Lucas A; Do Porto, Dario Fernández; Pereira Ramos, Pablo Ivan; Nicolás, Marisa Fabiana; Sosa, Ezequiel; Radusky, Leandro; Lanzarotti, Esteban; Turjanski, Adrián G; Marti, Marcelo A

2016-03-01

Current Tuberculosis treatment is long and expensive, faces the increasing burden of MDR/XDR strains and lack of effective treatment against latent form, resulting in an urgent need of new anti-TB drugs. Key to TB biology is its capacity to fight the host's RNOS mediated attack. RNOS are known to display a concentration dependent mycobactericidal activity, which leads to the following hypothesis "if we know which proteins are targeted by RNOS and kill TB, we we might be able to inhibit them with drugs resulting in a synergistic bactericidal effect". Based on this idea, we performed an Mtb metabolic network whole proteome analysis of potential RNOS sensitive and relevant targets which includes target druggability and essentiality criteria. Our results, available at http://tuberq.proteinq.com.ar yield new potential TB targets, like I3PS, while also providing and updated view of previous proposals becoming an important tool for researchers looking for new ways of killing TB. Copyright © 2015 Elsevier Ltd. All rights reserved.

High Throughput Screening for Inhibitors of Mycobacterium tuberculosis H37Rv

PubMed Central

ANANTHAN, SUBRAMANIAM; FAALEOLEA, ELLEN R.; GOLDMAN, ROBERT C.; HOBRATH, JUDITH V.; KWONG, CECIL D.; LAUGHON, BARBARA E.; MADDRY, JOSEPH A.; MEHTA, ALKA; RASMUSSEN, LYNN; REYNOLDS, ROBERT C.; SECRIST, JOHN A.; SHINDO, NICE; SHOWE, DUSTIN N.; SOSA, MELINDA I.; SULING, WILLIAM J.; WHITE, E. LUCILE

2009-01-01

SUMMARY There is an urgent need for the discovery and development of new antitubercular agents that target new biochemical pathways and treat drug resistant forms of the disease. One approach to addressing this need is through high throughput screening of medicinally relevant libraries against the whole bacterium in order to discover a variety of new, active scaffolds that will stimulate new biological research and drug discovery. Through the Tuberculosis Antimicrobial Acquisition and Coordinating Facility (www.taacf.org), a large, medicinally relevant chemical library was screened against M. tuberculosis strain H37Rv. The screening methods and a medicinal chemistry analysis of the results are reported herein. PMID:19758845

High-throughput identification and dendritic cell-based functional validation of MHC class I-restricted Mycobacterium tuberculosis epitopes

PubMed Central

Nair, Smita K.; Tomaras, Georgia D.; Sales, Ana Paula; Boczkowski, David; Chan, Cliburn; Plonk, Kelly; Cai, Yongting; Dannull, Jens; Kepler, Thomas B.; Pruitt, Scott K.; Weinhold, Kent J.

2014-01-01

Emergence of drug-resistant strains of the pathogen Mycobacterium tuberculosis (Mtb) and the ineffectiveness of BCG in curtailing Mtb infection makes vaccine development for tuberculosis an important objective. Identifying immunogenic CD8+ T cell peptide epitopes is necessary for peptide-based vaccine strategies. We present a three-tiered strategy for identifying and validating immunogenic peptides: first, identify peptides that form stable complexes with class I MHC molecules; second, determine whether cytotoxic T lymphocytes (CTLs) raised against the whole protein antigen recognize and lyse target cells pulsed with peptides that passed step 1; third, determine whether peptides that passed step 2, when administered in vivo as a vaccine in HLA-A2 transgenic mice, elicit CTLs that lyse target cells expressing the whole protein antigen. Our innovative approach uses dendritic cells transfected with Mtb antigen-encoding mRNA to drive antigen expression. Using this strategy, we have identified five novel peptide epitopes from the Mtb proteins Apa, Mtb8.4 and Mtb19. PMID:24755960

mHealth: Using Mobile Technology to Support Healthcare

PubMed Central

Okuboyejo, Senanu; Eyesan, Omatseyin

2014-01-01

Adherence to long-term therapy in outpatient setting is required to reduce the prevalence of chronic diseases such as HIV/AIDS, Diabetes, Tuberculosis and Malaria. This paper presents a mobile technology-based medical alert system for outpatient adherence in Nigeria. The system makes use of the SMS and voice features of mobile phones. The system has the potential of improving adherence to medication in outpatient setting by reminding patients of dosing schedules and attendance to scheduled appointments through SMS and voice calls. It will also inform patients of benefits and risks associated with adherence. Interventions aimed at improving adherence would provide significant positive return on investment through primary prevention (of risk factors) and secondary prevention of adverse health outcomes. PMID:24678384

Evidence-Based Design of Fixed-Dose Combinations: Principles and Application to Pediatric Anti-Tuberculosis Therapy.

PubMed

Svensson, Elin M; Yngman, Gunnar; Denti, Paolo; McIlleron, Helen; Kjellsson, Maria C; Karlsson, Mats O

2018-05-01

Fixed-dose combination formulations where several drugs are included in one tablet are important for the implementation of many long-term multidrug therapies. The selection of optimal dose ratios and tablet content of a fixed-dose combination and the design of individualized dosing regimens is a complex task, requiring multiple simultaneous considerations. In this work, a methodology for the rational design of a fixed-dose combination was developed and applied to the case of a three-drug pediatric anti-tuberculosis formulation individualized on body weight. The optimization methodology synthesizes information about the intended use population, the pharmacokinetic properties of the drugs, therapeutic targets, and practical constraints. A utility function is included to penalize deviations from the targets; a sequential estimation procedure was developed for stable estimation of break-points for individualized dosing. The suggested optimized pediatric anti-tuberculosis fixed-dose combination was compared with the recently launched World Health Organization-endorsed formulation. The optimized fixed-dose combination included 15, 36, and 16% higher amounts of rifampicin, isoniazid, and pyrazinamide, respectively. The optimized fixed-dose combination is expected to result in overall less deviation from the therapeutic targets based on adult exposure and substantially fewer children with underexposure (below half the target). The development of this design tool can aid the implementation of evidence-based formulations, integrating available knowledge and practical considerations, to optimize drug exposures and thereby treatment outcomes.

Mycobacterium smegmatis strain for detection of Mycobacterium tuberculosis by PCR used as internal control for inhibition of amplification and for quantification of bacteria.

PubMed Central

Kolk, A H; Noordhoek, G T; de Leeuw, O; Kuijper, S; van Embden, J D

1994-01-01

For the detection of Mycobacterium tuberculosis by PCR, the IS6110 sequence was used. A modified target was constructed by insertion of 56 nucleotides in the IS6110 insertion element of Mycobacterium bovis BCG. This modified insertion sequence was integrated into the genome of Mycobacterium smegmatis, a mycobacterium species which does not contain the IS6110 element. When DNA from the modified M. smegmatis 1008 strain was amplified with IS6110-specific primers INS1 and INS2, a band of 301 bp was seen on agarose gel, whereas the PCR product of M. tuberculosis complex DNA was a 245-bp fragment with these primers. The addition of a small number of M. smegmatis 1008 cells to clinical samples before DNA purification enables the detection of problems which may be due to the loss of DNA in the isolation procedure or to the presence of inhibitors. The presence of inhibitors of the amplification reaction can be confirmed by the addition of M. smegmatis 1008 DNA after the DNA isolation procedure. Furthermore, competition between the different target DNAs of M. smegmatis 1008 DNA and M. tuberculosis complex DNA enables the estimation of the number of IS6110 elements in the clinical sample. Images PMID:8051267

Metronidazole prevents reactivation of latent Mycobacterium tuberculosis infection in macaques

PubMed Central

Lin, Philana Ling; Dartois, Veronique; Johnston, Paul J.; Janssen, Christopher; Via, Laura; Goodwin, Michael B.; Klein, Edwin; Barry, Clifton E.; Flynn, JoAnne L.

2012-01-01

Targeting Mycobacterium tuberculosis bacilli in low-oxygen microenvironments, such as caseous granulomas, has been hypothesized to have the potential to shorten therapy for active tuberculosis (TB) and prevent reactivation of latent infection. We previously reported that upon low-dose M. tuberculosis infection, equal proportions of cynomolgus macaques develop active disease or latent infection and that latently infected animals reactivated upon neutralization of TNF. Using this model we now show that chemoprophylaxis of latently infected cynomolgus macaques with 6 mo of isoniazid (INH) effectively prevented anti-TNF antibody-induced reactivation. Similarly, 2-mo treatment of latent animals with a combination of INH and rifampicin (RIF) was highly effective at preventing reactivation disease in this model. Metronidazole (MTZ), which has activity only against anaerobic, nonreplicating bacteria, was as effective as either of these treatments in preventing reactivation of latent infection. Because hypoxic lesions also occur during active TB, we further showed that addition of MTZ to INH/RIF effectively treated animals with active TB within 2 mo. Healing lesions were associated with distinct changes in cellular pathology, with a shift toward increasingly fibrotic and calcified lesions. Our data in the nonhuman primate model of active and latent TB supports targeting bacteria in hypoxic environments for preventing reactivation of latent infection and possibly shortening the duration of therapy in active TB. PMID:22826237

Lipoarabinomannan and related glycoconjugates: structure, biogenesis and role in Mycobacterium tuberculosis physiology and host–pathogen interaction

PubMed Central

Mishra, Arun K; Driessen, Nicole N; Appelmelk, Ben J; Besra, Gurdyal S

2011-01-01

Approximately one third of the world's population is infected with Mycobacterium tuberculosis, the causative agent of tuberculosis. This bacterium has an unusual lipid-rich cell wall containing a vast repertoire of antigens, providing a hydrophobic impermeable barrier against chemical drugs, thus representing an attractive target for vaccine and drug development. Apart from the mycolyl–arabinogalactan–peptidoglycan complex, mycobacteria possess several immunomodulatory constituents, notably lipomannan and lipoarabinomannan. The availability of whole-genome sequences of M. tuberculosis and related bacilli over the past decade has led to the identification and functional characterization of various enzymes and the potential drug targets involved in the biosynthesis of these glycoconjugates. Both lipomannan and lipoarabinomannan possess highly variable chemical structures, which interact with different receptors of the immune system during host–pathogen interactions, such as Toll-like receptors-2 and C-type lectins. Recently, the availability of mutants defective in the synthesis of these glycoconjugates in mycobacteria and the closely related bacterium, Corynebacterium glutamicum, has paved the way for host–pathogen interaction studies, as well as, providing attenuated strains of mycobacteria for the development of new vaccine candidates. This review provides a comprehensive account of the structure, biosynthesis and immunomodulatory properties of these important glycoconjugates. PMID:21521247

Characterisation of methionine adenosyltransferase from Mycobacterium smegmatis and M. tuberculosis

PubMed Central

Berger, Bradley J; Knodel, Marvin H

2003-01-01

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 point for the synthesis of higher affinity purine-based inhibitors. PMID:12809568

A prospective blood RNA signature for tuberculosis disease risk

PubMed Central

Zak, Daniel E.; Penn-Nicholson, Adam; Scriba, Thomas J.; Thompson, Ethan; Suliman, Sara; Amon, Lynn M.; Mahomed, Hassan; Erasmus, Mzwandile; Whatney, Wendy; Hussey, Gregory D.; Abrahams, Deborah; Kafaar, Fazlin; Hawkridge, Tony; Verver, Suzanne; Hughes, E. Jane; Ota, Martin; Sutherland, Jayne; Howe, Rawleigh; Dockrell, Hazel M.; Boom, W. Henry; Thiel, Bonnie; Ottenhoff, Tom H.M.; Mayanja-Kizza, Harriet; Crampin, Amelia C; Downing, Katrina; Hatherill, Mark; Valvo, Joe; Shankar, Smitha; Parida, Shreemanta K; Kaufmann, Stefan H.E.; Walzl, Gerhard; Aderem, Alan; Hanekom, Willem A.

2016-01-01

Background Identification of blood biomarkers that prospectively predict progression of Mycobacterium tuberculosis infection to tuberculosis disease may lead to interventions that impact the epidemic. Methods Healthy, M. tuberculosis infected South African adolescents were followed for 2 years; blood was collected every 6 months. A prospective signature of risk was derived from whole blood RNA-Sequencing data by comparing participants who ultimately developed active tuberculosis disease (progressors) with those who remained healthy (matched controls). After adaptation to multiplex qRT-PCR, the signature was used to predict tuberculosis disease in untouched adolescent samples and in samples from independent cohorts of South African and Gambian adult progressors and controls. The latter participants were household contacts of adults with active pulmonary tuberculosis disease. Findings Of 6,363 adolescents screened, 46 progressors and 107 matched controls were identified. A 16 gene signature of risk was identified. The signature predicted tuberculosis progression with a sensitivity of 66·1% (95% confidence interval, 63·2–68·9) and a specificity of 80·6% (79·2–82·0) in the 12 months preceding tuberculosis diagnosis. The risk signature was validated in an untouched group of adolescents (p=0·018 for RNA-Seq and p=0·0095 for qRT-PCR) and in the independent South African and Gambian cohorts (p values <0·0001 by qRT-PCR) with a sensitivity of 53·7% (42·6–64·3) and a specificity of 82·8% (76·7–86) in 12 months preceding tuberculosis. Interpretation The whole blood tuberculosis risk signature prospectively identified persons at risk of developing active tuberculosis, opening the possibility for targeted intervention to prevent the disease. Funding Bill and Melinda Gates Foundation, the National Institutes of Health, Aeras, the European Union and the South African Medical Research Council (detail at end of text). PMID:27017310

Identifying socioeconomic, epidemiological and operational scenarios for tuberculosis control in Brazil: an ecological study.

PubMed

Pelissari, Daniele Maria; Rocha, Marli Souza; Bartholomay, Patricia; Sanchez, Mauro Niskier; Duarte, Elisabeth Carmen; Arakaki-Sanchez, Denise; Dantas, Cíntia Oliveira; Jacobs, Marina Gasino; Andrade, Kleydson Bonfim; Codenotti, Stefano Barbosa; Andrade, Elaine Silva Nascimento; Araújo, Wildo Navegantes de; Costa, Fernanda Dockhorn; Ramalho, Walter Massa; Diaz-Quijano, Fredi Alexander

2018-06-06

To identify scenarios based on socioeconomic, epidemiological and operational healthcare factors associated with tuberculosis incidence in Brazil. Ecological study. The study was based on new patients with tuberculosis and epidemiological/operational variables of the disease from the Brazilian National Information System for Notifiable Diseases and the Mortality Information System. We also analysed socioeconomic and demographic variables. The units of analysis were the Brazilian municipalities, which in 2015 numbered 5570 but 5 were excluded due to the absence of socioeconomic information. Tuberculosis incidence rate in 2015. We evaluated as independent variables the socioeconomic (2010), epidemiological and operational healthcare indicators of tuberculosis (2014 or 2015) using negative binomial regression. Municipalities were clustered by the k-means method considering the variables identified in multiple regression models. We identified two clusters according to socioeconomic variables associated with the tuberculosis incidence rate (unemployment rate and household crowding): a higher socioeconomic scenario (n=3482 municipalities) with a mean tuberculosis incidence rate of 16.3/100 000 population and a lower socioeconomic scenario (2083 municipalities) with a mean tuberculosis incidence rate of 22.1/100 000 population. In a second stage of clusterisation, we defined four subgroups in each of the socioeconomic scenarios using epidemiological and operational variables such as tuberculosis mortality rate, AIDS case detection rate and proportion of vulnerable population among patients with tuberculosis. Some of the subscenarios identified were characterised by fragility in their information systems, while others were characterised by the concentration of tuberculosis cases in key populations. Clustering municipalities in scenarios allowed us to classify them according to the socioeconomic, epidemiological and operational variables associated with tuberculosis risk. This classification can support targeted evidence-based decisions such as monitoring data quality for improving the information system or establishing integrative social protective policies for key populations. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Rapid and sensitive method for simultaneous determination of first-line anti-tuberculosis drugs in human plasma by HPLC-MS/MS: Application to therapeutic drug monitoring.

PubMed

Gao, Shouhong; Wang, Zhipeng; Xie, Xinfang; You, Chunhua; Yang, Yang; Xi, Yanhai; Chen, Wansheng

2018-03-01

First-line anti-tuberculosis drugs are playing vital roles for curbing rapid spread of tuberculosis. Multidrug therapies are commonly applied in clinical to achieve better treatment outcomes. However, drug resistance and adverse reactions come along with this therapies and therapeutic drug monitoring is a feasible way to precaution them. For this reasons, a simple and sensitive method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) and single protein precipitation was developed and validated for simultaneously quantifying of pyrazinamide, isoniazid, ethambutol, streptomycin and rifampicin in human plasma. Optimized chromatographic separation was achieved on a ZORBAX SB-C 18 column with heptafluorobutyric acid, an ion-pair reagent, in the mobile phase at a flow rate of 0.3 mL/min. The mass detection was achieved using electrospray ionization in the positive ion mode with a multiple reaction monitoring mode. The lower limit of quantification (LLOQ) and dynamic range of pyrazinamide, isoniazid, ethambutol, streptomycin and rifampicin were 200-4000 ng/mL, 80-2000 ng/mL, 0.2-1000 ng/mL, 2000-200000 ng/mL and 200-4000 ng/mL, respectively. The Inter-day and intra-day accuracy and precision were within ±15.0% and less than 15%. The method had been successfully applied to simultaneous determination of four first-line Anti-tuberculosis drugs in plasma from tuberculosis patients. Copyright © 2017 Elsevier Ltd. All rights reserved.

Neutral-red reaction is related to virulence and cell wall methyl-branched lipids in Mycobacterium tuberculosis.

PubMed

Cardona, P-J; Soto, C Y; Martín, C; Giquel, B; Agustí, G; Andreu, Núria; Guirado, E; Sirakova, T; Kolattukudy, P; Julián, E; Luquin, M

2006-01-01

Searching for virulence marking tests for Mycobacterium tuberculosis, Dubos and Middlebrook reported in 1948 that in an alkaline aqueous solution of neutral-red, the cells of the virulent H37Rv M. tuberculosis strain fixed the dye and became red in color, whereas the cells of the avirulent H37Ra M. tuberculosis strain remained unstained. In the 1950 and 1960s, fresh isolates of M. tuberculosis were tested for this neutral-red cytochemical reaction and it was reported that they were neutral-red positive, whereas other mycobacteria of diverse environmental origins that were non-pathogenic for guinea pigs were neutral-red negative. However, neutral-red has not really been proven to be a virulence marker. To test if virulence is in fact correlated to neutral-red, we studied a clinical isolate of M. tuberculosis that was originally neutral-red positive but, after more than 1 year passing through culture mediums, turned neutral-red negative. We found that, in comparison to the original neutral-red positive strain, this neutral-red negative variant was attenuated in two murine models of experimental tuberculosis. Lipid analysis showed that this neutral-red negative natural mutant lost the capacity to synthesize pthiocerol dimycocerosates, a cell wall methyl-branched lipid that has been related to virulence in M. tuberculosis. We also studied the neutral-red of different gene-targeted M. tuberculosis mutants unable to produce pthiocerol dimycocerosates or other cell wall methyl-branched lipids such as sulfolipids, and polyacyltrehaloses. We found a negative neutral-red reaction in mutants that were deficient in more than one type of methyl-branched lipids. We conclude that neutral-red is indeed a marker of virulence and it indicates important perturbations in the external surface of M. tuberculosis cells.

An adenosine triphosphate-independent proteasome activator contributes to the virulence of Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jastrab, Jordan B.; Wang, Tong; Murphy, J. Patrick

Mycobacterium tuberculosis encodes a proteasome that is highly similar to eukaryotic proteasomes and is required to cause lethal infections in animals. The only pathway known to target proteins for proteasomal degradation in bacteria is pupylation, which is functionally analogous to eukaryotic ubiquitylation. However, evidence suggests that the M. tuberculosis proteasome contributes to pupylation-independent pathways as well. To identify new proteasome cofactors that might contribute to such pathways, we isolated proteins that bound to proteasomes overproduced in M. tuberculosis and found a previously uncharacterized protein, Rv3780, which formed rings and capped M. tuberculosis proteasome core particles. Rv3780 enhanced peptide and proteinmore » degradation by proteasomes in an adenosine triphosphate (ATP)-independent manner. We identified putative Rv3780-dependent proteasome substrates and found that Rv3780 promoted robust degradation of the heat shock protein repressor, HspR. Importantly, an M. tuberculosis Rv3780 mutant had a general growth defect, was sensitive to heat stress, and was attenuated for growth in mice. Collectively, these data demonstrate that ATP-independent proteasome activators are not confined to eukaryotes and can contribute to the virulence of one the world’s most devastating pathogens.« less

Spatial pattern and temporal trend of mortality due to tuberculosis 10

PubMed Central

de Queiroz, Ana Angélica Rêgo; Berra, Thaís Zamboni; Garcia, Maria Concebida da Cunha; Popolin, Marcela Paschoal; Belchior, Aylana de Souza; Yamamura, Mellina; dos Santos, Danielle Talita; Arroyo, Luiz Henrique; Arcêncio, Ricardo Alexandre

2018-01-01

ABSTRACT Objectives: To describe the epidemiological profile of mortality due to tuberculosis (TB), to analyze the spatial pattern of these deaths and to investigate the temporal trend in mortality due to tuberculosis in Northeast Brazil. Methods: An ecological study based on secondary mortality data. Deaths due to TB were included in the study. Descriptive statistics were calculated and gross mortality rates were estimated and smoothed by the Local Empirical Bayesian Method. Prais-Winsten’s regression was used to analyze the temporal trend in the TB mortality coefficients. The Kernel density technique was used to analyze the spatial distribution of TB mortality. Results: Tuberculosis was implicated in 236 deaths. The burden of tuberculosis deaths was higher amongst males, single people and people of mixed ethnicity, and the mean age at death was 51 years. TB deaths were clustered in the East, West and North health districts, and the tuberculosis mortality coefficient remained stable throughout the study period. Conclusions: Analyses of the spatial pattern and temporal trend in mortality revealed that certain areas have higher TB mortality rates, and should therefore be prioritized in public health interventions targeting the disease. PMID:29742272

An adenosine triphosphate-independent proteasome activator contributes to the virulence of Mycobacterium tuberculosis

DOE PAGES

Jastrab, Jordan B.; Wang, Tong; Murphy, J. Patrick; ...

2015-03-23

Mycobacterium tuberculosis encodes a proteasome that is highly similar to eukaryotic proteasomes and is required to cause lethal infections in animals. The only pathway known to target proteins for proteasomal degradation in bacteria is pupylation, which is functionally analogous to eukaryotic ubiquitylation. However, evidence suggests that the M. tuberculosis proteasome contributes to pupylation-independent pathways as well. To identify new proteasome cofactors that might contribute to such pathways, we isolated proteins that bound to proteasomes overproduced in M. tuberculosis and found a previously uncharacterized protein, Rv3780, which formed rings and capped M. tuberculosis proteasome core particles. Rv3780 enhanced peptide and proteinmore » degradation by proteasomes in an adenosine triphosphate (ATP)-independent manner. We identified putative Rv3780-dependent proteasome substrates and found that Rv3780 promoted robust degradation of the heat shock protein repressor, HspR. Importantly, an M. tuberculosis Rv3780 mutant had a general growth defect, was sensitive to heat stress, and was attenuated for growth in mice. Collectively, these data demonstrate that ATP-independent proteasome activators are not confined to eukaryotes and can contribute to the virulence of one the world’s most devastating pathogens.« less

Transmission of Extensively Drug-Resistant Tuberculosis in South Africa

PubMed Central

Shah, N. Sarita; Auld, Sara C.; Brust, James C.M.; Mathema, Barun; Ismail, Nazir; Moodley, Pravi; Mlisana, Koleka; Allana, Salim; Campbell, Angela; Mthiyane, Thuli; Morris, Natashia; Mpangase, Primrose; van der Meulen, Hermina; Omar, Shaheed V.; Brown, Tyler S.; Narechania, Apurva; Shaskina, Elena; Kapwata, Thandi; Kreiswirth, Barry; Gandhi, Neel R.

2017-01-01

BACKGROUND Drug-resistant tuberculosis threatens recent gains in the treatment of tuberculosis and human immunodeficiency virus (HIV) infection worldwide. A widespread epidemic of extensively drug-resistant (XDR) tuberculosis is occurring in South Africa, where cases have increased substantially since 2002. The factors driving this rapid increase have not been fully elucidated, but such knowledge is needed to guide public health interventions. METHODS We conducted a prospective study involving 404 participants in KwaZulu-Natal Province, South Africa, with a diagnosis of XDR tuberculosis between 2011 and 2014. Interviews and medical-record reviews were used to elicit information on the participants’ history of tuberculosis and HIV infection, hospitalizations, and social networks. Mycobacterium tuberculosis isolates underwent insertion sequence (IS)6110 restriction-fragment– length polymorphism analysis, targeted gene sequencing, and whole-genome sequencing. We used clinical and genotypic case definitions to calculate the proportion of cases of XDR tuberculosis that were due to inadequate treatment of multidrug-resistant (MDR) tuberculosis (i.e., acquired resistance) versus those that were due to transmission (i.e., transmitted resistance). We used social-network analysis to identify community and hospital locations of transmission. RESULTS Of the 404 participants, 311 (77%) had HIV infection; the median CD4+ count was 340 cells per cubic millimeter (interquartile range, 117 to 431). A total of 280 participants (69%) had never received treatment for MDR tuberculosis. Genotypic analysis in 386 participants revealed that 323 (84%) belonged to 1 of 31 clusters. Clusters ranged from 2 to 14 participants, except for 1 large cluster of 212 participants (55%) with a LAM4/KZN strain. Person-to-person or hospital-based epidemiologic links were identified in 123 of 404 participants (30%). CONCLUSIONS The majority of cases of XDR tuberculosis in KwaZulu-Natal, South Africa, an area with a high tuberculosis burden, were probably due to transmission rather than to inadequate treatment of MDR tuberculosis. These data suggest that control of the epidemic of drug-resistant tuberculosis requires an increased focus on interrupting transmission. (Funded by the National Institute of Allergy and Infectious Diseases and others.) PMID:28099825

Cost-effectiveness of post-landing latent tuberculosis infection control strategies in new migrants to Canada.

PubMed

Campbell, Jonathon R; Johnston, James C; Sadatsafavi, Mohsen; Cook, Victoria J; Elwood, R Kevin; Marra, Fawziah

2017-01-01

The majority of tuberculosis in migrants to Canada occurs due to reactivation of latent TB infection. Risk of tuberculosis in those with latent tuberculosis infection can be significantly reduced with treatment. Presently, only 2.4% of new migrants are flagged for post-landing surveillance, which may include latent tuberculosis infection screening; no other migrants receive routine latent tuberculosis infection screening. To aid in reducing the tuberculosis burden in new migrants to Canada, we determined the cost-effectiveness of using different latent tuberculosis infection interventions in migrants under post-arrival surveillance and in all new migrants. A discrete event simulation model was developed that focused on a Canadian permanent resident cohort after arrival in Canada, utilizing a ten-year time horizon, healthcare system perspective, and 1.5% discount rate. Latent tuberculosis infection interventions were evaluated in the population under surveillance (N = 6100) and the total cohort (N = 260,600). In all evaluations, six different screening and treatment combinations were compared to the base case of tuberculin skin test screening followed by isoniazid treatment only in the population under surveillance. Quality adjusted life years, incident tuberculosis cases, and costs were recorded for each intervention and incremental cost-effectiveness ratios were calculated in relation to the base case. In the population under surveillance (N = 6100), using an interferon-gamma release assay followed by rifampin was dominant compared to the base case, preventing 4.90 cases of tuberculosis, a 4.9% reduction, adding 4.0 quality adjusted life years, and saving $353,013 over the ensuing ten-years. Latent tuberculosis infection screening in the total population (N = 260,600) was not cost-effective when compared to the base case, however could potentially prevent 21.8% of incident tuberculosis cases. Screening new migrants under surveillance with an interferon-gamma release assay and treating with rifampin is cost saving, but will not significantly impact TB incidence. Universal latent tuberculosis infection screening and treatment is cost-prohibitive. Research into using risk factors to target screening post-landing may provide alternate solutions.

Human Granuloma In Vitro Model, for TB Dormancy and Resuscitation

PubMed Central

Kapoor, Nidhi; Pawar, Santosh; Sirakova, Tatiana D.; Deb, Chirajyoti; Warren, William L.; Kolattukudy, Pappachan E.

2013-01-01

Tuberculosis (TB) is responsible for death of nearly two million people in the world annually. Upon infection, Mycobacterium tuberculosis (Mtb) causes formation of granuloma where the pathogen goes into dormant state and can live for decades before resuscitation to develop active disease when the immune system of the host is weakened and/or suppressed. In an attempt to better understand host-pathogen interactions, several groups have been developing in vitro models of human tuberculosis granuloma. However, to date, an in vitro granuloma model in which Mtb goes into dormancy and can subsequently resuscitate under conditions that mimic weakening of the immune system has not been reported. We describe the development of a biomimetic in vitro model of human tuberculosis granuloma using human primary leukocytes, in which the Mtb exhibited characteristics of dormant mycobacteria as demonstrated by (1) loss of acid-fastness, (2) accumulation of lipid bodies (3) development of rifampicin-tolerance and (4) gene expression changes. Further, when these micro granulomas were treated with immunosuppressant anti-tumor necrosis factor-alpha monoclonal antibodies (anti-TNFα mAbs), resuscitation of Mtb was observed as has been found in humans. In this human in vitro granuloma model triacylglycerol synthase 1deletion mutant (Δtgs1) with impaired ability to accumulate triacylglycerides (TG), but not the complemented mutant, could not go into dormancy. Deletion mutant of lipY, with compromised ability to mobilize the stored TG, but not the complemented mutant, was unable to come out of dormancy upon treatment with anti-TNFα mAbs. In conclusion, we have developed an in vitro human tuberculosis granuloma model that largely exhibits functional features of dormancy and resuscitation observed in human tuberculosis. PMID:23308269

Ga(III) Nanoparticles Inhibit Growth of both Mycobacterium tuberculosis and HIV and Release of Interleukin-6 (IL-6) and IL-8 in Coinfected Macrophages

PubMed Central

Choi, Seoung-ryoung; Britigan, Bradley E.

2017-01-01

ABSTRACT Treatment of individuals coinfected with human immunodeficiency virus (HIV) type 1 and Mycobacterium tuberculosis is challenging due to the prolonged treatment requirements, drug toxicity, and emergence of drug resistance. Mononuclear phagocytes (MP; macrophages) are one of the natural reservoirs for both HIV and M. tuberculosis. Here, the treatment of HIV and M. tuberculosis coinfection was studied by preloading human macrophages with MP-targeted gallium (Ga) nanoparticles to limit subsequent simultaneous infection with both HIV and M. tuberculosis. Ga nanoparticles provided sustained drug release for 15 days and significantly inhibited the replication of both HIV and M. tuberculosis. Addition of Ga nanoparticles to MP already infected with M. tuberculosis or HIV resulted in a significant decrease in the magnitude of these infections, but the magnitude was less than that achieved with nanoparticle preloading of the MP. In addition, macrophages that were coinfected with HIV and M. tuberculosis and that were loaded with Ga nanoparticles reduced the levels of interleukin-6 (IL-6) and IL-8 secretion for up to 15 days after drug loading. Ga nanoparticles also reduced the levels of IL-6 and IL-8 secretion by ionomycin- and lipopolysaccharide-induced macrophages, likely by modulating the IκB kinase-β/NF-κB pathway. Delivery of Ga nanoparticles to macrophages is a potent long-acting approach for suppressing HIV and M. tuberculosis coinfection of macrophages in vitro and sets the stage for the development of new approaches to the treatment of these important infections. PMID:28167548

Mycobacterium tuberculosis impairs dendritic cell functions through the serine hydrolase Hip1.

PubMed

Madan-Lala, Ranjna; Sia, Jonathan Kevin; King, Rebecca; Adekambi, Toidi; Monin, Leticia; Khader, Shabaana A; Pulendran, Bali; Rengarajan, Jyothi

2014-05-01

Mycobacterium tuberculosis is a highly successful human pathogen that primarily resides in host phagocytes, such as macrophages and dendritic cells (DCs), and interferes with their functions. Although multiple strategies used by M. tuberculosis to modulate macrophage responses have been discovered, interactions between M. tuberculosis and DCs are less well understood. DCs are the primary APCs of the immune system and play a central role in linking innate and adaptive immune responses to microbial pathogens. In this study, we show that M. tuberculosis impairs DC cytokine secretion, maturation, and Ag presentation through the cell envelope-associated serine hydrolase, Hip1. Compared to wild-type, a hip1 mutant strain of M. tuberculosis induced enhanced levels of the key Th1-inducing cytokine IL-12, as well as other proinflammatory cytokines (IL-23, IL-6, TNF-α, IL-1β, and IL-18) in DCs via MyD88- and TLR2/9-dependent pathways, indicating that Hip1 restricts optimal DC inflammatory responses. Infection with the hip1 mutant also induced higher levels of MHC class II and costimulatory molecules CD40 and CD86, indicating that M. tuberculosis impairs DC maturation through Hip1. Further, we show that M. tuberculosis promotes suboptimal Ag presentation, as DCs infected with the hip1 mutant showed increased capacity to present Ag to OT-II- and early secreted antigenic target 6-specific transgenic CD4 T cells and enhanced Th1 and Th17 polarization. Overall, these data show that M. tuberculosis impairs DC functions and modulates the nature of Ag-specific T cell responses, with important implications for vaccination strategies.

The adaptor molecule CARD9 is essential for tuberculosis control

PubMed Central

Dorhoi, Anca; Desel, Christiane; Yeremeev, Vladimir; Pradl, Lydia; Brinkmann, Volker; Mollenkopf, Hans-Joachim; Hanke, Karin; Gross, Olaf; Ruland, Jürgen

2010-01-01

The cross talk between host and pathogen starts with recognition of bacterial signatures through pattern recognition receptors (PRRs), which mobilize downstream signaling cascades. We investigated the role of the cytosolic adaptor caspase recruitment domain family, member 9 (CARD9) in tuberculosis. This adaptor was critical for full activation of innate immunity by converging signals downstream of multiple PRRs. Card9−/− mice succumbed early after aerosol infection, with higher mycobacterial burden, pyogranulomatous pneumonia, accelerated granulocyte recruitment, and higher abundance of proinflammatory cytokines and granulocyte colony-stimulating factor (G-CSF) in serum and lung. Neutralization of G-CSF and neutrophil depletion significantly prolonged survival, indicating that an exacerbated systemic inflammatory disease triggered lethality of Card9−/− mice. CARD9 deficiency had no apparent effect on T cell responses, but a marked impact on the hematopoietic compartment. Card9−/− granulocytes failed to produce IL-10 after Mycobaterium tuberculosis infection, suggesting that an absent antiinflammatory feedback loop accounted for granulocyte-dominated pathology, uncontrolled bacterial replication, and, ultimately, death of infected Card9−/− mice. Our data provide evidence that deregulated innate responses trigger excessive lung inflammation and demonstrate a pivotal role of CARD9 signaling in autonomous innate host defense against tuberculosis. PMID:20351059

2-aminoimidazoles potentiate ß-lactam antimicrobial activity against Mycobacterium tuberculosis by reducing ß-lactamase secretion and increasing cell envelope permeability

PubMed Central

Obregón-Henao, Andrés; Ackart, David F.; Podell, Brendan K.; Belardinelli, Juan M.; Jackson, Mary; Nguyen, Tuan V.; Blackledge, Meghan S.; Melander, Roberta J.; Melander, Christian; Johnson, Benjamin K.; Abramovitch, Robert B.

2017-01-01

There is an urgent need to develop new drug treatment strategies to control the global spread of drug-sensitive and multidrug-resistant Mycobacterium tuberculosis (M. tuberculosis). The ß-lactam class of antibiotics is among the safest and most widely prescribed antibiotics, but they are not effective against M. tuberculosis due to intrinsic resistance. This study shows that 2-aminoimidazole (2-AI)-based small molecules potentiate ß-lactam antibiotics against M. tuberculosis. Active 2-AI compounds significantly reduced the minimal inhibitory and bactericidal concentrations of ß-lactams by increasing M. tuberculosis cell envelope permeability and decreasing protein secretion including ß-lactamase. Metabolic labeling and transcriptional profiling experiments revealed that 2-AI compounds impair mycolic acid biosynthesis, export and linkage to the mycobacterial envelope, counteracting an important defense mechanism reducing permeability to external agents. Additionally, other important constituents of the M. tuberculosis outer membrane including sulfolipid-1 and polyacyltrehalose were also less abundant in 2-AI treated bacilli. As a consequence of 2-AI treatment, M. tuberculosis displayed increased sensitivity to SDS, increased permeability to nucleic acid staining dyes, and rapid binding of cell wall targeting antibiotics. Transcriptional profiling analysis further confirmed that 2-AI induces transcriptional regulators associated with cell envelope stress. 2-AI based small molecules potentiate the antimicrobial activity of ß-lactams by a mechanism that is distinct from specific inhibitors of ß-lactamase activity and therefore may have value as an adjunctive anti-TB treatment. PMID:28749949

Investing in improved performance of national tuberculosis programs reduces the tuberculosis burden: analysis of 22 high-burden countries, 2002-2009.

PubMed

Akachi, Yoko; Zumla, Alimuddin; Atun, Rifat

2012-05-15

To assess the impact of investment in national tuberculosis programs (NTPs) on NTP performance and tuberculosis burden in 22 high-burden countries, as determined by the World Health Organization (WHO). Estimates of annual tuberculosis burden and NTP performance indicators and control variables during 2002-2009 were obtained from the Organization for Economic Cooperation and Development, the WHO, the World Bank, and the Penn World Table for the 22 high-burden countries. Panel data analysis was performed using the outcome variables tuberculosis incidence, prevalence, and mortality and the key explanatory variables Partnership case detection rate and treatment success rate, controlling for gross domestic product per capita, population structure, and human immunodeficiency virus (HIV) prevalence. A $1 per capita (general population) higher NTP budget (including domestic and external sources) was associated with a 1.9% (95% confidence interval, .12%-3.6%) higher estimated case detection rate the following year for the 22 high-burden countries between 2002 and 2009. In the final models, which corrected for autocorrelation and heteroskedasticity, achieving the STOP TB Partnership case detection rate target of >70% was associated with significantly (P < .01) lower tuberculosis incidence, prevalence, and mortality the following year, even when controlling for general economic development and HIV prevalence as potential confounding variables. Increased investment in NTPs was significantly associated with improved performance and with a downward trend in the tuberculosis burden in the 22 high-burden countries during 2002-2009.

Docking into Mycobacterium tuberculosis Thioredoxin Reductase Protein Yields Pyrazolone Lead Molecules for Methicillin-Resistant Staphylococcus aureus

PubMed Central

Sweeney, Noreena L.; Lipker, Lauren; Hanson, Alicia M.; Bohl, Chris J.; Engel, Katie E.; Kalous, Kelsey S.; Stemper, Mary E.; Sem, Daniel S.; Schwan, William R.

2017-01-01

The thioredoxin/thioredoxin reductase system (Trx/TrxR) is an attractive drug target because of its involvement in a number of important physiological processes, from DNA synthesis to regulating signal transduction. This study describes the finding of pyrazolone compounds that are active against Staphylococcus aureus. Initially, the project was focused on discovering small molecules that may have antibacterial properties targeting the Mycobacterium tuberculosis thioredoxin reductase. This led to the discovery of a pyrazolone scaffold-containing compound series that showed bactericidal capability against S. aureus strains, including drug-resistant clinical isolates. The findings support continued development of the pyrazolone compounds as potential anti-S. aureus antibiotics. PMID:28134858

Preparation of immunochromatographic strips for rapid detection of early secreted protein ESAT-6 and culture filtrate protein CFP-10 from Mycobacterium tuberculosis

PubMed Central

Wu, Xiaoxin; Wang, Yeping; Weng, Tianhao; Hu, Chenyu; Wang, Frederick X.C.; Wu, Zhigang; Yu, Dongshan; Lu, Huoquan; Yao, Hangping

2017-01-01

Abstract The early secreted protein early secretory antigenic target 6(ESAT-6) and the culture filtrate protein 10 (CFP-10) are 2 antigens that are specific to Mycobacterium tuberculosis. These 2 antigens are good targets for tuberculosis (TB) detection. To rapidly diagnose TB across a variety of samples, we developed colloidal gold immunochromatographic strips (ICSs) based on ESAT-6 and CFP-10. The strips were evaluated using 233 samples, including sputum, plasma, and pleural effusion samples. The positive detection rates for ICSs for ESAT-6 and CFP-10 in sputum (culture-positive for M tuberculosis) were 100% and 91.2%, respectively. The positive detection rates for ICSs for ESAT-6 and CFP-10 in plasma were 34.1% and 29.4%, respectively. The positive detection rates for ICSs for ESAT-6 and CFP-10 in pleural effusion were 64.7% and 55.9%, respectively. Experimental analysis of culture supernatant showing that the ICS developed for ESAT-6 had a sensitivity of 100% and a specificity of 91.2%. While the ICS developed for CFP-10 had a sensitivity of 91.2% and a specificity of 88.2%. The validity of the test is limited by source of sample. The technique is sensitive and specific for samples in sputum and culture media but not for plasma or pleural effusion samples. Detection of M tuberculosis using ICSs is rapid, simple, and relatively effective; thus, ICSs are a potential screening tool for TB. PMID:29390519

Preparation of immunochromatographic strips for rapid detection of early secreted protein ESAT-6 and culture filtrate protein CFP-10 from Mycobacterium tuberculosis.

PubMed

Wu, Xiaoxin; Wang, Yeping; Weng, Tianhao; Hu, Chenyu; Wang, Frederick X C; Wu, Zhigang; Yu, Dongshan; Lu, Huoquan; Yao, Hangping

2017-12-01

The early secreted protein early secretory antigenic target 6(ESAT-6) and the culture filtrate protein 10 (CFP-10) are 2 antigens that are specific to Mycobacterium tuberculosis. These 2 antigens are good targets for tuberculosis (TB) detection.To rapidly diagnose TB across a variety of samples, we developed colloidal gold immunochromatographic strips (ICSs) based on ESAT-6 and CFP-10.The strips were evaluated using 233 samples, including sputum, plasma, and pleural effusion samples.The positive detection rates for ICSs for ESAT-6 and CFP-10 in sputum (culture-positive for M tuberculosis) were 100% and 91.2%, respectively. The positive detection rates for ICSs for ESAT-6 and CFP-10 in plasma were 34.1% and 29.4%, respectively. The positive detection rates for ICSs for ESAT-6 and CFP-10 in pleural effusion were 64.7% and 55.9%, respectively. Experimental analysis of culture supernatant showing that the ICS developed for ESAT-6 had a sensitivity of 100% and a specificity of 91.2%. While the ICS developed for CFP-10 had a sensitivity of 91.2% and a specificity of 88.2%.The validity of the test is limited by source of sample. The technique is sensitive and specific for samples in sputum and culture media but not for plasma or pleural effusion samples. Detection of M tuberculosis using ICSs is rapid, simple, and relatively effective; thus, ICSs are a potential screening tool for TB. Copyright © 2017 The Authors. Published by Wolters Kluwer Health, Inc. All rights reserved.

A high-throughput screen against pantothenate synthetase (PanC) identifies 3-biphenyl-4-cyanopyrrole-2-carboxylic acids as a new class of inhibitor with activity against Mycobacterium tuberculosis.

PubMed

Kumar, Anuradha; Casey, Allen; Odingo, Joshua; Kesicki, Edward A; Abrahams, Garth; Vieth, Michal; Masquelin, Thierry; Mizrahi, Valerie; Hipskind, Philip A; Sherman, David R; Parish, Tanya

2013-01-01

The enzyme pantothenate synthetase, PanC, is an attractive drug target in Mycobacterium tuberculosis. It is essential for the in vitro growth of M. tuberculosis and for survival of the bacteria in the mouse model of infection. PanC is absent from mammals. We developed an enzyme-based assay to identify inhibitors of PanC, optimized it for high-throughput screening, and tested a large and diverse library of compounds for activity. Two compounds belonging to the same chemical class of 3-biphenyl-4- cyanopyrrole-2-carboxylic acids had activity against the purified recombinant protein, and also inhibited growth of live M. tuberculosis in manner consistent with PanC inhibition. Thus we have identified a new class of PanC inhibitors with whole cell activity that can be further developed.

Genomic analysis of globally diverse Mycobacterium tuberculosis strains provides insights into emergence and spread of multidrug resistance

PubMed Central

Manson, Abigail L.; Cohen, Keira A.; Abeel, Thomas; Desjardins, Christopher A.; Armstrong, Derek T.; Barry, Clifton E.; Brand, Jeannette; Chapman, Sinéad B.; Cho, Sang-Nae; Gabrielian, Andrei; Gomez, James; Jodals, Andreea M.; Joloba, Moses; Jureen, Pontus; Lee, Jong Seok; Malinga, Lesibana; Maiga, Mamoudou; Nordenberg, Dale; Noroc, Ecaterina; Romancenco, Elena; Salazar, Alex; Ssengooba, Willy; Velayati, A. A.; Winglee, Kathryn; Zalutskaya, Aksana; Via, Laura E.; Cassell, Gail H.; Dorman, Susan E.; Ellner, Jerrold; Farnia, Parissa; Galagan, James E.; Rosenthal, Alex; Crudu, Valeriu; Homorodean, Daniela; Hsueh, Po-Ren; Narayanan, Sujatha; Pym, Alexander S.; Skrahina, Alena; Swaminathan, Soumya; Van der Walt, Martie; Alland, David; Bishai, William R.; Cohen, Ted; Hoffner, Sven; Birren, Bruce W.; Earl, Ashlee M.

2017-01-01

Multidrug-resistant tuberculosis (MDR-TB), caused by drug resistant strains of Mycobacterium tuberculosis, is an increasingly serious problem worldwide. In this study, we examined a dataset of 5,310 M. tuberculosis whole genome sequences from five continents. Despite great diversity with respect to geographic point of isolation, genetic background and drug resistance, patterns of drug resistance emergence were conserved globally. We have identified harbinger mutations that often precede MDR. In particular, the katG S315T mutation, conferring resistance to isoniazid, overwhelmingly arose before rifampicin resistance across all lineages, geographic regions, and time periods. Molecular diagnostics that include markers for rifampicin resistance alone will be insufficient to identify pre-MDR strains. Incorporating knowledge of pre-MDR polymorphisms, particularly katG S315, into molecular diagnostics will enable targeted treatment of patients with pre-MDR-TB to prevent further development of MDR-TB. PMID:28092681

Mycobacterium biofilms: factors involved in development, dispersal, and therapeutic strategies against biofilm-relevant pathogens.

PubMed

Xiang, Xiaohong; Deng, Wanyan; Liu, Minqiang; Xie, Jianping

2014-01-01

Many bacteria can develop biofilm (BF), a multicellular structure largely combining bacteria and their extracellular polymeric substances (EPS). The formation of biofilm results in an alternative existence in which microbes ensure their survival in adverse environments. Biofilm-relevant infections are more persistent, resistant to most antibiotics, and more recalcitrant to host immunity. Mycobacterium tuberculosis, the causative agent of tuberculosis, can develop biofilm, though whether M. tuberculosis can form biofilm within tuberculosis patients has yet to be determined. Here, we summarize the factors involved in the development and dispersal of mycobacterial biofilms, as well as underlying regulatory factors and inhibitors against biofilm to deepen our understanding of their development and to elucidate potential novel modes of action for future antibiotics. Key factors in biofilm formation identified as drug targets represent a novel and promising avenue for developing better antibiotics.

Enterobacterial Repetitive Intergenic Consensus Sequences as Molecular Targets for Typing of Mycobacterium tuberculosis Strains

PubMed Central

Sechi, Leonardo A.; Zanetti, Stefania; Dupré, Ilaria; Delogu, Giovanni; Fadda, Giovanni

1998-01-01

The presence of enterobacterial repetitive intergenic consensus (ERIC) sequences was demonstrated for the first time in the genome of Mycobacterium tuberculosis; these sequences have been found in transcribed regions of the chromosomes of gram-negative bacteria. In this study genetic diversity among clinical isolates of M. tuberculosis was determined by PCR with ERIC primers (ERIC-PCR). The study isolates comprised 71 clinical isolates collected from Sardinia, Italy. ERIC-PCR was able to identify 59 distinct profiles. The results obtained were compared with IS6110 and PCR-GTG fingerprinting. We found that the level of differentiation obtained by ERIC-PCR is greater than that obtained by IS6110 fingerprinting and comparable to that obtained by PCR-GTG. This method of fingerprinting is rapid and sensitive and can be applied to the study of the epidemiology of M. tuberculosis infections, especially when IS6110 fingerprinting is not of any help. PMID:9431935

Paper-based tuberculosis diagnostic devices with colorimetric gold nanoparticles

NASA Astrophysics Data System (ADS)

Tsai, Tsung-Ting; Shen, Shu-Wei; Cheng, Chao-Min; Chen, Chien-Fu

2013-08-01

A colorimetric sensing strategy employing gold nanoparticles and a paper assay platform has been developed for tuberculosis diagnosis. Unmodified gold nanoparticles and single-stranded detection oligonucleotides are used to achieve rapid diagnosis without complicated and time-consuming thiolated or other surface-modified probe preparation processes. To eliminate the use of sophisticated equipment for data analysis, the color variance for multiple detection results was simultaneously collected and concentrated on cellulose paper with the data readout transmitted for cloud computing via a smartphone. The results show that the 2.6 nM tuberculosis mycobacterium target sequences extracted from patients can easily be detected, and the turnaround time after the human DNA is extracted from clinical samples was approximately 1 h.

Comparison of gene expression profiles between pansensitive and multidrug-resistant strains of Mycobacterium tuberculosis.

PubMed

Peñuelas-Urquides, K; González-Escalante, L; Villarreal-Treviño, L; Silva-Ramírez, B; Gutiérrez-Fuentes, D J; Mojica-Espinosa, R; Rangel-Escareño, C; Uribe-Figueroa, L; Molina-Salinas, G M; Dávila-Velderrain, J; Castorena-Torres, F; Bermúdez de León, M; Said-Fernández, S

2013-09-01

Mycobacterium tuberculosis has developed resistance to anti-tuberculosis first-line drugs. Multidrug-resistant strains complicate the control of tuberculosis and have converted it into a worldwide public health problem. Mutational studies of target genes have tried to envisage the resistance in clinical isolates; however, detection of these mutations in some cases is not sufficient to identify drug resistance, suggesting that other mechanisms are involved. Therefore, the identification of new markers of susceptibility or resistance to first-line drugs could contribute (1) to specifically diagnose the type of M. tuberculosis strain and prescribe an appropriate therapy, and (2) to elucidate the mechanisms of resistance in multidrug-resistant strains. In order to identify specific genes related to resistance in M. tuberculosis, we compared the gene expression profiles between the pansensitive H37Rv strain and a clinical CIBIN:UMF:15:99 multidrug-resistant isolate using microarray analysis. Quantitative real-time PCR confirmed that in the clinical multidrug-resistant isolate, the esxG, esxH, rpsA, esxI, and rpmI genes were upregulated, while the lipF, groES, and narG genes were downregulated. The modified genes could be involved in the mechanisms of resistance to first-line drugs in M. tuberculosis and could contribute to increased efficiency in molecular diagnosis approaches of infections with drug-resistant strains.

Strengthening the Tuberculosis Specimen Referral Network in Uganda: The Role of Public-Private Partnerships.

PubMed

Joloba, Moses; Mwangi, Christina; Alexander, Heather; Nadunga, Diana; Bwanga, Freddie; Modi, Nelson; Downing, Robert; Nabasirye, Agnes; Adatu, Francis E; Shrivastava, Ritu; Gadde, Renuka; Nkengasong, John N

2016-04-15

Diagnosis of multidrug-resistant tuberculosis and prompt initiation of effective treatment rely on access to rapid and reliable drug-susceptibility testing. Efficient specimen transport systems and appropriate training on specimen referral contribute to optimal and timely access to tuberculosis diagnostic services. With support and technical assistance from a public-private partnership (PPP) between Becton Dickinson and the US President's Emergency Plan for AIDS Relief, the Uganda National TB Reference Laboratory (NTRL) and National TB and Leprosy Program redesigned the tuberculosis specimen transport network and trained healthcare workers with the goal of improving multidrug-resistant tuberculosis detection. Between 2008 and 2011, the PPP mapped 93% of health facilities and trained 724 healthcare and postal staff members covering 72% of districts. Strengthening the tuberculosis specimen referral system increased referrals from presumptive multidrug-resistant tuberculosis cases by >10-fold, with 94% of specimens reaching the NTRL within the established target transport time. This study demonstrates the potential of PPP collaborations with ministries of health to positively influence patient care by strengthening laboratory systems through increased access to drug-susceptibility testing in Uganda. Ongoing efforts to integrate specimen transport networks will maximize resources and improve patient management. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Detection of Mycobacterium tuberculosis complex by nested polymerase chain reaction in pulmonary and extrapulmonary specimens* ,**

PubMed Central

Furini, Adriana Antônia da Cruz; Pedro, Heloisa da Silveira Paro; Rodrigues, Jean Francisco; Montenegro, Lilian Maria Lapa; Machado, Ricardo Luiz Dantas; Franco, Célia; Schindler, Haiana Charifker; Batista, Ida Maria Foschiani Dias; Rossit, Andrea Regina Baptista

2013-01-01

OBJECTIVE: To compare the performance of nested polymerase chain reaction (NPCR) with that of cultures in the detection of the Mycobacterium tuberculosis complex in pulmonary and extrapulmonary specimens. METHODS: We analyzed 20 and 78 pulmonary and extrapulmonary specimens, respectively, of 67 hospitalized patients suspected of having tuberculosis. An automated microbial system was used for the identification of Mycobacterium spp. cultures, and M. tuberculosis IS6110 was used as the target sequence in the NPCR. The kappa statistic was used in order to assess the level of agreement among the results. RESULTS: Among the 67 patients, 6 and 5, respectively, were diagnosed with pulmonary and extrapulmonary tuberculosis, and the NPCR was positive in all of the cases. Among the 98 clinical specimens, smear microscopy, culture, and NPCR were positive in 6.00%, 8.16%, and 13.26%, respectively. Comparing the results of NPCR with those of cultures (the gold standard), we found that NPCR had a sensitivity and specificity of 100% and 83%, respectively, in pulmonary specimens, compared with 83% and 96%, respectively, in extrapulmonary specimens, with good concordance between the tests (kappa, 0.50 and 0.6867, respectively). CONCLUSIONS: Although NPCR proved to be a very useful tool for the detection of M. tuberculosis complex, clinical, epidemiological, and other laboratory data should also be considered in the diagnosis and treatment of pulmonary and extrapulmonary tuberculosis. PMID:24473765

Can inhibitor-resistant substitutions in the Mycobacterium tuberculosis β-Lactamase BlaC lead to clavulanate resistance?: a biochemical rationale for the use of β-lactam-β-lactamase inhibitor combinations.

PubMed

Kurz, Sebastian G; Wolff, Kerstin A; Hazra, Saugata; Bethel, Christopher R; Hujer, Andrea M; Smith, Kerri M; Xu, Yan; Tremblay, Lee W; Blanchard, John S; Nguyen, Liem; Bonomo, Robert A

2013-12-01

The current emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis calls for novel treatment strategies. Recently, BlaC, the principal β-lactamase of Mycobacterium tuberculosis, was recognized as a potential therapeutic target. The combination of meropenem and clavulanic acid, which inhibits BlaC, was found to be effective against even extensively drug-resistant M. tuberculosis strains when tested in vitro. Yet there is significant concern that drug resistance against this combination will also emerge. To investigate the potential of BlaC to evolve variants resistant to clavulanic acid, we introduced substitutions at important amino acid residues of M. tuberculosis BlaC (R220, A244, S130, and T237). Whereas the substitutions clearly led to in vitro clavulanic acid resistance in enzymatic assays but at the expense of catalytic activity, transformation of variant BlaCs into an M. tuberculosis H37Rv background revealed that impaired inhibition of BlaC did not affect inhibition of growth in the presence of ampicillin and clavulanate. From these data we propose that resistance to β-lactam-β-lactamase inhibitor combinations will likely not arise from structural alteration of BlaC, therefore establishing confidence that this therapeutic modality can be part of a successful treatment regimen against M. tuberculosis.

Molecular Quantum Similarity, Chemical Reactivity and Database Screening of 3D Pharmacophores of the Protein Kinases A, B and G from Mycobacterium tuberculosis.

PubMed

Morales-Bayuelo, Alejandro

2017-06-21

Mycobacterium tuberculosis remains one of the world's most devastating pathogens. For this reason, we developed a study involving 3D pharmacophore searching, selectivity analysis and database screening for a series of anti-tuberculosis compounds, associated with the protein kinases A, B, and G. This theoretical study is expected to shed some light onto some molecular aspects that could contribute to the knowledge of the molecular mechanics behind interactions of these compounds, with anti-tuberculosis activity. Using the Molecular Quantum Similarity field and reactivity descriptors supported in the Density Functional Theory, it was possible to measure the quantification of the steric and electrostatic effects through the Overlap and Coulomb quantitative convergence (alpha and beta) scales. In addition, an analysis of reactivity indices using global and local descriptors was developed, identifying the binding sites and selectivity on these anti-tuberculosis compounds in the active sites. Finally, the reported pharmacophores to PKn A, B and G, were used to carry out database screening, using a database with anti-tuberculosis drugs from the Kelly Chibale research group (http://www.kellychibaleresearch.uct.ac.za/), to find the compounds with affinity for the specific protein targets associated with PKn A, B and G. In this regard, this hybrid methodology (Molecular Mechanic/Quantum Chemistry) shows new insights into drug design that may be useful in the tuberculosis treatment today.

Predictors of knowledge about tuberculosis: results from SANHANES I, a national, cross-sectional household survey in South Africa.

PubMed

Naidoo, Pamela; Simbayi, Leickness; Labadarios, Demetre; Ntsepe, Yoliswa; Bikitsha, Nwabisa; Khan, Gadija; Sewpaul, Ronel; Moyo, Sizulu; Rehle, Thomas

2016-03-18

South Africa is one of the 22 high tuberculosis burden countries that contribute 80% of the global tuberculosis cases. Tuberculosis is infectious and due to its rapid and easy transmission route poses a threat to population health. Considering the importance of social and psychological factors in influencing health outcomes, appraising knowledge and awareness of tuberculosis, remain vital for effective tuberculosis control. The main aim of this study was to investigate the factors that predict knowledge about tuberculosis among 18-64 year old adults in South Africa. A cross-sectional survey method was used. Multi-stage disproportionate, stratified cluster sampling was used to select households within enumeration areas stratified by province and locality type. Based on the Human Sciences Research Council 2007 master sample, 500 Enumerator Areas representative of the socio-demographic profile of South Africa were identified and a random sample of 20 households was randomly selected from each Enumerator Area, yielding an overall sample of 10,000 households. The tuberculosis module contained in the South African National Health And Nutrition Examination Survey I was the only module that examined the social determinants of an infectious disease. This module was questionnaire-based with no biomarkers obtained to screen for the presence of tuberculosis disease among the participants. Data was collected by administering a researcher developed individual level questionnaire. Simple and multiple linear regression was used to determine the independent variables associated with tuberculosis knowledge. Half the sample (52.6%) was female and the majority of the respondents were black African (76.5%). More than two thirds (68.0%) resided in urban areas, 56.9% did not complete high school and half were not in formal employment. Significant predictors of tuberculosis knowledge were race, sex, completion of high school, being in employment, having a diagnosis of the disease in ones' life-time and learning about tuberculosis from television, brochures, health workers, and teachers. To reduce the burden of tuberculosis in South Africa, media campaigns targeting both rural and urban communities should include conveying accurate information about the disease. Policy makers should also address structural barriers that vulnerable communities face.

Uridine monophosphate kinase as potential target for tuberculosis: from target to lead identification.

PubMed

Arvind, Akanksha; Jain, Vaibhav; Saravanan, Parameswaran; Mohan, C Gopi

2013-12-01

Mycobacterium tuberculosis (Mtb) is a causative agent of tuberculosis (TB) disease, which has affected approximately 2 billion people worldwide. Due to the emergence of resistance towards the existing drugs, discovery of new anti-TB drugs is an important global healthcare challenge. To address this problem, there is an urgent need to identify new drug targets in Mtb. In the present study, the subtractive genomics approach has been employed for the identification of new drug targets against TB. Screening the Mtb proteome using the Database of Essential Genes (DEG) and human proteome resulted in the identification of 60 key proteins which have no eukaryotic counterparts. Critical analysis of these proteins using Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways database revealed uridine monophosphate kinase (UMPK) enzyme as a potential drug target for developing novel anti-TB drugs. Homology model of Mtb-UMPK was constructed for the first time on the basis of the crystal structure of E. coli-UMPK, in order to understand its structure-function relationships, and which would in turn facilitate to perform structure-based inhibitor design. Furthermore, the structural similarity search was carried out using physiological inhibitor UTP of Mtb-UMPK to virtually screen ZINC database. Retrieved hits were further screened by implementing several filters like ADME and toxicity followed by molecular docking. Finally, on the basis of the Glide docking score and the mode of binding, 6 putative leads were identified as inhibitors of this enzyme which can potentially emerge as future drugs for the treatment of TB.

Improved BM212 MmpL3 Inhibitor Analogue Shows Efficacy in Acute Murine Model of Tuberculosis Infection

PubMed Central

Alfonso, Salvatore; Cocozza, Martina; Porretta, Giulio Cesare; Ballell, Lluís; Rullas, Joaquin; Ortega, Fátima; De Logu, Alessandro; Agus, Emanuela; La Rosa, Valentina; Pasca, Maria Rosalia; De Rossi, Edda; Wae, Baojie; Franzblau, Scott G.; Manetti, Fabrizio; Botta, Maurizio; Biava, Mariangela

2013-01-01

1,5-Diphenyl pyrroles were previously identified as a class of compounds endowed with high in vitro efficacy against M. tuberculosis. To improve the physical chemical properties and drug-like parameters of this class of compounds, a medicinal chemistry effort was undertaken. By selecting the optimal substitution patterns for the phenyl rings at N1 and C5 and by replacing the thiomorpholine moiety with a morpholine one, a new series of compounds was produced. The replacement of the sulfur with oxygen gave compounds with lower lipophilicity and improved in vitro microsomal stability. Moreover, since the parent compound of this family has been shown to target MmpL3, mycobacterial mutants resistant to two compounds have been isolated and characterized by sequencing the mmpL3 gene; all the mutants showed point mutations in this gene. The best compound identified to date was progressed to dose-response studies in an acute murine TB infection model. The resulting ED99 of 49 mg/Kg is within the range of commonly employed tuberculosis drugs, demonstrating the potential of this chemical series. The in vitro and in vivo target validation evidence presented here adds further weight to MmpL3 as a druggable target of interest for anti-tubercular drug discovery. PMID:23437287

Mycobacteria, Metals, and the Macrophage

PubMed Central

Niederweis, Michael; Wolschendorf, Frank; Mitra, Avishek; Neyrolles, Olivier

2015-01-01

Summary Mycobacterium tuberculosis is a facultative intracellular pathogen that thrives inside host macrophages. A key trait of M. tuberculosis is to exploit and manipulate metal cation trafficking inside infected macrophages to ensure survival and replication inside the phagosome. Here we describe the recent fascinating discoveries that the mammalian immune system responds to infections with M. tuberculosis by overloading the phagosome with copper and zinc, two metals which are essential nutrients in small quantities but are toxic in excess. M. tuberculosis has developed multi-faceted resistance mechanisms to protect itself from metal toxicity including control of uptake, sequestration inside the cell, oxidation, and efflux. The host response to infections combines this metal poisoning strategy with nutritional immunity mechanisms that deprive M. tuberculosis from metals such as iron and manganese to prevent bacterial replication. Both immune mechanisms rely on the translocation of metal transporter proteins to the phagosomal membrane during the maturation process of the phagosome. This review summarizes these recent findings and discusses how metal-targeted approaches might complement existing TB chemotherapeutic regimens with novel anti-infective therapies. PMID:25703564

Comparative analyses of nonpathogenic, opportunistic, and totally pathogenic mycobacteria reveal genomic and biochemical variabilities and highlight the survival attributes of Mycobacterium tuberculosis.

PubMed

Rahman, Syed Asad; Singh, Yadvir; Kohli, Sakshi; Ahmad, Javeed; Ehtesham, Nasreen Z; Tyagi, Anil K; Hasnain, Seyed E

2014-11-04

Mycobacterial evolution involves various processes, such as genome reduction, gene cooption, and critical gene acquisition. Our comparative genome size analysis of 44 mycobacterial genomes revealed that the nonpathogenic (NP) genomes were bigger than those of opportunistic (OP) or totally pathogenic (TP) mycobacteria, with the TP genomes being smaller yet variable in size--their genomic plasticity reflected their ability to evolve and survive under various environmental conditions. From the 44 mycobacterial species, 13 species, representing TP, OP, and NP, were selected for genomic-relatedness analyses. Analysis of homologous protein-coding genes shared between Mycobacterium indicus pranii (NP), Mycobacterium intracellulare ATCC 13950 (OP), and Mycobacterium tuberculosis H37Rv (TP) revealed that 4,995 (i.e., ~95%) M. indicaus pranii proteins have homology with M. intracellulare, whereas the homologies among M. indicus pranii, M. intracellulare ATCC 13950, and M. tuberculosis H37Rv were significantly lower. A total of 4,153 (~79%) M. indicus pranii proteins and 4,093 (~79%) M. intracellulare ATCC 13950 proteins exhibited homology with the M. tuberculosis H37Rv proteome, while 3,301 (~82%) and 3,295 (~82%) M. tuberculosis H37Rv proteins showed homology with M. indicus pranii and M. intracellulare ATCC 13950 proteomes, respectively. Comparative metabolic pathway analyses of TP/OP/NP mycobacteria showed enzymatic plasticity between M. indicus pranii (NP) and M. intracellulare ATCC 13950 (OP), Mycobacterium avium 104 (OP), and M. tuberculosis H37Rv (TP). Mycobacterium tuberculosis seems to have acquired novel alternate pathways with possible roles in metabolism, host-pathogen interactions, virulence, and intracellular survival, and by implication some of these could be potential drug targets. The complete sequence analysis of Mycobacterium indicus pranii, a novel species of Mycobacterium shown earlier to have strong immunomodulatory properties and currently in use for the treatment of leprosy, places it evolutionarily at the point of transition to pathogenicity. With the purpose of establishing the importance of M. indicus pranii in providing insight into the virulence mechanism of tuberculous and nontuberculous mycobacteria, we carried out comparative genomic and proteomic analyses of 44 mycobacterial species representing nonpathogenic (NP), opportunistic (OP), and totally pathogenic (TP) mycobacteria. Our results clearly placed M. indicus pranii as an ancestor of the M. avium complex. Analyses of comparative metabolic pathways between M. indicus pranii (NP), M. tuberculosis (TP), and M. intracellulare (OP) pointed to the presence of novel alternative pathways in M. tuberculosis with implications for pathogenesis and survival in the human host and identification of new drug targets. Copyright © 2014 Rahman et al.

Antigen 85C Inhibition Restricts Mycobacterium tuberculosis Growth through Disruption of Cord Factor Biosynthesis

PubMed Central

Warrier, Thulasi; Tropis, Marielle; Werngren, Jim; Diehl, Anne; Gengenbacher, Martin; Schlegel, Brigitte; Schade, Markus; Oschkinat, Hartmut; Daffe, Mamadou; Hoffner, Sven; Eddine, Ali Nasser

2012-01-01

The antigen 85 (Ag85) protein family, consisting of Ag85A, -B, and -C, is vital for Mycobacterium tuberculosis due to its role in cell envelope biogenesis. The mycoloyl transferase activity of these proteins generates trehalose dimycolate (TDM), an envelope lipid essential for M. tuberculosis virulence, and cell wall arabinogalactan-linked mycolic acids. Inhibition of these enzymes through substrate analogs hinders growth of mycobacteria, but a link to mycolic acid synthesis has not been established. In this study, we characterized a novel inhibitor of Ag85C, 2-amino-6-propyl-4,5,6,7-tetrahydro-1-benzothiophene-3-carbonitrile (I3-AG85). I3-AG85 was isolated from a panel of four inhibitors that exhibited structure- and dose-dependent inhibition of M. tuberculosis division in broth culture. I3-AG85 also inhibited M. tuberculosis survival in infected primary macrophages. Importantly, it displayed an identical MIC against the drug-susceptible H37Rv reference strain and a panel of extensively drug-resistant/multidrug-resistant M. tuberculosis strains. Nuclear magnetic resonance analysis indicated binding of I3-AG85 to Ag85C, similar to its binding to the artificial substrate octylthioglucoside. Quantification of mycolic acid-linked lipids of the M. tuberculosis envelope showed a specific blockade of TDM synthesis. This was accompanied by accumulation of trehalose monomycolate, while the overall mycolic acid abundance remained unchanged. Inhibition of Ag85C activity also disrupted the integrity of the M. tuberculosis envelope. I3-AG85 inhibited the division of and reduced TDM synthesis in an M. tuberculosis strain deficient in Ag85C. Our results indicate that Ag85 proteins are promising targets for novel antimycobacterial drug design. PMID:22290959

Structure based drug discovery for designing leads for the non-toxic metabolic targets in multi drug resistant Mycobacterium tuberculosis.

PubMed

Kaur, Divneet; Mathew, Shalu; Nair, Chinchu G S; Begum, Azitha; Jainanarayan, Ashwin K; Sharma, Mukta; Brahmachari, Samir K

2017-12-21

The problem of drug resistance and bacterial persistence in tuberculosis is a cause of global alarm. Although, the UN's Sustainable Development Goals for 2030 has targeted a Tb free world, the treatment gap exists and only a few new drug candidates are in the pipeline. In spite of large information from medicinal chemistry to 'omics' data, there has been a little effort from pharmaceutical companies to generate pipelines for the development of novel drug candidates against the multi drug resistant Mycobacterium tuberculosis. In the present study, we describe an integrated methodology; utilizing systems level information to optimize ligand selection to lower the failure rates at the pre-clinical and clinical levels. In the present study, metabolic targets (Rv2763c, Rv3247c, Rv1094, Rv3607c, Rv3048c, Rv2965c, Rv2361c, Rv0865, Rv0321, Rv0098, Rv0390, Rv3588c, Rv2244, Rv2465c and Rv2607) in M. tuberculosis, identified using our previous Systems Biology and data-intensive genome level analysis, have been used to design potential lead molecules, which are likely to be non-toxic. Various in silico drug discovery tools have been utilized to generate small molecular leads for each of the 15 targets with available crystal structures. The present study resulted in identification of 20 novel lead molecules including 4 FDA approved drugs (droxidropa, tetroxoprim, domperidone and nemonapride) which can be further taken for drug repurposing. This comprehensive integrated methodology, with both experimental and in silico approaches, has the potential to not only tackle the MDR form of Mtb but also the most important persister population of the bacterium, with a potential to reduce the failures in the Tb drug discovery. We propose an integrated approach of systems and structural biology for identifying targets that address the high attrition rate issue in lead identification and drug development We expect that this system level analysis will be applicable for identification of drug candidates to other pathogenic organisms as well.

The Expanding Diversity of Mycobacterium tuberculosis Drug Targets.

PubMed

Wellington, Samantha; Hung, Deborah T

2018-05-11

After decades of relative inactivity, a large increase in efforts to discover antitubercular therapeutics has brought insights into the biology of Mycobacterium tuberculosis (Mtb) and promising new drugs such as bedaquiline, which inhibits ATP synthase, and the nitroimidazoles delamanid and pretomanid, which inhibit both mycolic acid synthesis and energy production. Despite these advances, the drug discovery pipeline remains underpopulated. The field desperately needs compounds with novel mechanisms of action capable of inhibiting multi- and extensively drug -resistant Mtb (M/XDR-TB) and, potentially, nonreplicating Mtb with the hope of shortening the duration of required therapy. New knowledge about Mtb, along with new methods and technologies, has driven exploration into novel target areas, such as energy production and central metabolism, that diverge from the classical targets in macromolecular synthesis. Here, we review new small molecule drug candidates that act on these novel targets to highlight the methods and perspectives advancing the field. These new targets bring with them the aspiration of shortening treatment duration as well as a pipeline of effective regimens against XDR-TB, positioning Mtb drug discovery to become a model for anti-infective discovery.

Ebselen and analogs as inhibitors of Bacillus anthracis thioredoxin reductase and bactericidal antibacterials targeting Bacillus species, Staphylococcus aureus and Mycobacterium tuberculosis.

PubMed

Gustafsson, Tomas N; Osman, Harer; Werngren, Jim; Hoffner, Sven; Engman, Lars; Holmgren, Arne

2016-06-01

Bacillus anthracis is the causative agent of anthrax, a disease associated with a very high mortality rate in its invasive forms. We studied a number of ebselen analogs as inhibitors of B. anthracis thioredoxin reductase and their antibacterial activity on Bacillus subtilis, Staphylococcus aureus, Bacillus cereus and Mycobacterium tuberculosis. The most potent compounds in the series gave IC(50) values down to 70 nM for the pure enzyme and minimal inhibitory concentrations (MICs) down to 0.4 μM (0.12 μg/ml) for B. subtilis, 1.5 μM (0.64 μg/ml) for S. aureus, 2 μM (0.86 μg/ml) for B. cereus and 10 μg/ml for M. tuberculosis. Minimal bactericidal concentrations (MBCs) were found at 1-1.5 times the MIC, indicating a general, class-dependent, bactericidal mode of action. The combined bacteriological and enzymological data were used to construct a preliminary structure-activity-relationship for the benzoisoselenazol class of compounds. When S. aureus and B. subtilis were exposed to ebselen, we were unable to isolate resistant mutants on both solid and in liquid medium suggesting a high resistance barrier. These results suggest that ebselen and analogs thereof could be developed into a novel antibiotic class, useful for the treatment of infections caused by B. anthracis, S. aureus, M. tuberculosis and other clinically important bacteria. Furthermore, the high barrier against resistance development is encouraging for further drug development. We have characterized the thioredoxin system from B. anthracis as a novel drug target and ebselen and analogs thereof as a potential new class of antibiotics targeting several important human pathogens. Copyright © 2016 Elsevier B.V. All rights reserved.

Ethical Issues in Tuberculosis Control

PubMed Central

Elbek, Osman

2015-01-01

Tuberculosis (TB) remains a major global public health problem as also defined by the World Health Organization (WHO). On the other hand, the incidence of TB worldwide decreases at a lower rate than the intended targets, and it is seen that the targets set for 2015 will not be achieved at the global level. According to the WHO, failure to achieve the targets in TB control results from “resource constraints”, “conflict and instability” and “generalized human immunodeficiency virus epidemics”. This article is aimed to maintain an ethical debate in TB control and to investigate the WHO’s TB control policy and question the reasons for failure of this policy. Within the scope of this article; the TB of TB control was problematized at macro political level within the context of philosophy, while the approach of health care staff to TB patients was analysed at micro level within the context of professional ethics. PMID:29404082

Bioenergetics of Mycobacterium: An Emerging Landscape for Drug Discovery

PubMed Central

Iqbal, Iram Khan; Bajeli, Sapna; Akela, Ajit Kumar

2018-01-01

Mycobacterium tuberculosis (Mtb) exhibits remarkable metabolic flexibility that enables it to survive a plethora of host environments during its life cycle. With the advent of bedaquiline for treatment of multidrug-resistant tuberculosis, oxidative phosphorylation has been validated as an important target and a vulnerable component of mycobacterial metabolism. Exploiting the dependence of Mtb on oxidative phosphorylation for energy production, several components of this pathway have been targeted for the development of new antimycobacterial agents. This includes targeting NADH dehydrogenase by phenothiazine derivatives, menaquinone biosynthesis by DG70 and other compounds, terminal oxidase by imidazopyridine amides and ATP synthase by diarylquinolines. Importantly, oxidative phosphorylation also plays a critical role in the survival of persisters. Thus, inhibitors of oxidative phosphorylation can synergize with frontline TB drugs to shorten the course of treatment. In this review, we discuss the oxidative phosphorylation pathway and development of its inhibitors in detail. PMID:29473841

Evaporative concentration on a paper-based device to concentrate analytes in a biological fluid.

PubMed

Wong, Sharon Y; Cabodi, Mario; Rolland, Jason; Klapperich, Catherine M

2014-12-16

We report the first demonstration of using heat on a paper device to rapidly concentrate a clinically relevant analyte of interest from a biological fluid. Our technology relies on the application of localized heat to a paper strip to evaporate off hundreds of microliters of liquid to concentrate the target analyte. This method can be used to enrich for a target analyte that is present at low concentrations within a biological fluid to enhance the sensitivity of downstream detection methods. We demonstrate our method by concentrating the tuberculosis-specific glycolipid, lipoarabinomannan (LAM), a promising urinary biomarker for the detection and diagnosis of tuberculosis. We show that the heat does not compromise the subsequent immunodetectability of LAM, and in 20 min, the tuberculosis biomarker was concentrated by nearly 20-fold in simulated urine. Our method requires only 500 mW of power, and sample flow is self-driven via capillary action. As such, our technology can be readily integrated into portable, battery-powered, instrument-free diagnostic devices intended for use in low-resource settings.

Thiophenecarboxamide Derivatives Activated by EthA Kill Mycobacterium tuberculosis by Inhibiting the CTP Synthetase PyrG

PubMed Central

Mori, Giorgia; Chiarelli, Laurent R.; Esposito, Marta; Makarov, Vadim; Bellinzoni, Marco; Hartkoorn, Ruben C.; Degiacomi, Giulia; Boldrin, Francesca; Ekins, Sean; de Jesus Lopes Ribeiro, Ana Luisa; Marino, Leonardo B.; Centárová, Ivana; Svetlíková, Zuzana; Blaško, Jaroslav; Kazakova, Elena; Lepioshkin, Alexander; Barilone, Nathalie; Zanoni, Giuseppe; Porta, Alessio; Fondi, Marco; Fani, Renato; Baulard, Alain R.; Mikušová, Katarína; Alzari, Pedro M.; Manganelli, Riccardo; de Carvalho, Luiz Pedro S.; Riccardi, Giovanna; Cole, Stewart T.; Pasca, Maria Rosalia

2015-01-01

Summary To combat the emergence of drug-resistant strains of Mycobacterium tuberculosis, new antitubercular agents and novel drug targets are needed. Phenotypic screening of a library of 594 hit compounds uncovered two leads that were active against M. tuberculosis in its replicating, non-replicating, and intracellular states: compounds 7947882 (5-methyl-N-(4-nitrophenyl)thiophene-2-carboxamide) and 7904688 (3-phenyl-N-[(4-piperidin-1-ylphenyl)carbamothioyl]propanamide). Mutants resistant to both compounds harbored mutations in ethA (rv3854c), the gene encoding the monooxygenase EthA, and/or in pyrG (rv1699) coding for the CTP synthetase, PyrG. Biochemical investigations demonstrated that EthA is responsible for the activation of the compounds, and by mass spectrometry we identified the active metabolite of 7947882, which directly inhibits PyrG activity. Metabolomic studies revealed that pharmacological inhibition of PyrG strongly perturbs DNA and RNA biosynthesis, and other metabolic processes requiring nucleotides. Finally, the crystal structure of PyrG was solved, paving the way for rational drug design with this newly validated drug target. PMID:26097035

Crystallization and X-ray diffraction studies of a complete bacterial fatty-acid synthase type I

DOE Office of Scientific and Technical Information (OSTI.GOV)

Enderle, Mathias; Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried; McCarthy, Andrew

Bacterial and fungal type I fatty-acid synthases (FAS I) are evolutionarily connected, as bacterial FAS I is considered to be the ancestor of fungal FAS I. In this work, the production, crystallization and X-ray diffraction data analysis of a bacterial FAS I are reported. While a deep understanding of the fungal and mammalian multi-enzyme type I fatty-acid synthases (FAS I) has been achieved in recent years, the bacterial FAS I family, which is narrowly distributed within the Actinomycetales genera Mycobacterium, Corynebacterium and Nocardia, is still poorly understood. This is of particular relevance for two reasons: (i) although homologous to fungalmore » FAS I, cryo-electron microscopic studies have shown that bacterial FAS I has unique structural and functional properties, and (ii) M. tuberculosis FAS I is a drug target for the therapeutic treatment of tuberculosis (TB) and therefore is of extraordinary importance as a drug target. Crystals of FAS I from C. efficiens, a homologue of M. tuberculosis FAS I, were produced and diffracted X-rays to about 4.5 Å resolution.« less

Humoral immune profiling of mycobacterial antigen recognition in sarcoidosis and Löfgren's syndrome using high-content peptide microarrays.

PubMed

Ferrara, Giovanni; Valentini, Davide; Rao, Martin; Wahlström, Jan; Grunewald, Johan; Larsson, Lars-Olof; Brighenti, Susanna; Dodoo, Ernest; Zumla, Alimuddin; Maeurer, Markus

2017-03-01

Sarcoidosis is considered an idiopathic granulomatous disease, although similar immunological and clinical features with tuberculosis (TB) suggest mycobacterial involvement in its pathogenesis. High-content peptide microarrays (HCPM) may help to decipher mycobacteria-specific antibody reactivity in sarcoidosis. Serum samples from patients with sarcoidosis, Löfgren's syndrome, and TB, as well as from healthy individuals (12/group), were tested on HCPM containing 5964 individual peptides spanning 154 Mycobacterium tuberculosis proteins displayed as 15-amino acid stretches. Inclusion/exclusion and significance analyses were performed according to published methods. Each study group recognized 68-78% M. tuberculosis peptides at least once. M. tuberculosis epitope recognition by sarcoidosis patient sera was 42.7%, and by TB patient sera was 39.1%. Seven and 16 peptides were recognized in 9/12 (75%) and 8/12 (67%) sarcoidosis patient sera but not in TB patient sera, respectively. Nine (75%) and eight (67%) out of twelve TB patient sera, respectively recognized M. tuberculosis peptides that were not recognized in sarcoidosis patient sera. Specific IgG recognition patterns for M. tuberculosis antigens in sarcoidosis patients re-affirm mycobacterial involvement in sarcoidosis, providing biologically relevant targets for future studies pertaining to diagnostics and immunotherapy. Copyright © 2017. Published by Elsevier Ltd.

Four-gene Pan-African Blood Signature Predicts Progression to Tuberculosis.

PubMed

Suliman, Sara; Thompson, Ethan; Sutherland, Jayne; Weiner Rd, January; Ota, Martin O C; Shankar, Smitha; Penn-Nicholson, Adam; Thiel, Bonnie; Erasmus, Mzwandile; Maertzdorf, Jeroen; Duffy, Fergal J; Hill, Philip C; Hughes, E Jane; Stanley, Kim; Downing, Katrina; Fisher, Michelle L; Valvo, Joe; Parida, Shreemanta K; van der Spuy, Gian; Tromp, Gerard; Adetifa, Ifedayo M O; Donkor, Simon; Howe, Rawleigh; Mayanja-Kizza, Harriet; Boom, W Henry; Dockrell, Hazel; Ottenhoff, Tom H M; Hatherill, Mark; Aderem, Alan; Hanekom, Willem A; Scriba, Thomas J; Kaufmann, Stefan He; Zak, Daniel E; Walzl, Gerhard

2018-04-06

Contacts of tuberculosis (TB) patients constitute an important target population for preventative measures as they are at high risk of infection with Mycobacterium tuberculosis and progression to disease. We investigated biosignatures with predictive ability for incident tuberculosis. In a case-control study nested within the Grand Challenges 6-74 longitudinal HIV-negative African cohort of exposed household contacts, we employed RNA sequencing, polymerase chain reaction (PCR) and the Pair Ratio algorithm in a training/test set approach. Overall, 79 progressors, who developed tuberculosis between 3 and 24 months following exposure, and 328 matched non-progressors, who remained healthy during 24 months of follow-up, were investigated. A four-transcript signature (RISK4), derived from samples in a South African and Gambian training set, predicted progression up to two years before onset of disease in blinded test set samples from South Africa, The Gambia and Ethiopia with little population-associated variability and also validated on an external cohort of South African adolescents with latent Mycobacterium tuberculosis infection. By contrast, published diagnostic or prognostic tuberculosis signatures predicted on samples from some but not all 3 countries, indicating site-specific variability. Post-hoc meta-analysis identified a single gene pair, C1QC/TRAV27, that would consistently predict TB progression in household contacts from multiple African sites but not in infected adolescents without known recent exposure events. Collectively, we developed a simple whole blood-based PCR test to predict tuberculosis in household contacts from diverse African populations, with potential for implementation in national TB contact investigation programs.

Automated Peak Detection and Matching Algorithm for Gas Chromatography–Differential Mobility Spectrometry

PubMed Central

Fong, Sim S.; Rearden, Preshious; Kanchagar, Chitra; Sassetti, Christopher; Trevejo, Jose; Brereton, Richard G.

2013-01-01

A gas chromatography–differential mobility spectrometer (GC-DMS) involves a portable and selective mass analyzer that may be applied to chemical detection in the field. Existing approaches examine whole profiles and do not attempt to resolve peaks. A new approach for peak detection in the 2D GC-DMS chromatograms is reported. This method is demonstrated on three case studies: a simulated case study; a case study of headspace gas analysis of Mycobacterium tuberculosis (MTb) cultures consisting of three matching GC-DMS and GC-MS chromatograms; a case study consisting of 41 GC-DMS chromatograms of headspace gas analysis of MTb culture and media. PMID:21204557

Recently disclosed chemical entities as potential candidates for management of tuberculosis.

PubMed

Stec, Jozef; Abourashed, Ehab A

2015-01-01

Tuberculosis (TB) is one of the deadliest infectious diseases worldwide. The drug discovery process of novel, safe and effective agents to combat TB involves identification of new molecular targets and novel chemical scaffolds. The current anti-TB drug pipeline includes several small molecules with more to follow as new candidates are disclosed. This review highlights the most significant findings described in 78 international, European and US patents for chemically diverse compounds as prospective anti-TB medications. Main points of emphasis include chemical classification, in vitro and in vivo activity, ADME/Tox profile and mycobacterial target as described in each patent. The collective mass of compounds disclosed in the reviewed patents introduces new candidates as potential therapeutic agents for TB infections.

Host-Directed Therapeutics as a Novel Approach for Tuberculosis Treatment.

PubMed

Kim, Ye-Ram; Yang, Chul-Su

2017-09-28

Despite significant efforts to improve the treatment of tuberculosis (TB), it remains a prevalent infectious disease worldwide owing to the limitations of current TB therapeutic regimens. Recent work on novel TB treatment strategies has suggested that directly targeting host factors may be beneficial for TB treatment. Such strategies, termed host-directed therapeutics (HDTs), focus on host-pathogen interactions. HDTs may be more effective than the currently approved TB drugs, which are limited by the long durations of treatment needed and the emergence of drug-resistant strains. Targets of HDTs include host factors such as cytokines, immune checkpoints, immune cell functions, and essential enzyme activities. This review article discusses examples of potentially promising HDTs and introduces novel approaches for their development.

Incidence of tuberculosis among school-going adolescents in South India.

PubMed

Uppada, Dharma Rao; Selvam, Sumithra; Jesuraj, Nelson; Lau, Esther L; Doherty, T Mark; Grewal, Harleen M S; Vaz, Mario; Lindtjørn, Bernt

2016-07-26

Tuberculosis (TB) incidence data in vaccine target populations, particularly adolescents, are important for designing and powering vaccine clinical trials. Little is known about the incidence of tuberculosis among adolescents in India. The objective of current study is to estimate the incidence of pulmonary tuberculosis (PTB) disease among adolescents attending school in South India using two different surveillance methods (active and passive) and to compare the incidence between the two groups. The study was a prospective cohort study with a 2-year follow-up period. The study was conducted in Palamaner, Chittoor District of Andhra Pradesh, South India from February 2007 to July 2010. A random sampling procedure was used to select a subset of schools to enable approximately 8000 subjects to be available for randomization in the study. A stratified randomization procedure was used to assign the selected schools to either active or passive surveillance. Participants who met the criteria for being exposed to TB were referred to the diagnostic ward for pulmonary tuberculosis confirmation. A total number of 3441 males and 3202 females between the ages 11 and less than 18 years were enrolled into the study. Of the 3102 participants in the active surveillance group, four subjects were diagnosed with definite tuberculosis, four subjects with probable tuberculosis, and 71 subjects had non-tuberculous Mycobacteria (NTM) isolated from their sputum. Of the 3541 participants in the passive surveillance group, four subjects were diagnosed with definite tuberculosis, two subjects with probable tuberculosis, and 48 subjects had non-tuberculosis Mycobacteria isolated from their sputum. The incidence of definite + probable TB was 147.60 / 100,000 person years in the active surveillance group and 87 / 100,000 person years in the passive surveillance group. The incidence of pulmonary tuberculosis among adolescents in our study is lower than similar studies conducted in South Africa and Eastern Uganda - countries with a higher incidence of tuberculosis and human immunodeficiency virus (HIV) than India. The study data will inform sample design for vaccine efficacy trials among adolescents in India.

Alcohol, Hospital Discharge, and Socioeconomic Risk Factors for Default from Multidrug Resistant Tuberculosis Treatment in Rural South Africa: A Retrospective Cohort Study

PubMed Central

Kendall, Emily A.; Theron, Danie; Franke, Molly F.; van Helden, Paul; Victor, Thomas C.; Murray, Megan B.; Warren, Robin M.; Jacobson, Karen R.

2013-01-01

Background Default from multidrug-resistant tuberculosis (MDR-TB) treatment remains a major barrier to cure and epidemic control. We sought to identify patient risk factors for default from MDR-TB treatment and high-risk time periods for default in relation to hospitalization and transition to outpatient care. Methods We retrospectively analyzed a cohort of 225 patients who initiated MDR-TB treatment between 2007 through 2010 at a rural TB hospital in the Western Cape Province, South Africa. Results Fifty percent of patients were cured or completed treatment, 27% defaulted, 14% died, 4% failed treatment, and 5% transferred out. Recent alcohol use was common (63% of patients). In multivariable proportional hazards regression, older age (hazard ratio [HR]= 0.97 [95% confidence interval 0.94-0.99] per year of greater age), formal housing (HR=0.38 [0.19-0.78]), and steady employment (HR=0.41 [0.19-0.90]) were associated with decreased risk of default, while recent alcohol use (HR=2.1 [1.1-4.0]), recent drug use (HR=2.0 [1.0-3.6]), and Coloured (mixed ancestry) ethnicity (HR=2.3 [1.1-5.0]) were associated with increased risk of default (P<0.05). Defaults occurred throughout the first 18 months of the two-year treatment course but were especially frequent among alcohol users after discharge from the initial four-to-five-month in-hospital phase of treatment, with the highest default rates occurring among alcohol users within two months of discharge. Default rates during the first two months after discharge were also elevated for patients who received care from mobile clinics. Conclusions Among patients who were not cured or did not complete MDR-TB treatment, the majority defaulted from treatment. Younger, economically-unstable patients and alcohol and drug users were particularly at risk. For alcohol users as well as mobile-clinic patients, the early outpatient treatment phase is a high-risk period for default that could be targeted in efforts to increase treatment completion rates. PMID:24349518

Alcohol, hospital discharge, and socioeconomic risk factors for default from multidrug resistant tuberculosis treatment in rural South Africa: a retrospective cohort study.

PubMed

Kendall, Emily A; Theron, Danie; Franke, Molly F; van Helden, Paul; Victor, Thomas C; Murray, Megan B; Warren, Robin M; Jacobson, Karen R

2013-01-01

Default from multidrug-resistant tuberculosis (MDR-TB) treatment remains a major barrier to cure and epidemic control. We sought to identify patient risk factors for default from MDR-TB treatment and high-risk time periods for default in relation to hospitalization and transition to outpatient care. We retrospectively analyzed a cohort of 225 patients who initiated MDR-TB treatment between 2007 through 2010 at a rural TB hospital in the Western Cape Province, South Africa. Fifty percent of patients were cured or completed treatment, 27% defaulted, 14% died, 4% failed treatment, and 5% transferred out. Recent alcohol use was common (63% of patients). In multivariable proportional hazards regression, older age (hazard ratio [HR]= 0.97 [95% confidence interval 0.94-0.99] per year of greater age), formal housing (HR=0.38 [0.19-0.78]), and steady employment (HR=0.41 [0.19-0.90]) were associated with decreased risk of default, while recent alcohol use (HR=2.1 [1.1-4.0]), recent drug use (HR=2.0 [1.0-3.6]), and Coloured (mixed ancestry) ethnicity (HR=2.3 [1.1-5.0]) were associated with increased risk of default (P<0.05). Defaults occurred throughout the first 18 months of the two-year treatment course but were especially frequent among alcohol users after discharge from the initial four-to-five-month in-hospital phase of treatment, with the highest default rates occurring among alcohol users within two months of discharge. Default rates during the first two months after discharge were also elevated for patients who received care from mobile clinics. Among patients who were not cured or did not complete MDR-TB treatment, the majority defaulted from treatment. Younger, economically-unstable patients and alcohol and drug users were particularly at risk. For alcohol users as well as mobile-clinic patients, the early outpatient treatment phase is a high-risk period for default that could be targeted in efforts to increase treatment completion rates.

A High Fundamental Frequency (HFF)-based QCM Immunosensor for Tuberculosis Detection.

PubMed

Montoya, Angel; March, Carmen; Montagut, Yeison J; Moreno, Maria J; Manclus, Juan J; Arnau, Antonio; Jimenez, Yolanda; Jaramillo, Marisol; Marin, Paula A; Torres, Robinson A

2017-01-01

Tuberculosis, one of the oldest diseases affecting human beings, is still considered as a world public health problem by the World Health Organization. Therefore, there is a need for new and more powerful analytical methods for early illness diagnosis. With this idea in mind, the development of a High Fundamental Frequency (HFF) piezoelectric immunosensor for the sensitive detection of tuberculosis was undertaken. A 38 kDa protein secreted by Mycobacterium tuberculosis was first selected as the target biomarker. Then, specific monoclonal antibodies (MAbs) were obtained. Myc-31 MAb, which showed the highest affinity to the analyte, was employed to set up a reference enzyme-linked immunosorbent assay (ELISA) with a limit of detection of 14 ng mL-1 of 38 kDa antigen. For the development of the HFF piezoelectric immunosensor, 100 MHz quartz crystals were used as transducer elements. The gold electrode surface was functionalized by covalent immobilization of the target biomarker through mixed self-assembled monolayers (mSAM) of carboxylic alkane thiols. A competitive immunoassay based on Myc-31 MAb was integrated with the transducer as sensing bio-recognition event. Reliable assay signals were obtained using low concentrations of antigen for functionalization and MAb for the competitive immunoassay. Under optimized conditions, the HFF immunosensor calibration curve for 38 kDa determination showed a limit of detection as low as 11 ng mL-1 of the biomarker. The high detectability attained by this immunosensor, in the picomolar range, makes it a promising tool for the easy, direct and sensitive detection of the tuberculosis biomarker in biological fluids such as sputum. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Rapid and accurate identification of Mycobacterium tuberculosis complex and common non-tuberculous mycobacteria by multiplex real-time PCR targeting different housekeeping genes.

PubMed

Nasr Esfahani, Bahram; Rezaei Yazdi, Hadi; Moghim, Sharareh; Ghasemian Safaei, Hajieh; Zarkesh Esfahani, Hamid

2012-11-01

Rapid and accurate identification of mycobacteria isolates from primary culture is important due to timely and appropriate antibiotic therapy. Conventional methods for identification of Mycobacterium species based on biochemical tests needs several weeks and may remain inconclusive. In this study, a novel multiplex real-time PCR was developed for rapid identification of Mycobacterium genus, Mycobacterium tuberculosis complex (MTC) and the most common non-tuberculosis mycobacteria species including M. abscessus, M. fortuitum, M. avium complex, M. kansasii, and the M. gordonae in three reaction tubes but under same PCR condition. Genetic targets for primer designing included the 16S rDNA gene, the dnaJ gene, the gyrB gene and internal transcribed spacer (ITS). Multiplex real-time PCR was setup with reference Mycobacterium strains and was subsequently tested with 66 clinical isolates. Results of multiplex real-time PCR were analyzed with melting curves and melting temperature (T (m)) of Mycobacterium genus, MTC, and each of non-tuberculosis Mycobacterium species were determined. Multiplex real-time PCR results were compared with amplification and sequencing of 16S-23S rDNA ITS for identification of Mycobacterium species. Sensitivity and specificity of designed primers were each 100 % for MTC, M. abscessus, M. fortuitum, M. avium complex, M. kansasii, and M. gordonae. Sensitivity and specificity of designed primer for genus Mycobacterium was 96 and 100 %, respectively. According to the obtained results, we conclude that this multiplex real-time PCR with melting curve analysis and these novel primers can be used for rapid and accurate identification of genus Mycobacterium, MTC, and the most common non-tuberculosis Mycobacterium species.

Cost-effectiveness and resource implications of aggressive action on tuberculosis in China, India, and South Africa: a combined analysis of nine models.

PubMed

Menzies, Nicolas A; Gomez, Gabriela B; Bozzani, Fiammetta; Chatterjee, Susmita; Foster, Nicola; Baena, Ines Garcia; Laurence, Yoko V; Qiang, Sun; Siroka, Andrew; Sweeney, Sedona; Verguet, Stéphane; Arinaminpathy, Nimalan; Azman, Andrew S; Bendavid, Eran; Chang, Stewart T; Cohen, Ted; Denholm, Justin T; Dowdy, David W; Eckhoff, Philip A; Goldhaber-Fiebert, Jeremy D; Handel, Andreas; Huynh, Grace H; Lalli, Marek; Lin, Hsien-Ho; Mandal, Sandip; McBryde, Emma S; Pandey, Surabhi; Salomon, Joshua A; Suen, Sze-Chuan; Sumner, Tom; Trauer, James M; Wagner, Bradley G; Whalen, Christopher C; Wu, Chieh-Yin; Boccia, Delia; Chadha, Vineet K; Charalambous, Salome; Chin, Daniel P; Churchyard, Gavin; Daniels, Colleen; Dewan, Puneet; Ditiu, Lucica; Eaton, Jeffrey W; Grant, Alison D; Hippner, Piotr; Hosseini, Mehran; Mametja, David; Pretorius, Carel; Pillay, Yogan; Rade, Kiran; Sahu, Suvanand; Wang, Lixia; Houben, Rein M G J; Kimerling, Michael E; White, Richard G; Vassall, Anna

2016-11-01

The post-2015 End TB Strategy sets global targets of reducing tuberculosis incidence by 50% and mortality by 75% by 2025. We aimed to assess resource requirements and cost-effectiveness of strategies to achieve these targets in China, India, and South Africa. We examined intervention scenarios developed in consultation with country stakeholders, which scaled up existing interventions to high but feasible coverage by 2025. Nine independent modelling groups collaborated to estimate policy outcomes, and we estimated the cost of each scenario by synthesising service use estimates, empirical cost data, and expert opinion on implementation strategies. We estimated health effects (ie, disability-adjusted life-years averted) and resource implications for 2016-35, including patient-incurred costs. To assess resource requirements and cost-effectiveness, we compared scenarios with a base case representing continued current practice. Incremental tuberculosis service costs differed by scenario and country, and in some cases they more than doubled existing funding needs. In general, expansion of tuberculosis services substantially reduced patient-incurred costs and, in India and China, produced net cost savings for most interventions under a societal perspective. In all three countries, expansion of access to care produced substantial health gains. Compared with current practice and conventional cost-effectiveness thresholds, most intervention approaches seemed highly cost-effective. Expansion of tuberculosis services seems cost-effective for high-burden countries and could generate substantial health and economic benefits for patients, although substantial new funding would be required. Further work to determine the optimal intervention mix for each country is necessary. Bill & Melinda Gates Foundation. Copyright © 2016 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY license. Published by Elsevier Ltd.. All rights reserved.

Structural Basis for Catalysis of a Tetrameric Class IIa Fructose 1,6-Bisphosphate Aldolase from Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pegan, Scott D.; Ruskseree, Kamolchanok; Franzblau, Scott G.

2009-03-04

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), currently infects one-third of the world's population in its latent form. The emergence of multidrug-resistant and extensive drug-resistant strains has highlighted the need for new pharmacological targets within M. tuberculosis. The class IIa fructose 1,6-bisphosphate aldolase (FBA) enzyme from M. tuberculosis (MtFBA) has been proposed as one such target since it is upregulated in latent TB. Since the structure of MtFBA has not been determined and there is little information available on its reaction mechanism, we sought to determine the X-ray structure of MtFBA in complex with its substrates. By lowering themore » pH of the enzyme in the crystalline state, we were able to determine a series of high-resolution X-ray structures of MtFBA bound to dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, and fructose 1,6-bisphosphate at 1.5, 2.1, and 1.3 {angstrom}, respectively. Through these structures, it was discovered that MtFBA belongs to a novel tetrameric class of type IIa FBAs. The molecular details at the interface of the tetramer revealed important information for better predictability of the quaternary structures among the FBAs based on their primary sequences. These X-ray structures also provide interesting and new details on the reaction mechanism of class II FBAs. Substrates and products were observed in geometries poised for catalysis; in addition, unexpectedly, the hydroxyl-enolate intermediate of dihydroxyacetone phosphate was also captured and resolved structurally. These concise new details offer a better understanding of the reaction mechanisms for FBAs in general and provide a structural basis for inhibitor design efforts aimed at this class of enzymes.« less

Structural basis for catalysis of a tetrameric, class IIa fructose 1,6-bisphosphate aldolase from M. tuberculosis

PubMed Central

Pegan, Scott D.; Rukseree, Kamolchanok; Franzblau, Scott G.; Mesecar, Andrew D.

2009-01-01

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), currently infects one third of the world’s population in its latent form. The emergence of multidrug resistant strains, MDR-TB and XDR-TB has highlighted the need for new pharmacological targets within M. tuberculosis. The Class IIa fructose 1,6-bisphosphate aldolase (FBA) enzyme from M. tuberculosis (MtFBA) has been proposed as one such target since its upregulated in latent TB. Since the structure of MtFBA had not been determined and since there was little information available on its reaction mechanism, we sought to determine the X-ray structure of MtFBA in complex with its substrates. By lowering the pH of the enzyme in the crystalline state, we were able to determine a series of high-resolution X-ray structures of MtFBA bound to dihydroxyacetonephosphate (DHAP), glyceraldehyde-3-phosphate (G3P), and fructose 1,6-bisphosphate (FBP) at 1.5 Å, 2.1 Å, and 1.3 Å respectively. Through these structures it was discovered that MtFBA belongs to a novel tetrameric class of the type IIa FBAs. The molecular details at the interface of the tetramer revealed important information for being able to better predict the quaternary structures among the FBAs based on their primary sequences. These X-ray structures also provide interesting and new details on the reaction mechanism of class II FBAs. Not only were the substrates and products observed in geometries poised for catalysis, but unexpectedly the hydroxyl enolate intermediate of DHAP was also captured and resolved structurally. These concise new details provide a better understanding of the reaction mechanisms for FBAs in general and provide a structural basis for inhibitor design efforts aimed at this class of enzymes. PMID:19167403

Trends in discovery of new drugs for tuberculosis therapy.

PubMed

Riccardi, Giovanna; Pasca, Maria Rosalia

2014-09-01

After the introduction of isoniazid and rifampicin, the second one discovered in the Lepetit Research Laboratories (Milan, Italy), under the supervision of Professor Piero Sensi, tuberculosis (TB) was considered an illness of the past. Unfortunately, this infectious disease is still a global health fear, due to the multidrug-resistant Mycobacterium tuberculosis and extensively circulating drug-resistant strains, as well as the unrecognized TB transmission, especially in regions with high HIV incidence. In the last few years, new antitubercular molecules appeared on the horizon both in preclinical and clinical stage of evaluation. In this review, we focus on a few of them and on their mechanism of action. Two new promising drug targets, DprE1 and MmpL3, are also discussed.

Cellular Immune Responses to Nine Mycobacterium tuberculosis Vaccine Candidates following Intranasal Vaccination

PubMed Central

Sable, Suraj B.; Cheruvu, Mani; Nandakumar, Subhadra; Sharma, Sunita; Bandyopadhyay, Kakali; Kellar, Kathryn L.; Posey, James E.; Plikaytis, Bonnie B.; Amara, Rama Rao; Shinnick, Thomas M.

2011-01-01

Background The identification of Mycobacterium tuberculosis vaccines that elicit a protective immune response in the lungs is important for the development of an effective vaccine against tuberculosis. Methods and Principal Findings In this study, a comparison of intranasal (i.n.) and subcutaneous (s.c.) vaccination with the BCG vaccine demonstrated that a single moderate dose delivered intranasally induced a stronger and sustained M. tuberculosis-specific T-cell response in lung parenchyma and cervical lymph nodes of BALB/c mice than vaccine delivered subcutaneously. Both BCG and a multicomponent subunit vaccine composed of nine M. tuberculosis recombinant proteins induced strong antigen-specific T-cell responses in various local and peripheral immune compartments. Among the nine recombinant proteins evaluated, the alanine proline rich antigen (Apa, Rv1860) was highly antigenic following i.n. BCG and immunogenic after vaccination with a combination of the nine recombinant antigens. The Apa-induced responses included induction of both type 1 and type 2 cytokines in the lungs as evaluated by ELISPOT and a multiplexed microsphere-based cytokine immunoassay. Of importance, i.n. subunit vaccination with Apa imparted significant protection in the lungs and spleen of mice against M. tuberculosis challenge. Despite observed differences in the frequencies and location of specific cytokine secreting T cells both BCG vaccination routes afforded comparable levels of protection in our study. Conclusion and Significance Overall, our findings support consideration and further evaluation of an intranasally targeted Apa-based vaccine to prevent tuberculosis. PMID:21799939

The endothelin system has a significant role in the pathogenesis and progression of Mycobacterium tuberculosis infection.

PubMed

Correa, Andre F; Bailão, Alexandre M; Bastos, Izabela M D; Orme, Ian M; Soares, Célia M A; Kipnis, Andre; Santana, Jaime M; Junqueira-Kipnis, Ana Paula

2014-12-01

Tuberculosis (TB) remains a major global health problem, and although multiple studies have addressed the relationship between Mycobacterium tuberculosis and the host on an immunological level, few studies have addressed the impact of host physiological responses. Proteases produced by bacteria have been associated with important alterations in the host tissues, and a limited number of these enzymes have been characterized in mycobacterial species. M. tuberculosis produces a protease called Zmp1, which appears to be associated with virulence and has a putative action as an endothelin-converting enzyme. Endothelins are a family of vasoactive peptides, of which 3 distinct isoforms exist, and endothelin 1 (ET-1) is the most abundant and the best-characterized isoform. The aim of this work was to characterize the Zmp1 protease and evaluate its role in pathogenicity. Here, we have shown that M. tuberculosis produces and secretes an enzyme with ET-1 cleavage activity. These data demonstrate a possible role of Zmp1 for mycobacterium-host interactions and highlights its potential as a drug target. Moreover, the results suggest that endothelin pathways have a role in the pathogenesis of M. tuberculosis infections, and ETA or ETB receptor signaling can modulate the host response to the infection. We hypothesize that a balance between Zmp1 control of ET-1 levels and ETA/ETB signaling can allow M. tuberculosis adaptation and survival in the lung tissues. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Sequence analysis of the drug‑resistant rpoB gene in the Mycobacterium tuberculosis L‑form among patients with pneumoconiosis complicated by tuberculosis.

PubMed

Lu, Jun; Jiang, Shan; Ye, Song; Deng, Yun; Ma, Shuai; Li, Chao-Pin

2014-04-01

The aim of the present study was to investigate the mutational characteristics of the drug‑resistant Mycobacterium tuberculosis L‑form of the rpoB gene isolated from patients with pneumoconiosis complicated by tuberculosis, in order to reduce the occurrence of the drug resistance of patients and gain a more complete information on the resistance of the Mycobacterium tuberculosis L‑form. A total of 42 clinically isolated strains of Mycobacterium tuberculosis L‑form were collected, including 31 drug‑resistant strains. The genomic DNA was extracted, then the target genes were amplified by polymerase chain reaction and the hot mutational regions of the rpoB gene were analyzed by direct sequencing. The results revealed that no rpoB gene mutation was present in 11 rifampicin (RFP)‑sensitive strains, while conformational changes were identified in 31 RFP‑resistant strains. The mutation rate was 93.55% (29/31) in the resistant strains, and was frequently concentrated in codons 531 (51.61%; 16/31) and 526 (32.26%; 10/31), mainly occurring by case substitutions, including 27 unit point mutations and two two‑point mutations. The novel mutation identified in codon 516 had not been previously reported. The substitution of highly‑conserved amino acids encoded by the rpoB gene resulted in the molecular mechanism responsible for RFP resistance in the Mycobacterium tuberculosis L‑form. This also demonstrated that the rpoB gene is diversiform.

Improving tuberculosis control through public-private collaboration in India: literature review.

PubMed

Dewan, Puneet K; Lal, S S; Lonnroth, Knut; Wares, Fraser; Uplekar, Mukund; Sahu, Suvanand; Granich, Reuben; Chauhan, Lakhbir Singh

2006-03-11

To review the characteristics of public-private mix projects in India and their effect on case notification and treatment outcomes for tuberculosis. Literature review. Review of surveillance records from Indian tuberculosis programme project, evaluation reports, and medical literature for public-private mix projects in India. Project characteristics, tuberculosis case notification of new patients with sputum smear results positive for acid fast bacilli, and treatment outcome. Of 24 identified public-private mix projects, data were available from 14 (58%), involving private practitioners, corporations, and non-governmental organisations. In all reviewed projects, the public sector tuberculosis programme provided training and supervision of private providers. Among the five projects with available data on historical controls, case notification rates were higher after implementation of a public-private mix project. Among seven projects involving private practitioners, 2796 of 12 147 (23%) new patients positive for acid fast bacilli were attributed to private providers. Corporate based and non-governmental organisations served as the main source for tuberculosis programme services in seven project areas, detecting 9967 new patients positive for acid fast bacilli. In nine of 12 projects with data on treatment outcomes, private providers exceeded the programme target of 85% treatment success for new patients positive for acid fast bacilli. Public-private mix activities were associated with increased case notification, while maintaining acceptable treatment outcomes. Collaborations between public and private providers of health care hold considerable potential to improve tuberculosis control in India.

Mycobacterium tuberculosis two-component systems and implications in novel vaccines and drugs.

PubMed

Zhou, PeiFu; Long, QuanXin; Zhou, YeXin; Wang, HongHai; Xie, JianPing

2012-01-01

Communication is vital for nearly all organisms to survive and thrive. For some particularly successful intracellular pathogens, a robust and precise signal transduction system is imperative for handling the complex, volatile, and harsh niche. The communication network of the etiology of tuberculosis, Mycobacterium tuberculosis (M.tb), namely two-component system (TCS), the eukaryotic-like Ser/Thr protein kinases(STPKs) system, the protein tyrosine kinase(PTK) system and the extracytoplasmic function σ(ECF-σ) system, determine how the pathogen responds to environmental fluctuations. At least 12 pair TCSs and four orphan proteins (three response regulators, Rv2884, Rv0260c, Rv0818, and one putative sensory transduction protein, Rv3143) can be found in the M.tb H37Rv genome. They regulate various aspects of M.tb, including virulence, dormancy, persistence, and drug resistance. This review focuses on the physiological roles of TCSs and the network of M.tb TCSs from a systems biology perspective. The implications of TCSs for better vaccine and new drug targets against tuberculosis are also examined.

A Mycobacterium tuberculosis cytochrome bd oxidase mutant is hypersensitive to bedaquiline.

PubMed

Berney, Michael; Hartman, Travis E; Jacobs, William R

2014-07-15

The new medicinal compound bedaquiline (BDQ) kills Mycobacterium tuberculosis by inhibiting F1Fo-ATP synthase. BDQ is bacteriostatic for 4 to 7 days and kills relatively slowly compared to other frontline tuberculosis (TB) drugs. Here we show that killing with BDQ can be improved significantly by inhibiting cytochrome bd oxidase, a non-proton-pumping terminal oxidase. BDQ was instantly bactericidal against a cytochrome bd oxidase null mutant of M. tuberculosis, and the rate of killing was increased by more than 50%. We propose that this exclusively bacterial enzyme should be a high-priority target for new drug discovery. Importance: A major drawback of current TB chemotherapy is its long duration. New drug regimens with rapid killing kinetics are desperately needed. Our study demonstrates that inhibition of a nonessential bacterial enzyme greatly improves the efficacy of the latest TB drug bedaquiline and emphasizes that screening for compounds with synergistic killing mechanisms is a promising strategy. Copyright © 2014 Berney et al.

The Inosine Monophosphate Dehydrogenase, GuaB2, Is a Vulnerable New Bactericidal Drug Target for Tuberculosis

PubMed Central

2016-01-01

VCC234718, a molecule with growth inhibitory activity against Mycobacterium tuberculosis (Mtb), was identified by phenotypic screening of a 15344-compound library. Sequencing of a VCC234718-resistant mutant identified a Y487C substitution in the inosine monophosphate dehydrogenase, GuaB2, which was subsequently validated to be the primary molecular target of VCC234718 in Mtb. VCC234718 inhibits Mtb GuaB2 with a Ki of 100 nM and is uncompetitive with respect to IMP and NAD+. This compound binds at the NAD+ site, after IMP has bound, and makes direct interactions with IMP; therefore, the inhibitor is by definition uncompetitive. VCC234718 forms strong pi interactions with the Y487 residue side chain from the adjacent protomer in the tetramer, explaining the resistance-conferring mutation. In addition to sensitizing Mtb to VCC234718, depletion of GuaB2 was bactericidal in Mtb in vitro and in macrophages. When supplied at a high concentration (≥125 μM), guanine alleviated the toxicity of VCC234718 treatment or GuaB2 depletion via purine salvage. However, transcriptional silencing of guaB2 prevented Mtb from establishing an infection in mice, confirming that Mtb has limited access to guanine in this animal model. Together, these data provide compelling validation of GuaB2 as a new tuberculosis drug target. PMID:27726334

Educating international students about tuberculosis and infections associated with travel to visit friends and relatives (VFR-travel).

PubMed

Gibney, Katherine B; Brass, Amanda; Hume, Sam C; Leder, Karin

2014-01-01

International students in Victoria, Australia, originate from over 140 different countries. They are over-represented in disease notifications for tuberculosis and travel-associated infections, including enteric fever, hepatitis A, and malaria. We describe a public health initiative aimed to increase awareness of these illnesses among international students and their support staff. We identified key agencies including student support advisors, medical practitioners, health insurers, and government and professional organisations. We developed health education materials targeting international students regarding tuberculosis and travel-related infections to be disseminated via a number of different media, including electronic and printed materials. We sought informal feedback from personnel in all interested agencies regarding the materials developed, their willingness to deliver these materials to international students, and their preferred media for disseminating these materials. Education institutions with dedicated international student support staff and on-campus health clinics were more easily engaged to provide feedback and disseminate the health education materials than institutions without such dedicated personnel. Response to contacting off-campus medical practices was poor. Delivery of educational materials via electronic and social media was preferred over face-to-face education. It is feasible to provide health education messages targeting international students for dissemination via appropriately-staffed educational institutions. This initiative could be expanded in terms of age-group, geographic range, and health issues to be targeted. Copyright © 2013 Elsevier Ltd. All rights reserved.

Targeting Mycobacterium tuberculosis Antigens to Dendritic Cells via the DC-Specific-ICAM3-Grabbing-Nonintegrin Receptor Induces Strong T-Helper 1 Immune Responses

PubMed Central

Velasquez, Lis Noelia; Stüve, Philipp; Gentilini, Maria Virginia; Swallow, Maxine; Bartel, Judith; Lycke, Nils Yngve; Barkan, Daniel; Martina, Mariana; Lujan, Hugo D.; Kalay, Hakan; van Kooyk, Yvette; Sparwasser, Tim D.; Berod, Luciana

2018-01-01

Tuberculosis remains a major global health problem and efforts to develop a more effective vaccine have been unsuccessful so far. Targeting antigens (Ags) to dendritic cells (DCs) in vivo has emerged as a new promising vaccine strategy. In this approach, Ags are delivered directly to DCs via antibodies that bind to endocytic cell-surface receptors. Here, we explored DC-specific-ICAM3-grabbing-nonintegrin (DC-SIGN) targeting as a potential vaccine against tuberculosis. For this, we made use of the hSIGN mouse model that expresses human DC-SIGN under the control of the murine CD11c promoter. We show that in vitro and in vivo delivery of anti-DC-SIGN antibodies conjugated to Ag85B and peptide 25 of Ag85B in combination with anti-CD40, the fungal cell wall component zymosan, and the cholera toxin-derived fusion protein CTA1-DD induces strong Ag-specific CD4+ T-cell responses. Improved anti-mycobacterial immunity was accompanied by increased frequencies of Ag-specific IFN-γ+ IL-2+ TNF-α+ polyfunctional CD4+ T cells in vaccinated mice compared with controls. Taken together, in this study we provide the proof of concept that the human DC-SIGN receptor can be efficiently exploited for vaccine purposes to promote immunity against mycobacterial infections. PMID:29662482

The Inosine Monophosphate Dehydrogenase, GuaB2, Is a Vulnerable New Bactericidal Drug Target for Tuberculosis.

PubMed

Singh, Vinayak; Donini, Stefano; Pacitto, Angela; Sala, Claudia; Hartkoorn, Ruben C; Dhar, Neeraj; Keri, Gyorgy; Ascher, David B; Mondésert, Guillaume; Vocat, Anthony; Lupien, Andréanne; Sommer, Raphael; Vermet, Hélène; Lagrange, Sophie; Buechler, Joe; Warner, Digby F; McKinney, John D; Pato, Janos; Cole, Stewart T; Blundell, Tom L; Rizzi, Menico; Mizrahi, Valerie

2017-01-13

VCC234718, a molecule with growth inhibitory activity against Mycobacterium tuberculosis (Mtb), was identified by phenotypic screening of a 15344-compound library. Sequencing of a VCC234718-resistant mutant identified a Y487C substitution in the inosine monophosphate dehydrogenase, GuaB2, which was subsequently validated to be the primary molecular target of VCC234718 in Mtb. VCC234718 inhibits Mtb GuaB2 with a K i of 100 nM and is uncompetitive with respect to IMP and NAD + . This compound binds at the NAD + site, after IMP has bound, and makes direct interactions with IMP; therefore, the inhibitor is by definition uncompetitive. VCC234718 forms strong pi interactions with the Y487 residue side chain from the adjacent protomer in the tetramer, explaining the resistance-conferring mutation. In addition to sensitizing Mtb to VCC234718, depletion of GuaB2 was bactericidal in Mtb in vitro and in macrophages. When supplied at a high concentration (≥125 μM), guanine alleviated the toxicity of VCC234718 treatment or GuaB2 depletion via purine salvage. However, transcriptional silencing of guaB2 prevented Mtb from establishing an infection in mice, confirming that Mtb has limited access to guanine in this animal model. Together, these data provide compelling validation of GuaB2 as a new tuberculosis drug target.

Structural basis of DNA sequence recognition by the response regulator PhoP in Mycobacterium tuberculosis.

PubMed

He, Xiaoyuan; Wang, Liqin; Wang, Shuishu

2016-04-15

The transcriptional regulator PhoP is an essential virulence factor in Mycobacterium tuberculosis, and it presents a target for the development of new anti-tuberculosis drugs and attenuated tuberculosis vaccine strains. PhoP binds to DNA as a highly cooperative dimer by recognizing direct repeats of 7-bp motifs with a 4-bp spacer. To elucidate the PhoP-DNA binding mechanism, we determined the crystal structure of the PhoP-DNA complex. The structure revealed a tandem PhoP dimer that bound to the direct repeat. The surprising tandem arrangement of the receiver domains allowed the four domains of the PhoP dimer to form a compact structure, accounting for the strict requirement of a 4-bp spacer and the highly cooperative binding of the dimer. The PhoP-DNA interactions exclusively involved the effector domain. The sequence-recognition helix made contact with the bases of the 7-bp motif in the major groove, and the wing interacted with the adjacent minor groove. The structure provides a starting point for the elucidation of the mechanism by which PhoP regulates the virulence of M. tuberculosis and guides the design of screening platforms for PhoP inhibitors.

Antitubercular Nanocarrier Combination Therapy: Formulation Strategies and in Vitro Efficacy for Rifampicin and SQ641

PubMed Central

2015-01-01

Tuberculosis (TB) remains a major global health concern, and new therapies are needed to overcome the problems associated with dosing frequency, patient compliance, and drug resistance. To reduce side effects associated with systemic drug distribution and improve drug concentration at the target site, stable therapeutic nanocarriers (NCs) were prepared and evaluated for efficacy in vitro in Mycobacterium tuberculosis-infected macrophages. Rifampicin (RIF), a current, broad-spectrum antibiotic used in TB therapy, was conjugated by degradable ester bonds to form hydrophobic prodrugs. NCs encapsulating various ratios of nonconjugated RIF and the prodrugs showed the potential ability to rapidly deliver and knockdown intracellular M. tuberculosis by nonconjugated RIF and to obtain sustained release of RIF by hydrolysis of the RIF prodrug. NCs of the novel antibiotic SQ641 and a combination NC with cyclosporine A were formed by flash nanoprecipitation. Delivery of SQ641 in NC form resulted in significantly improved activity compared to that of the free drug against intracellular M. tuberculosis. A NC formulation with a three-compound combination of SQ641, cyclosporine A, and vitamin E inhibited intracellular replication of M. tuberculosis significantly better than SQ641 alone or isoniazid, a current first-line anti-TB drug. PMID:25811733

Solution Structures of Mycobacterium tuberculosis Thioredoxin C and Models of the Intact Thioredoxin System Suggest New Approaches to Inhibitor and Drug Design

PubMed Central

Olson, Andrew L.; Neumann, Terrence S.; Cai, Sheng; Sem, Daniel S.

2012-01-01

Here we report the NMR solution structures of Mycobacterium tuberculosis (M. tuberculosis) thioredoxin C in both oxidized and reduced states, with discussion of structural changes that occur in going between redox states. The NMR solution structure of the oxidized TrxC corresponds closely to that of the crystal structure, except in the C-terminal region. It appears that crystal packing effects have caused an artifactual shift in the α4 helix in the previously reported crystal structure, compared to the solution structure. Based on these TrxC structures, chemical shift mapping, a previously reported crystal structure of the M. tuberculosis thioredoxin reductase (not bound to a Trx) and structures for intermediates in the E. coli thioredoxin catalytic cycle, we have modeled the complete M. tuberculosis thioredoxin system for the various steps in the catalytic cycle. These structures and models reveal pockets at the TrxR/TrxC interface in various steps in the catalytic cycle, which can be targeted in the design of uncompetitive inhibitors as potential anti-mycobacterial agents, or as chemical genetic probes of function. PMID:23229911

Anti-mycobacterial activity of marine fungus-derived 4-deoxybostrycin and nigrosporin.

PubMed

Wang, Cong; Wang, Juan; Huang, Yuhong; Chen, Hong; Li, Yan; Zhong, Lili; Chen, Yi; Chen, Shengping; Wang, Jun; Kang, Juling; Peng, Yi; Yang, Bin; Lin, Yongcheng; She, Zhigang; Lai, Xiaomin

2013-01-29

4-Deoxybostrycin is a natural anthraquinone compound isolated from the Mangrove endophytic fungus Nigrospora sp. collected from the South China Sea. Nigrosporin is the deoxy-derivative of 4-deoxybostrycin. They were tested against mycobacteria, especially Mycobacterium tuberculosis. In the Kirby-Bauer disk diffusion susceptibility test, they both had inhibition zone sizes of over 25 mm. The results of the absolute concentration susceptibility test suggested that they had inhibitory effects against mycobacteria. Moreover, 4-deoxybostrycin exhibited good inhibition which was even better than that of first line anti-tuberculosis (TB) drugs against some clinical multidrug-resistant (MDR) M. tuberculosis strains. The gene expression profile of M. tuberculosis H37Rv after treatment with 4-deoxybostrycin was compared with untreated bacteria. One hundred and nineteen out of 3,875 genes were significantly different in M. tuberculosis exposed to 4-deoxybostrycin from control. There were 46 functionally known genes which are involved in metabolism, information storage and processing and cellular processes. The differential expressions of six genes were further confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). The present study provides a useful experiment basis for exploitation of correlative new drugs against TB and for finding out new targets of anti-mycobacterial therapy.

SU-E-J-252: A Motion Algorithm to Extract Physical and Motion Parameters of a Mobile Target in Cone-Beam Computed Tomographic Imaging Retrospective to Image Reconstruction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ali, I; Ahmad, S; Alsbou, N

Purpose: A motion algorithm was developed to extract actual length, CT-numbers and motion amplitude of a mobile target imaged with cone-beam-CT (CBCT) retrospective to image-reconstruction. Methods: The motion model considered a mobile target moving with a sinusoidal motion and employed three measurable parameters: apparent length, CT number level and gradient of a mobile target obtained from CBCT images to extract information about the actual length and CT number value of the stationary target and motion amplitude. The algorithm was verified experimentally with a mobile phantom setup that has three targets with different sizes manufactured from homogenous tissue-equivalent gel material embeddedmore » into a thorax phantom. The phantom moved sinusoidal in one-direction using eight amplitudes (0–20mm) and a frequency of 15-cycles-per-minute. The model required imaging parameters such as slice thickness, imaging time. Results: This motion algorithm extracted three unknown parameters: length of the target, CT-number-level, motion amplitude for a mobile target retrospective to CBCT image reconstruction. The algorithm relates three unknown parameters to measurable apparent length, CT-number-level and gradient for well-defined mobile targets obtained from CBCT images. The motion model agreed with measured apparent lengths which were dependent on actual length of the target and motion amplitude. The cumulative CT-number for a mobile target was dependent on CT-number-level of the stationary target and motion amplitude. The gradient of the CT-distribution of mobile target is dependent on the stationary CT-number-level, actual target length along the direction of motion, and motion amplitude. Motion frequency and phase did not affect the elongation and CT-number distributions of mobile targets when imaging time included several motion cycles. Conclusion: The motion algorithm developed in this study has potential applications in diagnostic CT imaging and radiotherapy to extract actual length, size and CT-numbers distorted by motion in CBCT imaging. The model provides further information about motion of the target.« less

The key DNA-binding residues in the C-terminal domain of Mycobacterium tuberculosis DNA gyrase A subunit (GyrA)

PubMed Central

Huang, You-Yi; Deng, Jiao-Yu; Gu, Jing; Zhang, Zhi-Ping; Maxwell, Anthony; Bi, Li-Jun; Chen, Yuan-Yuan; Zhou, Ya-Feng; Yu, Zi-Niu; Zhang, Xian-En

2006-01-01

As only the type II topoisomerase is capable of introducing negative supercoiling, DNA gyrase is involved in crucial cellular processes. Although the other domains of DNA gyrase are better understood, the mechanism of DNA binding by the C-terminal domain of the DNA gyrase A subunit (GyrA-CTD) is less clear. Here, we investigated the DNA-binding sites in the GyrA-CTD of Mycobacterium tuberculosis gyrase through site-directed mutagenesis. The results show that Y577, R691 and R745 are among the key DNA-binding residues in M.tuberculosis GyrA-CTD, and that the third blade of the GyrA-CTD is the main DNA-binding region in M.tuberculosis DNA gyrase. The substitutions of Y577A, D669A, R691A, R745A and G729W led to the loss of supercoiling and relaxation activities, although they had a little effect on the drug-dependent DNA cleavage and decatenation activities, and had no effect on the ATPase activity. Taken together, these results showed that the GyrA-CTD is essential to DNA gyrase of M.tuberculosis, and promote the idea that the M.tuberculosis GyrA-CTD is a new potential target for drug design. It is the first time that the DNA-binding sites in GyrA-CTD have been identified. PMID:17038336

Evaluation of a nested-PCR for mycobacterium tuberculosis detection in blood and urine samples.

PubMed

da Cruz, Heidi Lacerda Alves; de Albuquerque Montenegro, Rosana; de Araújo Lima, Juliana Falcão; da Rocha Poroca, Diogo; da Costa Lima, Juliana Figueirêdo; Maria Lapa Montenegro, Lílian; Crovella, Sergio; Charifker Schindler, Haiana

2011-01-01

The polymerase chain reaction (PCR) and its variations, such as the nested-PCR, have been described as promising techniques for rapid diagnosis of tuberculosis (TB). With the aim of evaluating the usefulness of a nested-PCR method on samples of blood and urine of patients suspected of tuberculosis we analyzed 192 clinical samples, using as a molecular target the insertion element IS6110 specific of M. tuberculosis genome. Nested-PCR method showed higher sensitivity in patients with extrapulmonary tuberculosis (47.8% and 52% in blood and urine) when compared to patients with the pulmonary form of the disease (sensitivity of 29% and 26.9% in blood and urine), regardless of the type of biological sample used. The nested-PCR is a rapid technique that, even if not showing a good sensitivity, should be considered as a helpful tool especially in the extrapulmonary cases or in cases where confirmatory diagnosis is quite difficult to be achieved by routine methods. The performance of PCR-based techniques should be considered and tested in future works on other types of biological specimens besides sputum, like blood and urine, readily obtainable in most cases. The improving of M. tuberculosis nested-PCR detection in TB affected patients will give the possibility of an earlier detection of bacilli thus interrupting the transmission chain of the disease.

An Evaluation of Systematic Tuberculosis Screening at Private Facilities in Karachi, Pakistan

PubMed Central

Creswell, Jacob; Khowaja, Saira; Codlin, Andrew; Hashmi, Rabia; Rasheed, Erum; Khan, Mubashir; Durab, Irfan; Mergenthaler, Christina; Hussain, Owais; Khan, Faisal; Khan, Aamir J.

2014-01-01

Background In Pakistan, like many Asian countries, a large proportion of healthcare is provided through the private sector. We evaluated a systematic screening strategy to identify people with tuberculosis in private facilities in Karachi and assessed the approaches' ability to diagnose patients earlier in their disease progression. Methods and Findings Lay workers at 89 private clinics and a large hospital outpatient department screened all attendees for tuberculosis using a mobile phone-based questionnaire during one year. The number needed to screen to detect a case of tuberculosis was calculated. To evaluate early diagnosis, we tested for differences in cough duration and smear grading by screening facility. 529,447 people were screened, 1,010 smear-positive tuberculosis cases were detected and 942 (93.3%) started treatment, representing 58.7% of all smear-positive cases notified in the intervention area. The number needed to screen to detect a smear-positive case was 124 (prevalence 806/100,000) at the hospital and 763 (prevalence 131/100,000) at the clinics; however, ten times the number of individuals were screened in clinics. People with smear-positive TB detected at the hospital were less likely to report cough lasting 2–3 weeks (RR 0.66 95%CI [0.49–0.90]) and more likely to report cough duration >3 weeks (RR 1.10 95%CI [1.03–1.18]). Smear-positive cases at the clinics were less likely to have a +3 grade (RR 0.76 95%CI [0.63–0.92]) and more likely to have +1 smear grade (RR 1.24 95%CI [1.02–1.51]). Conclusions Tuberculosis screening at private facilities is acceptable and can yield large numbers of previously undiagnosed cases. Screening at general practitioner clinics may find cases earlier than at hospitals although more people must be screened to identify a case of tuberculosis. Limitations include lack of culture testing, therefore underestimating true TB prevalence. Using more sensitive and specific screening and diagnostic tests such as chest x-ray and Xpert MTB/RIF may improve results. PMID:24705600

An evaluation of systematic tuberculosis screening at private facilities in Karachi, Pakistan.

PubMed

Creswell, Jacob; Khowaja, Saira; Codlin, Andrew; Hashmi, Rabia; Rasheed, Erum; Khan, Mubashir; Durab, Irfan; Mergenthaler, Christina; Hussain, Owais; Khan, Faisal; Khan, Aamir J

2014-01-01

In Pakistan, like many Asian countries, a large proportion of healthcare is provided through the private sector. We evaluated a systematic screening strategy to identify people with tuberculosis in private facilities in Karachi and assessed the approaches' ability to diagnose patients earlier in their disease progression. Lay workers at 89 private clinics and a large hospital outpatient department screened all attendees for tuberculosis using a mobile phone-based questionnaire during one year. The number needed to screen to detect a case of tuberculosis was calculated. To evaluate early diagnosis, we tested for differences in cough duration and smear grading by screening facility. 529,447 people were screened, 1,010 smear-positive tuberculosis cases were detected and 942 (93.3%) started treatment, representing 58.7% of all smear-positive cases notified in the intervention area. The number needed to screen to detect a smear-positive case was 124 (prevalence 806/100,000) at the hospital and 763 (prevalence 131/100,000) at the clinics; however, ten times the number of individuals were screened in clinics. People with smear-positive TB detected at the hospital were less likely to report cough lasting 2-3 weeks (RR 0.66 95%CI [0.49-0.90]) and more likely to report cough duration >3 weeks (RR 1.10 95%CI [1.03-1.18]). Smear-positive cases at the clinics were less likely to have a +3 grade (RR 0.76 95%CI [0.63-0.92]) and more likely to have +1 smear grade (RR 1.24 95%CI [1.02-1.51]). Tuberculosis screening at private facilities is acceptable and can yield large numbers of previously undiagnosed cases. Screening at general practitioner clinics may find cases earlier than at hospitals although more people must be screened to identify a case of tuberculosis. Limitations include lack of culture testing, therefore underestimating true TB prevalence. Using more sensitive and specific screening and diagnostic tests such as chest x-ray and Xpert MTB/RIF may improve results.

Identification of small molecular ligands as potent inhibitors of fatty acid metabolism in Mycobacterium tuberculosis

NASA Astrophysics Data System (ADS)

Malikanti, Ramesh; Vadija, Rajender; Veeravarapu, Hymavathi; Mustyala, Kiran Kumar; Malkhed, Vasavi; Vuruputuri, Uma

2017-12-01

Tuberculosis (Tb) is one of the major health challenges for the global scientific community. The 3-hydroxy butyryl-CoA dehydrogenase (Fad B2) protein belongs to 3-hydroxyl acetyl-CoA dehydrogenase family, which plays a key role in the fatty acid metabolism and β-oxidation in the cell membrane of Mycobacterium tuberculosis (Mtb). In the present study the Fad B2 protein is targeted for the identification of potential drug candidates for tuberculosis. The 3D model of the target protein Fad B2, was generated using homology modeling approach and was validated. The plausible binding site of the Fad B2 protein was identified from computational binding pocket prediction tools, which ranges from ASN120 to VAL150 amino acid residues. Virtual screening was carried out with the databases, Ligand box UOS and hit definder, at the binding site region. 133 docked complex structures were generated as an output. The identified ligands show good glide scores and glide energies. All the ligand molecules contain benzyl amine pharmacophore in common, which show specific and selective binding interactions with the SER122 and ASN146 residues of the Fad B2 protein. The ADME properties of all the ligand molecules were observed to be within the acceptable range. It is suggested from the result of the present study that the docked molecular structures with a benzyl amine pharmacophore act as potential ligands for Fad B2 protein binding and as leads in Tb drug discovery.

Small RNA profiling in Mycobacterium tuberculosis identifies MrsI as necessary for an anticipatory iron sparing response.

PubMed

Gerrick, Elias R; Barbier, Thibault; Chase, Michael R; Xu, Raylin; François, Josie; Lin, Vincent H; Szucs, Matthew J; Rock, Jeremy M; Ahmad, Rushdy; Tjaden, Brian; Livny, Jonathan; Fortune, Sarah M

2018-06-19

One key to the success of Mycobacterium tuberculosis as a pathogen is its ability to reside in the hostile environment of the human macrophage. Bacteria adapt to stress through a variety of mechanisms, including the use of small regulatory RNAs (sRNAs), which posttranscriptionally regulate bacterial gene expression. However, very little is currently known about mycobacterial sRNA-mediated riboregulation. To date, mycobacterial sRNA discovery has been performed primarily in log-phase growth, and no direct interaction between any mycobacterial sRNA and its targets has been validated. Here, we performed large-scale sRNA discovery and expression profiling in M. tuberculosis during exposure to five pathogenically relevant stresses. From these data, we identified a subset of sRNAs that are highly induced in multiple stress conditions. We focused on one of these sRNAs, ncRv11846, here renamed mycobacterial regulatory sRNA in iron (MrsI). We characterized the regulon of MrsI and showed in mycobacteria that it regulates one of its targets, bfrA , through a direct binding interaction. MrsI mediates an iron-sparing response that is required for optimal survival of M. tuberculosis under iron-limiting conditions. However, MrsI is induced by multiple host-like stressors, which appear to trigger MrsI as part of an anticipatory response to impending iron deprivation in the macrophage environment. Copyright © 2018 the Author(s). Published by PNAS.

Identification of a New DNA Region Specific for Members of Mycobacterium tuberculosis Complex

PubMed Central

Magdalena, Juana; Vachée, Anne; Supply, Philip; Locht, Camille

1998-01-01

The successful use of DNA amplification for the detection of tuberculous mycobacteria crucially depends on the choice of the target sequence, which ideally should be present in all tuberculous mycobacteria and absent from all other bacteria. In the present study we developed a PCR procedure based on the intergenic region (IR) separating two genes encoding a recently identified mycobacterial two-component system named SenX3-RegX3. The senX3-regX3 IR is composed of a novel type of repetitive sequence, called mycobacterial interspersed repetitive units (MIRUs). In a survey of 116 Mycobacterium tuberculosis strains characterized by different IS6110 restriction fragment length polymorphisms, 2 Mycobacterium africanum strains, 3 Mycobacterium bovis strains (including 2 BCG strains), and 1 Mycobacterium microti strain, a specific PCR fragment was amplified in all cases. This collection included M. tuberculosis strains that lack IS6110 or mtp40, two target sequences that have previously been used for the detection of M. tuberculosis. No PCR fragment was amplified when DNA from other organisms was used, giving a sensitivity of 100% and a specificity of 100% in the confidence limit of this study. The numbers of MIRUs were found to vary among strains, resulting in six different groups of strains on the basis of the size of the amplified PCR fragment. However, the vast majority of the strains (approximately 90%) fell within the same group, containing two 77-bp MIRUs followed by one 53-bp MIRU. PMID:9542912

Identification of a novel inhibitor of isocitrate lyase as a potent antitubercular agent against both active and non-replicating Mycobacterium tuberculosis.

PubMed

Liu, Yishuang; Zhou, Shuang; Deng, Qi; Li, Xinghua; Meng, Jianzhou; Guan, Yan; Li, Chuanyou; Xiao, Chunling

2016-03-01

Screen and identify novel inhibitors of isocitrate lyase (ICL) as potent antitubercular agents against Mycobacterium tuberculosis and determine their inhibitory characteristics, antitubercular activities and mechanisms of action. Recombinant ICL of M. tuberculosis was expressed and purified, which was used for high-throughput screening (HTS) and the following experiments. A total of 71,765 compounds were screened to identify ICL inhibitors which were then evaluated for their roles as potent antitubercular agents. To determine the inhibitory characteristics of the agents against latent M. tuberculosis in persistent infections, a macrophage model (mouse J774A.1 cell) infected with Mycobacterium marinum BAA-535 strain was built and assessed. The potent antitubercular agents were identified using the macrophage model. Then, the inhibitory intensity and mode of the agents that exhibit on ICL protein of M. tuberculosis were analyzed, and the interaction mechanisms were preliminarily clarified according to the parameters of enzyme kinetics, circular dichroism experiments, fluorescence quenching assay, and molecular docking. The previously established ICL inhibitor screening model was evaluated to be suitable for HTS assay. Of the 71,765 compounds, 13 of them were identified to inhibit ICL effectively and stably. IMBI-3 demonstrated the most significant inhibitory activity with IC50 of 30.9 μmol/L. Its minimum inhibitory concentration (MIC) for M. tuberculosis, including extensively drug-resistant tuberculosis (XDR-TB) and multidrug-resistant tuberculosis (MDR-TB), were determined in the range of 0.25-1 μg/mL. When IMBI-3 is used in combination with isoniazid, the colony-forming units (CFU) counting of latent M. tuberculosis in J774A.1 macrophage cells decreased significantly as IMBI-3 concentration increased. The inhibition mode of IMBI-3 on ICL was probably competitive inhibition with an inhibition constant (Ki) of approximate 1.85 μmol/L. The interaction between IMBI-3 and ICL of M. tuberculosis was also confirmed by circular dichroism experiments and fluorescence quenching assay. And seven possible active amino acids of ICL of M. tuberculosis were identified in the active site through molecular docking. IMBI-3, a novel potent antitubercular agent targeting ICL of M. tuberculosis, was identified and evaluated. It inhibited both log-phase M. tuberculosis in vitro and dormant M. tuberculosis in macrophages. It was the first representative compound of this family with the ICL enzyme inhibition and antimycobacterial activities. Copyright © 2016 Elsevier Ltd. All rights reserved.

Directly observed treatment is associated with reduced default among foreign tuberculosis patients in Thailand.

PubMed

Kapella, B K; Anuwatnonthakate, A; Komsakorn, S; Moolphate, S; Charusuntonsri, P; Limsomboon, P; Wattanaamornkiat, W; Nateniyom, S; Varma, J K

2009-02-01

Thailand's Tuberculosis (TB) Active Surveillance Network in four provinces in Thailand. As treatment default is common in mobile and foreign populations, we evaluated risk factors for default among non-Thai TB patients in Thailand. Observational cohort study using TB program data. Analysis was restricted to patients with an outcome categorized as cured, completed, failure or default. We used multivariate analysis to identify factors associated with default, including propensity score analysis, to adjust for factors associated with receiving directly observed treatment (DOT). During October 2004-September 2006, we recorded data for 14359 TB patients, of whom 995 (7%) were non-Thais. Of the 791 patients analyzed, 313 (40%) defaulted. In multivariate analysis, age>or=45 years (RR 1.47, 95%CI 1.25-1.74), mobility (RR 2.36, 95%CI 1.77-3.14) and lack of DOT (RR 2.29, 95%CI 1.45-3.61) were found to be significantly associated with default among non-Thais. When controlling for propensity to be assigned DOT, the risk of default remained increased in those not assigned DOT (RR 1.99, 95%CI 1.03-3.85). In non-Thai TB patients, DOT was the only modifiable factor associated with default. Using DOT may help improve TB treatment outcomes in non-Thai TB patients.

Active Tuberculosis Case Finding Interventions Among Immigrants, Refugees and Asylum Seekers in Italy

PubMed Central

Schepisi, Monica Sañé; Gualano, Gina; Piselli, Pierluca; Mazza, Marta; D’Angelo, Donatella; Fasciani, Francesca; Barbieri, Alberto; Rocca, Giorgia; Gnolfo, Filippo; Olivani, Piefranco; Ferrarese, Maurizio; Codecasa, Luigi Ruffo; Palmieri, Fabrizio; Girardi, Enrico

2016-01-01

In Italy tuberculosis (TB) is largely concentrated in vulnerable groups such as migrants and in urban settings. We analyzed three TB case finding interventions conducted at primary centers and mobile clinics for regular/irregular immigrants and refugees/asylum seekers performed over a four-year period (November 2009-March 2014) at five different sites in Rome and one site in Milan, Italy. TB history and presence of symptoms suggestive of active TB were investigated by verbal screening through a structured questionnaire in migrants presenting for any medical condition to out-patient and mobile clinics. Individuals reporting TB history or symptoms were referred to a TB clinic for diagnostic workup. Among 6347 migrants enrolled, 891 (14.0%) reported TB history or symptoms suggestive of active TB and 546 (61.3%) were referred to the TB clinic. Of them, 254 (46.5%) did not present for diagnostic evaluation. TB was diagnosed in 11 individuals representing 0.17% of those screened and 3.76% of those evaluated. The overall yield of this intervention was in the range reported for other TB screening programs for migrants, although we recorded an unsatisfactory adherence to diagnostic workup. Possible advantages of this intervention include low cost and reduced burden of medical procedures for the screened population. PMID:27403270

Tuberculosis of hip: A current concept review

PubMed Central

Saraf, Shyam Kumar; Tuli, Surendra Mohan

2015-01-01

Tuberculosis (TB) of the hip is second to spine only hence a good number of cases are visiting the medical facilities every year. Many present in the advanced stage of the disease due to delayed diagnosis. In early stages of TB of hip, there is a diagnostic dilemma when plain X-rays are negative. In the present time, diagnostic modalities have improved from the days when diagnosis was based essentially on clinicoradiological presentation alone. By the time definite radiological changes appear on plain X-ray, the disease has moderately advanced. The modern diagnostic facilities like ultrasonography (USG) or magnetic resonance imaging of the hip joint, USG guided aspiration of synovial fluid and obtaining the material for polymerase chain reaction and tissue diagnosis must be utilized. In the treatment, current emphasis is more on mobility with stability at hip. Joint debridement, skeletal traction, and mobilization exercises may give more satisfying results as compared to the immobilization by hip spica. Adults with advanced arthritis and healed infection should be informed and discussed the various treatment modalities including the joint replacement. More and more surgeons are taking up the challenge of putting the total hip replacement in the active stage of the disease. Until the long term results in active disease are well established, we recommend it for the healed disease only in selected cases. PMID:25593352

Tuberculosis of hip: A current concept review.

PubMed

Saraf, Shyam Kumar; Tuli, Surendra Mohan

2015-01-01

Tuberculosis (TB) of the hip is second to spine only hence a good number of cases are visiting the medical facilities every year. Many present in the advanced stage of the disease due to delayed diagnosis. In early stages of TB of hip, there is a diagnostic dilemma when plain X-rays are negative. In the present time, diagnostic modalities have improved from the days when diagnosis was based essentially on clinicoradiological presentation alone. By the time definite radiological changes appear on plain X-ray, the disease has moderately advanced. The modern diagnostic facilities like ultrasonography (USG) or magnetic resonance imaging of the hip joint, USG guided aspiration of synovial fluid and obtaining the material for polymerase chain reaction and tissue diagnosis must be utilized. In the treatment, current emphasis is more on mobility with stability at hip. Joint debridement, skeletal traction, and mobilization exercises may give more satisfying results as compared to the immobilization by hip spica. Adults with advanced arthritis and healed infection should be informed and discussed the various treatment modalities including the joint replacement. More and more surgeons are taking up the challenge of putting the total hip replacement in the active stage of the disease. Until the long term results in active disease are well established, we recommend it for the healed disease only in selected cases.

Current use and acceptability of novel diagnostic tests for active tuberculosis: a worldwide survey

PubMed Central

Amicosante, Massimo; D’Ambrosio, Lia; Munoz, Marcela; Mello, Fernanda Carvalho de Queiroz; Tebruegge, Marc; Chegou, Novel Njweipi; Seghrouchni, Fouad; Centis, Rosella; Goletti, Delia; Bothamley, Graham; Migliori, Giovanni Battista

2017-01-01

ABSTRACT Objective: To determine the current use and potential acceptance (by tuberculosis experts worldwide) of novel rapid tests for the diagnosis of tuberculosis that are in line with World Health Organization target product profiles. Methods: A multilingual survey was disseminated online between July and November of 2016. Results: A total of 723 individuals from 114 countries responded to the survey. Smear microscopy was the most commonly used rapid tuberculosis test (available to 90.9% of the respondents), followed by molecular assays (available to 70.7%). Only a small proportion of the respondents in middle- and low-income countries had access to interferon-gamma-release assays. Serological and lateral flow immunoassays were used by more than a quarter (25.4%) of the respondents. Among the respondents who had access to molecular tests, 46.7% were using the Xpert assay overall, that proportion being higher in lower middle-income countries (55.6%) and low-income countries (76.6%). The data also suggest that there was some alignment of pricing for molecular assays. Respondents stated they would accept novel rapid tuberculosis tests if available, including molecular assays (acceptable to 86.0%) or biomarker-based serological assays (acceptable to 81.7%). Simple biomarker-based assays were more commonly deemed acceptable in middle- and low-income countries. Conclusions: Second-generation molecular assays have become more widely available in high- and low-resource settings. However, the development of novel rapid tuberculosis tests continues to be considered important by tuberculosis experts. Our data also underscore the need for additional training and education of end users. PMID:29160384

Costs of novel tuberculosis diagnostics--will countries be able to afford it?

PubMed

Pantoja, Andrea; Kik, Sandra V; Denkinger, Claudia M

2015-04-01

Four priority target product profiles for the development of diagnostic tests for tuberculosis were identified: 1) Rapid sputum-based (RSP), 2) non-sputum Biomarker-based (BMT), 3) triage test followed by confirmatory test (TT), and 4) drug-susceptibility testing (DST). We assessed the cost of the new tests in suitable strategies and of the conventional diagnosis of tuberculosis as per World Health Organization guidelines, in 36 high tuberculosis and MDR burden countries. Costs were then compared to the available funding for tuberculosis at country level. Costs of diagnosing tuberculosis using RSP ranged US$93-187 million/year; if RSP unit cost is of US$2-4 it would be lower/similar cost than conventional strategy with sputum smear microscopy (US$ 119 million/year). Using BMT (with unit cost of US$2-4) would cost US$70-121 million/year and be lower/comparable cost than conventional diagnostics. Using TT with TPP characteristics (unit cost of US$1-2) followed by Xpert would reduce diagnostic costs up to US$36 million/year. Costs of using different novel DST strategies for the diagnosis of drug resistance would be higher compared with conventional diagnosis. Introducing a TT or a biomarker test with optimal characteristics would be affordable from a cost and affordability perspective at the current available funding for tuberculosis. Additional domestic or donor funding would be needed in most countries to achieve affordability for other new diagnostic tests. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Current use and acceptability of novel diagnostic tests for active tuberculosis: a worldwide survey.

PubMed

Amicosante, Massimo; D'Ambrosio, Lia; Munoz, Marcela; Mello, Fernanda Carvalho de Queiroz; Tebruegge, Marc; Chegou, Novel Njweipi; Seghrouchni, Fouad; Centis, Rosella; Goletti, Delia; Bothamley, Graham; Migliori, Giovanni Battista

2017-01-01

To determine the current use and potential acceptance (by tuberculosis experts worldwide) of novel rapid tests for the diagnosis of tuberculosis that are in line with World Health Organization target product profiles. A multilingual survey was disseminated online between July and November of 2016. A total of 723 individuals from 114 countries responded to the survey. Smear microscopy was the most commonly used rapid tuberculosis test (available to 90.9% of the respondents), followed by molecular assays (available to 70.7%). Only a small proportion of the respondents in middle- and low-income countries had access to interferon-gamma-release assays. Serological and lateral flow immunoassays were used by more than a quarter (25.4%) of the respondents. Among the respondents who had access to molecular tests, 46.7% were using the Xpert assay overall, that proportion being higher in lower middle-income countries (55.6%) and low-income countries (76.6%). The data also suggest that there was some alignment of pricing for molecular assays. Respondents stated they would accept novel rapid tuberculosis tests if available, including molecular assays (acceptable to 86.0%) or biomarker-based serological assays (acceptable to 81.7%). Simple biomarker-based assays were more commonly deemed acceptable in middle- and low-income countries. Second-generation molecular assays have become more widely available in high- and low-resource settings. However, the development of novel rapid tuberculosis tests continues to be considered important by tuberculosis experts. Our data also underscore the need for additional training and education of end users.

Pathogenic mechanisms of intracellular bacteria.

PubMed

Niller, Hans Helmut; Masa, Roland; Venkei, Annamária; Mészáros, Sándor; Minarovits, Janos

2017-06-01

We wished to overview recent data on a subset of epigenetic changes elicited by intracellular bacteria in human cells. Reprogramming the gene expression pattern of various host cells may facilitate bacterial growth, survival, and spread. DNA-(cytosine C5)-methyltransferases of Mycoplasma hyorhinis targeting cytosine-phosphate-guanine (CpG) dinucleotides and a Mycobacterium tuberculosis methyltransferase targeting non-CpG sites methylated the host cell DNA and altered the pattern of gene expression. Gene silencing by CpG methylation and histone deacetylation, mediated by cellular enzymes, also occurred in M. tuberculosis-infected macrophages. M. tuberculosis elicited cell type-specific epigenetic changes: it caused increased DNA methylation in macrophages, but induced demethylation, deposition of euchromatic histone marks and activation of immune-related genes in dendritic cells. A secreted transposase of Acinetobacter baumannii silenced a cellular gene, whereas Mycobacterium leprae altered the epigenotype, phenotype, and fate of infected Schwann cells. The 'keystone pathogen' oral bacterium Porphyromonas gingivalis induced local DNA methylation and increased the level of histone acetylation in host cells. These epigenetic changes at the biofilm-gingiva interface may contribute to the development of periodontitis. Epigenetic regulators produced by intracellular bacteria alter the epigenotype and gene expression pattern of host cells and play an important role in pathogenesis.

Evaluation of Mycobacterium tuberculosis Early Secreted Antigenic Target 6 Recombinant Protein as a Diagnostic Marker in Skin Test.

PubMed

Moradi, Jale; Mosavari, Nader; Ebrahimi, Mahmoud; Arefpajohi, Reza; Tebianian, Majid

2015-02-01

Tuberculosis (TB) is the leading infectious disease in the developing world. Delayed-type hypersensitivity skin test diagnoses TB using tuberculin purified protein derivative (PPD), but this test is incapable of distinguishing Mycobacterium tuberculosis (MTB) infection from bacillus Calmette-Guérin (BCG) vaccination or an infection caused by nontuberculous mycobacteria (NTM). This study was performed to evaluate the use of recombinant early secretory antigenic target 6 (rESAT-6), a secretory protein found only in MTB, Mycobacterium bovis, and few other mycobacterial species, as a skin marker for MTB in guinea pigs. We prepared recombinant MTB ESAT-6 and evaluated its use as a specific antigen for MTB in guinea pigs. Our results show that the purified MTB rESAT-6 antigen is capable of inducing a positive reaction only in guinea pigs sensitized to MTB. No such reaction was observed in the animals sensitized to M. bovis, BCG vaccination, or NTM (Mycobacterium avium). Our study results confirm that the ESAT-6 antigen is more specific to MTB infection than PPD and could be used in more specific skin tests for detection of MTB in large animals and in humans.

Inhibition of Mycobacterium tuberculosis Methionine Aminopeptidases by Bengamide Derivatives

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, Jing-Ping; Yuan, Xiu-Hua; Yuan, Hai

Methionine aminopeptidase (MetAP) carries out an essential function of protein N-terminal processing in many bacteria and is a promising target for the development of novel antitubercular agents. Natural bengamides potently inhibit the proliferation of mammalian cells by targeting MetAP enzymes, and the X-ray crystal structure of human type 2 MetAP in complex with a bengamide derivative reveals the key interactions at the active site. By preserving the interactions with the conserved residues inside the binding pocket while exploring the differences between bacterial and human MetAPs around the binding pocket, seven bengamide derivatives were synthesized and evaluated for inhibition of MtMetAP1amore » and MtMetAP1c in different metalloforms, inhibition of M. tuberculosis growth in replicating and non-replicating states, and inhibition of human K562 cell growth. Potent inhibition of MtMetAP1a and MtMetAP1c and modest growth inhibition of M. tuberculosis were observed for some of these derivatives. Crystal structures of MtMetAP1c in complex with two of the derivatives provided valuable structural information for improvement of these inhibitors for potency and selectivity.« less

Treatment outcomes of drug-resistant tuberculosis patients in Kenya.

PubMed

Mibei, D J; Kiarie, J W; Wairia, A; Kamene, M; Okumu, M E

2016-11-01

Successful treatment of drug-resistant tuberculosis (DR-TB) is crucial in preventing disease transmission and reducing related morbidity and mortality. A standardised DR-TB treatment regimen is used in Kenya. Although patients on treatment are monitored, no evaluation of factors affecting treatment outcomes has yet been performed. To analyse treatment outcomes of DR-TB patients in Kenya and factors associated with successful outcome. Retrospective analysis of secondary data from Kenya's National Tuberculosis, Leprosy and Lung disease programme. DR-TB data from the national database for January to December 2012 were reviewed. Of 205 DR-TB patients included in the analysis, 169 (82.4%) had a successful treatment outcome, 18 (9%) died and 18 (9%) were lost to follow-up. Only sex (P = 0.006) and human immunodeficiency virus (HIV) status (P = 0.008) were predictors of successful treatment. Females were more likely to attain treatment success (OR 3.86, 95%CI 1.47-10.12), and HIV-negative status increased the likelihood of successful treatment (OR 3.53, 95%CI 1.4-8.9). Treatment success rates were higher than World Health Organization targets. Targeted policies for HIV-positive patients and males will improve treatment outcomes in these groups.

Will the European Union reach the United Nations Millennium declaration target of a 50% reduction of tuberculosis mortality between 1990 and 2015?

PubMed

van der Werf, Marieke J; Bonfigli, Sandro; Hruba, Frantiska

2017-07-06

The Millennium Development Goals (MDG) provide targets for 2015. MDG 6 includes a target to reduce the tuberculosis (TB) death rate by 50% compared with 1990. We aimed to assess whether this target was reached by the European Union (EU) and European Economic Area countries. We used Eurostat causes of death data to assess whether the target was reached in the EU. We calculated the reduction in reported and adjusted death rates and the annual average percentage decline based on the available data. Between 1999 and 2014, the TB death rate decreased by 50%, the adjusted death rate by 56% and the annual average percentage decline was 5.43% (95% confidence interval 4.94-6.74) for the EU. Twenty of 26 countries reporting >5 TB deaths in the first reporting year reached the target of 50% reduction in adjusted death rate. The EU reached the MDG target of a 50% reduction of the TB death rate and also the annual average percentage decline was larger than the 2.73% needed to reach the target. The World Health Organization 'End TB Strategy' requires a further reduction of the number of TB deaths of 35% by 2020 compared to 2015, which will challenge TB prevention and care services in the EU.

'Team up against TB': promoting involvement in Thibela TB, a trial of community-wide tuberculosis preventive therapy.

PubMed

Grant, Alison D; Coetzee, Leonie; Fielding, Katherine L; Lewis, James J; Ntshele, Smanga; Luttig, Mariëtha M; Mngadi, Kathryn T; Muller, Dorothy; Popane, Flora; Mdluli, John; Mngadi, Nkosinathi; Sefuthi, Clement; Clark, David A; Churchyard, Gavin

2010-11-01

To describe a programme of community education and mobilization to promote uptake in a cluster-randomized trial of tuberculosis preventive therapy offered to all members of intervention clusters. Gold mines in South Africa, where tuberculosis incidence is extremely high, despite conventional control measures. All employees in intervention clusters (mine shaft and associated hostel) were invited to enrol. Cumulative enrolment in the study in intervention clusters. Key steps in communicating information relevant to the study included extensive consultation with key stakeholders; working with a communication company to develop a project 'brand'; developing a communication strategy tailored to each intervention site; and involving actors from a popular television comedy series to help inform communities about the study. One-to-one communications used peer educators along with study staff, and participant advisory groups facilitated two-way communication between study staff and participants. By contrast, treatment 'buddies' and text messaging to promote adherence proved less successful. Mean cumulative enrolment in the first four intervention clusters was 61.9%, increasing to 83.0% in the final four clusters. A tailored communication strategy can facilitate a high level of enrolment in a community health intervention.

Identification of Mycobacterium tuberculosis in Clinical Specimens of Patients Suspected of Having Extrapulmonary Tuberculosis by Application of Nested PCR on Five Different Genes

PubMed Central

Khosravi, Azar D.; Alami, Ameneh; Meghdadi, Hossein; Hosseini, Atta A.

2017-01-01

Definitive and rapid diagnosis of extrapulmonary tuberculosis (EPTB) is challenging since conventional techniques have limitations due to the paucibacillary nature of the disease. To increase the sensitivity of detection of Mycobacterium tuberculosis (MTB) in EPTB specimens, we performed a nested PCR assay targeting several genes of MTB on EPTB specimens. A total of 100 clinical specimens from suspected cases of EPTB were processed. Standard staining for acid fast bacilli (AFB) was performed as the preliminary screening test. Extracted DNAs from specimens were subjected to Nested PCR technique for the detection of five different MTB target genes of IS6110, IS1081, hsp65kd, mbp64, and mtp40. On performing AFB staining, only 13% of specimens were positive, of which ascites fluid (33.3%), followed by pleural effusion (30.8%) showed the greatest AFB positivity rate. We demonstrated slight improvement in yields in lymph node which comprised the majority of specimens in this study, by employing PCR targeted to IS6110- and hsp65-genes in comparison to AFB staining. However, the yields in ascites fluid and pleural effusion were not substantially improved by PCR, but those from bone and wound were, as in nested PCR employing either gene, the same positivity rate were obtained for ascites fluid (33.3%), while for pleural effusion specimens only IS1081 based PCR showed identical positivity rate with AFB stain (30.8%). The results for bone and wound specimens, however, demonstrated an improved yield mainly by employing IS1081 gene. Here, we report higher detection rate of EPTB in clinical specimens using five different targeted MTB genes. This nested PCR approach facilitates the comparison and the selection of the most frequently detected genes. Of course this study demonstrated the priority of IS1081 followed by mtp40 and IS6110, among the five tested genes and indicates the effectiveness of any of the three genes in the design of an efficient nested-PCR test that facilitates an early diagnosis of paucibacillary EPTB cases, which are difficult to diagnose with the available standard. PMID:28144587

Identification of Mycobacterium tuberculosis in Clinical Specimens of Patients Suspected of Having Extrapulmonary Tuberculosis by Application of Nested PCR on Five Different Genes.

PubMed

Khosravi, Azar D; Alami, Ameneh; Meghdadi, Hossein; Hosseini, Atta A

2017-01-01

Definitive and rapid diagnosis of extrapulmonary tuberculosis (EPTB) is challenging since conventional techniques have limitations due to the paucibacillary nature of the disease. To increase the sensitivity of detection of Mycobacterium tuberculosis (MTB) in EPTB specimens, we performed a nested PCR assay targeting several genes of MTB on EPTB specimens. A total of 100 clinical specimens from suspected cases of EPTB were processed. Standard staining for acid fast bacilli (AFB) was performed as the preliminary screening test. Extracted DNAs from specimens were subjected to Nested PCR technique for the detection of five different MTB target genes of IS6110, IS1081, hsp65kd, mbp64 , and mtp40 . On performing AFB staining, only 13% of specimens were positive, of which ascites fluid (33.3%), followed by pleural effusion (30.8%) showed the greatest AFB positivity rate. We demonstrated slight improvement in yields in lymph node which comprised the majority of specimens in this study, by employing PCR targeted to IS6110 - and hsp65-genes in comparison to AFB staining. However, the yields in ascites fluid and pleural effusion were not substantially improved by PCR, but those from bone and wound were, as in nested PCR employing either gene, the same positivity rate were obtained for ascites fluid (33.3%), while for pleural effusion specimens only IS1081 based PCR showed identical positivity rate with AFB stain (30.8%). The results for bone and wound specimens, however, demonstrated an improved yield mainly by employing IS1081 gene. Here, we report higher detection rate of EPTB in clinical specimens using five different targeted MTB genes. This nested PCR approach facilitates the comparison and the selection of the most frequently detected genes. Of course this study demonstrated the priority of IS1081 followed by mtp40 and IS6110 , among the five tested genes and indicates the effectiveness of any of the three genes in the design of an efficient nested-PCR test that facilitates an early diagnosis of paucibacillary EPTB cases, which are difficult to diagnose with the available standard.

Screening of a Novel Fragment Library with Functional Complexity against Mycobacterium tuberculosis InhA.

PubMed

Prati, Federica; Zuccotto, Fabio; Fletcher, Daniel; Convery, Maire A; Fernandez-Menendez, Raquel; Bates, Robert; Encinas, Lourdes; Zeng, Jingkun; Chung, Chun-Wa; De Dios Anton, Paco; Mendoza-Losana, Alfonso; Mackenzie, Claire; Green, Simon R; Huggett, Margaret; Barros, David; Wyatt, Paul G; Ray, Peter C

2018-04-06

Our findings reported herein provide support for the benefits of including functional group complexity (FGC) within fragments when screening against protein targets such as Mycobacterium tuberculosis InhA. We show that InhA fragment actives with FGC maintained their binding pose during elaboration. Furthermore, weak fragment hits with functional group handles also allowed for facile fragment elaboration to afford novel and potent InhA inhibitors with good ligand efficiency metrics for optimization. © 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Molecular characterization of the human immunodeficiency virus type 1 among children in Lima, Peru.

PubMed

Carrión, A G; Laguna-Torres, V A; Soto-Castellares, G; Castillo, M; Salazar, E; Negron, E; Kolevic, L; Montano, S M; Sánchez, J L; Bautista, C T; Oberhelman, R A; Kochel, T J

2009-08-01

In Peru, there is a lack of information on molecular analysis in pediatric human immunodeficiency virus (HIV) infection. At present, the mother-to-child transmission rate is estimated at approximately 2-4%. The objective of this study was to assess the molecular epidemiology of HIV-1 in infected children. Children with suspected or confirmed pulmonary tuberculosis were evaluated at two public hospitals between 2002 and 2007. Whole blood samples were obtained from 90 HIV-positive children, who were confirmed to be positive by enzyme-linked immunosorbent assay and Western blot. The specimens were subjected to envelope heteroduplex mobility assay (env HMA) followed by gag and pol gene region sequence analysis. Subtype B was found in 88 (98%) of 90 children and 2 (2%) children were subtype BF recombinants. This is the first report of recombinant HIV strains in HIV-infected children in Peru. Understanding the origin, diversity, and spread of HIV strains worldwide will be necessary for the development of an effective vaccine that targets pediatric populations throughout the world.

Immunogenic Properties of Lactobacillus plantarum Producing Surface-Displayed Mycobacterium tuberculosis Antigens

PubMed Central

Kleiveland, Charlotte R.; Minic, Rajna; Moen, Lars F.; Øverland, Lise; Tjåland, Rannei; Carlsen, Harald; Lea, Tor; Eijsink, Vincent G. H.

2016-01-01

ABSTRACT Tuberculosis (TB) remains among the most deadly diseases in the world. The only available vaccine against tuberculosis is the bacille Calmette-Guérin (BCG) vaccine, which does not ensure full protection in adults. There is a global urgency for the development of an effective vaccine for preventing disease transmission, and it requires novel approaches. We are exploring the use of lactic acid bacteria (LAB) as a vector for antigen delivery to mucosal sites. Here, we demonstrate the successful expression and surface display of a Mycobacterium tuberculosis fusion antigen (comprising Ag85B and ESAT-6, referred to as AgE6) on Lactobacillus plantarum. The AgE6 fusion antigen was targeted to the bacterial surface using two different anchors, a lipoprotein anchor directing the protein to the cell membrane and a covalent cell wall anchor. AgE6-producing L. plantarum strains using each of the two anchors induced antigen-specific proliferative responses in lymphocytes purified from TB-positive donors. Similarly, both strains induced immune responses in mice after nasal or oral immunization. The impact of the anchoring strategies was reflected in dissimilarities in the immune responses generated by the two L. plantarum strains in vivo. The present study comprises an initial step toward the development of L. plantarum as a vector for M. tuberculosis antigen delivery. IMPORTANCE This work presents the development of Lactobacillus plantarum as a candidate mucosal vaccine against tuberculosis. Tuberculosis remains one of the top infectious diseases worldwide, and the only available vaccine, bacille Calmette-Guérin (BCG), fails to protect adults and adolescents. Direct antigen delivery to mucosal sites is a promising strategy in tuberculosis vaccine development, and lactic acid bacteria potentially provide easy, safe, and low-cost delivery vehicles for mucosal immunization. We have engineered L. plantarum strains to produce a Mycobacterium tuberculosis fusion antigen and to anchor this antigen to the bacterial cell wall or to the cell membrane. The recombinant strains elicited proliferative antigen-specific T-cell responses in white blood cells from tuberculosis-positive humans and induced specific immune responses after nasal and oral administrations in mice. PMID:27815271

Evaluation of profile and functionality of memory T cells in pulmonary tuberculosis.

PubMed

Tonaco, Marcela M; Moreira, Jôsimar D; Nunes, Fernanda F C; Loures, Cristina M G; Souza, Larissa R; Martins, Janaina M; Silva, Henrique R; Porto, Arthur Henrique R; Toledo, Vicente Paulo C P; Miranda, Silvana S; Guimarães, Tânia Mara P D

2017-12-01

The cells T CD4+ T and CD8+ can be subdivided into phenotypes naïve, T of central memory, T of effector memory and effector, according to the expression of surface molecules CD45RO and CD27. The T lymphocytes are cells of long life with capacity of rapid expansion and function, after a new antigenic exposure. In tuberculosis, it was found that specific memory T cells are present, however, gaps remain about the role of such cells in the disease immunology. In this study, the phenotypic profile was analyzed and characterized the functionality of CD4+ T lymphocytes and CD8+ T cells of memory and effector, in response to specific stimuli in vitro, in patients with active pulmonary TB, compared to individuals with latent infection with Mycobacterium tuberculosis the ones treated with pulmonary TB. It was observed that the group of patients with active pulmonary tuberculosis was the one which presented the highest proportion of cells T CD4+ of central memory IFN-ɣ+ e TNF-α+, suggesting that in TB, these T of central memory cells would have a profile of protective response, being an important target of study for the development of more effective vaccines; this group also developed lower proportion of CD8+ T effector lymphocytes than the others, a probable cause of specific and less effective response against the bacillus in these individuals; the ones treated for pulmonary tuberculosis were those who developed higher proportion of T CD4+ of memory central IL-17+ cells, indicating that the stimulation of long duration, with high antigenic load, followed by elimination of the pathogen, contribute to more significant generation of such cells; individuals with latent infection by M. tuberculosis and treated for pulmonary tuberculosis, showed greater response of CD8+ T effector lymphocytes IFN-ɣ+ than the controls, suggesting that these cells, as well as CD4+ T lymphocytes, have crucial role of protection against M. tuberculosis. These findings have contributed to a better understanding of the immunologic changes in M. tuberculosis infection and the development of new strategies for diagnosis and prevention of tuberculosis. Copyright © 2017. Published by Elsevier B.V.

Immunogenic Properties of Lactobacillus plantarum Producing Surface-Displayed Mycobacterium tuberculosis Antigens.

PubMed

Kuczkowska, Katarzyna; Kleiveland, Charlotte R; Minic, Rajna; Moen, Lars F; Øverland, Lise; Tjåland, Rannei; Carlsen, Harald; Lea, Tor; Mathiesen, Geir; Eijsink, Vincent G H

2017-01-15

Tuberculosis (TB) remains among the most deadly diseases in the world. The only available vaccine against tuberculosis is the bacille Calmette-Guérin (BCG) vaccine, which does not ensure full protection in adults. There is a global urgency for the development of an effective vaccine for preventing disease transmission, and it requires novel approaches. We are exploring the use of lactic acid bacteria (LAB) as a vector for antigen delivery to mucosal sites. Here, we demonstrate the successful expression and surface display of a Mycobacterium tuberculosis fusion antigen (comprising Ag85B and ESAT-6, referred to as AgE6) on Lactobacillus plantarum The AgE6 fusion antigen was targeted to the bacterial surface using two different anchors, a lipoprotein anchor directing the protein to the cell membrane and a covalent cell wall anchor. AgE6-producing L. plantarum strains using each of the two anchors induced antigen-specific proliferative responses in lymphocytes purified from TB-positive donors. Similarly, both strains induced immune responses in mice after nasal or oral immunization. The impact of the anchoring strategies was reflected in dissimilarities in the immune responses generated by the two L. plantarum strains in vivo The present study comprises an initial step toward the development of L. plantarum as a vector for M. tuberculosis antigen delivery. This work presents the development of Lactobacillus plantarum as a candidate mucosal vaccine against tuberculosis. Tuberculosis remains one of the top infectious diseases worldwide, and the only available vaccine, bacille Calmette-Guérin (BCG), fails to protect adults and adolescents. Direct antigen delivery to mucosal sites is a promising strategy in tuberculosis vaccine development, and lactic acid bacteria potentially provide easy, safe, and low-cost delivery vehicles for mucosal immunization. We have engineered L. plantarum strains to produce a Mycobacterium tuberculosis fusion antigen and to anchor this antigen to the bacterial cell wall or to the cell membrane. The recombinant strains elicited proliferative antigen-specific T-cell responses in white blood cells from tuberculosis-positive humans and induced specific immune responses after nasal and oral administrations in mice. Copyright © 2016 American Society for Microbiology.

Development and Evaluation of Chitosan Microparticles Based Dry Powder Inhalation Formulations of Rifampicin and Rifabutin.

PubMed

Pai, Rohan V; Jain, Rajesh R; Bannalikar, Anilkumar S; Menon, Mala D

2016-04-01

The lung is the primary entry site and target for Mycobacterium tuberculosis; more than 80% of the cases reported worldwide are of pulmonary tuberculosis. Hence, direct delivery of anti-tubercular drugs to the lung would be beneficial in reducing both, the dose required, as well as the duration of therapy for pulmonary tuberculosis. In the present study, microsphere-based dry powder inhalation systems of the anti-tubercular drugs, rifampicin and rifabutin, were developed and evaluated, with a view to achieve localized and targeted delivery of these drugs to the lung. The drug-loaded chitosan microparticles were prepared by an ionic gelation method, followed by spray-drying to obtain respirable particles. The microparticles were evaluated for particle size and drug release. The drug-loaded microparticles were then adsorbed onto an inhalable lactose carrier and characterized for in vitro lung deposition on an Andersen Cascade Impactor (ACI) followed by in vitro uptake study in U937 human macrophage cell lines. In vivo toxicity of the developed formulations was evaluated using Sprague Dawley rats. Both rifampicin and rifabutin-loaded microparticles had MMAD close to 5 μm and FPF values of 21.46% and 29.97%, respectively. In vitro release study in simulated lung fluid pH 7.4 showed sustained release for 12 hours for rifampicin microparticles and up to 96 hours for rifabutin microparticles, the release being dependent on both swelling of the polymer and solubility of the drugs in the dissolution medium. In vitro uptake studies in U937 human macrophage cell line suggested that microparticles were internalized within the macrophages. In vivo acute toxicity study of the microparticles in Sprague Dawley rats revealed no significant evidence for local adverse effects. Thus, spray-dried microparticles of the anti-tubercular drugs, rifampicin and rifabutin, could prove to be an improved, targeted, and efficient system for treatment of tuberculosis.

ESCMID Study Group for Infections in Compromised Hosts (ESGICH) Consensus Document on the safety of targeted and biological therapies: an infectious diseases perspective (Soluble immune effector molecules [I]: anti-tumor necrosis factor-α agents).

PubMed

Baddley, J W; Cantini, F; Goletti, D; Gómez-Reino, J J; Mylonakis, E; San-Juan, R; Fernández-Ruiz, M; Torre-Cisneros, J

2018-06-01

The present review is part of the ESCMID Study Group for Infections in Compromised Hosts (ESGICH) Consensus Document on the safety of targeted and biological therapies. To review, from an Infectious Diseases perspective, the safety profile of agents targeting tumour necrosis factor-α (TNF-α) and to suggest preventive recommendations. Computer-based MEDLINE searches with MeSH terms pertaining to each agent or therapeutic family. Preclinical and clinical evidence indicate that anti-TNF-α therapy (infliximab, adalimumab, golimumab, certolizumab pegol and etanercept) is associated with a two-to four-fold increase in the risk of active tuberculosis and other granulomatous conditions (mostly resulting from the reactivation of a latent infection). In addition, it may lead to the occurrence of other serious infections (bacterial, fungal, opportunistic and certain viral infections). These associated risks seem to be lower for etanercept than other agents. Screening for latent tuberculosis infection should be performed before starting anti-TNF-α therapy, followed by anti-tuberculosis therapy if appropriate. Screening for chronic hepatitis B virus (HBV) infection is also recommended, and antiviral prophylaxis may be warranted for hepatitis B surface antigen-positive individuals. No benefit is expected from the use of antibacterial, anti-Pneumocystis or antifungal prophylaxis. Pneumococcal and age-appropriate antiviral vaccinations (i.e. influenza) should be administered. Live-virus vaccines (i.e. varicella-zoster virus or measles-mumps-rubella) may be contraindicated in people receiving anti-TNF-α therapy, although additional data are needed before definitive recommendations can be made. Prevention measures should be implemented to reduce the risk of latent tuberculosis or HBV reactivation among individuals receiving anti-TNF-α therapy. Copyright © 2018 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Optimal Clinical Doses of Faropenem, Linezolid, and Moxifloxacin in Children With Disseminated Tuberculosis: Goldilocks

PubMed Central

Srivastava, Shashikant; Deshpande, Devyani; Pasipanodya, Jotam; Nuermberger, Eric; Swaminathan, Soumya; Gumbo, Tawanda

2016-01-01

Background. When treated with the same antibiotic dose, children achieve different 0- to 24-hour area under the concentration-time curves (AUC0–24) because of maturation and between-child physiological variability on drug clearance. Children are also infected by Mycobacterium tuberculosis isolates with different antibiotic minimum inhibitory concentrations (MICs). Thus, each child will achieve different AUC0–24/MIC ratios when treated with the same dose. Methods. We used 10 000-subject Monte Carlo experiments to identify the oral doses of linezolid, moxifloxacin, and faropenem that would achieve optimal target exposures associated with optimal efficacy in children with disseminated tuberculosis. The linezolid and moxifloxacin exposure targets were AUC0–24/MIC ratios of 62 and 122, and a faropenem percentage of time above MIC >60%, in combination therapy. A linezolid AUC0–24 of 93.4 mg × hour/L was target for toxicity. Population pharmacokinetic parameters of each drug and between-child variability, as well as MIC distribution, were used, and the cumulative fraction of response (CFR) was calculated. We also considered drug penetration indices into meninges, bone, and peritoneum. Results. The linezolid dose of 15 mg/kg in full-term neonates and infants aged up to 3 months and 10 mg/kg in toddlers, administered once daily, achieved CFR ≥ 90%, with <10% achieving linezolid AUC0–24 associated with toxicity. The moxifloxacin dose of 25 mg/kg/day achieved a CFR > 90% in infants, but the optimal dose was 20 mg/kg/day in older children. The faropenem medoxomil optimal dosage was 30 mg/kg 3–4 times daily. Conclusions. The regimen and doses of linezolid, moxifloxacin, and faropenem identified are proposed to be adequate for all disseminated tuberculosis syndromes, whether drug-resistant or -susceptible. PMID:27742641

Cytosolic Proteome Profiling of Aminoglycosides Resistant Mycobacterium tuberculosis Clinical Isolates Using MALDI-TOF/MS.

PubMed

Sharma, Divakar; Lata, Manju; Singh, Rananjay; Deo, Nirmala; Venkatesan, Krishnamurthy; Bisht, Deepa

2016-01-01

Emergence of extensively drug resistant tuberculosis (XDR-TB) is the consequence of the failure of second line TB treatment. Aminoglycosides are the important second line anti-TB drugs used to treat the multi drug resistant tuberculosis (MDR-TB). Main known mechanism of action of aminoglycosides is to inhibit the protein synthesis by inhibiting the normal functioning of ribosome. Primary target of aminoglycosides are the ribosomal RNA and its associated proteins. Various mechanisms have been proposed for aminoglycosides resistance but still some are unsolved. As proteins are involved in most of the biological processes, these act as a potential diagnostic markers and drug targets. In the present study we analyzed the purely cytosolic proteome of amikacin (AK) and kanamycin (KM) resistant Mycobacterium tuberculosis isolates by proteomic and bioinformatic approaches. Twenty protein spots were found to have over expressed in resistant isolates and were identified. Among these Rv3208A, Rv2623, Rv1360, Rv2140c, Rv1636, and Rv2185c are six proteins with unknown functions or undefined role. Docking results showed that AK and KM binds to the conserved domain (DUF, USP-A, Luciferase, PEBP and Polyketidecyclase/dehydrase domain) of these hypothetical proteins and over expression of these proteins might neutralize/modulate the effect of drug molecules. TBPred and GPS-PUP predicted cytoplasmic nature and potential pupylation sites within these identified proteins, respectively. String analysis also suggested that over expressed proteins along with their interactive partners might be involved in aminoglycosides resistance. Cumulative effect of these over expressed proteins could be involved in AK and KM resistance by mitigating the toxicity, repression of drug target and neutralizing affect. These findings need further exploitation for the expansion of newer therapeutics or diagnostic markers against AK and KM resistance so that an extreme condition like XDR-TB can be prevented.

SMSaúde: Design, Development, and Implementation of a Remote/Mobile Patient Management System to Improve Retention in Care for HIV/AIDS and Tuberculosis Patients.

PubMed

Nhavoto, José António; Grönlund, Åke; Chaquilla, Walter Ponce

2015-03-09

The widespread and low cost of mobile phones and the convenience of short message service (SMS) text messaging suggest potential suitability for use with alternative strategies for supporting retention in care and adherence to the treatment of various chronic diseases, such as HIV and tuberculosis (TB). Despite the growing body of literature reporting positive outcomes of SMS text message-based communication with patients, there is yet very little research about the integration of communication technologies and electronic medical records or electronic patient tracking systems. To design, develop, and implement an integrated mobile phone text messaging system used to follow up with patients with HIV and TB in treatment in Mozambique. Following the design science research methodology, we developed a Web-based system that provides support to patients. A case study involving three health care sites in Mozambique was a basis for discussing design issues for this kind of system. We used brainstorming techniques to solicit usability requirements, focus group meetings to discuss and define system architecture, and prototyping to test in real environments and to improve the system. We found six sets of system requirements that need to be addressed for success: data collection, telecommunication costs, privacy and data security, text message content, connectivity, and system scalability. A text messaging system was designed and implemented in three health facilities. These sites feed data into a central data repository, which can be used for analysis of operations and decision support. Based on the treatment schedule, the system automatically sent SMS text message appointment reminders, medication reminders, as well as motivational and educational messages to patients enrolled in antiretroviral therapy and TB treatment programs. We successfully defined the requirements for, designed, and implemented a mobile phone text messaging system to support HIV and TB treatments. Implementation of this system could improve patients' self-management skills and strengthen communication between patients and health care providers.

Novel approach based on one-tube nested PCR and a lateral flow strip for highly sensitive diagnosis of tuberculous meningitis.

PubMed

Sun, Yajuan; Chen, Jiajun; Li, Jia; Xu, Yawei; Jin, Hui; Xu, Na; Yin, Rui; Hu, Guohua

2017-01-01

Rapid and sensitive detection of Mycobacterium tuberculosis (M. Tb) in cerebrospinal fluid is crucial in the diagnosis of tuberculous meningitis (TBM), but conventional diagnostic technologies have limited sensitivity and specificity or are time-consuming. In this work, a novel, highly sensitive molecular diagnostic method, one-tube nested PCR-lateral flow strip test (OTNPCR-LFST), was developed for detecting M. tuberculosis. This one-tube nested PCR maintains the sensitivity of conventional two-step nested PCR and reduces both the chance of cross-contamination and the time required for analysis. The PCR product was detected by a lateral flow strip assay, which provided a basis for migration of the test to a point-of-care (POC) microfluidic format. The developed assay had an improved sensitivity compared with traditional PCR, and the limit of detection was up to 1 fg DNA isolated from M. tuberculosis. The assay was also specific for M. tuberculosis, and no cross-reactions were found in other non-target bacteria. The application of this technique to clinical samples was successfully evaluated, and OTNPCR-LFST showed 89% overall sensitivity and 100% specificity for TBM patients. This one-tube nested PCR-lateral flow strip assay is useful for detecting M. tuberculosis in TBM due to its rapidity, high sensitivity and simple manipulation.

Evidence for the cytotoxic effects of Mycobacterium tuberculosis phospholipase C towards macrophages.

PubMed

Bakala N'goma, J C; Schué, M; Carrière, F; Geerlof, A; Canaan, S

2010-12-01

Phospholipase Cs (PLCs) contribute importantly to the virulence and pathogenicity of several bacteria. It has been reported in previous studies that mutations in the four predicted plc genes of Mycobacterium tuberculosis inhibit the growth of these bacteria during the late phase of infection in mice. These enzymes have not yet been fully characterised, mainly because they are not easy to produce in large quantities. With a view to elucidating the role of all Mycobacterium tuberculosis phospholipase Cs (PLC-A, PLC-B, PLC-C and PLC-D), a large amount of active, soluble recombinant PLCs, were expressed and purified using Mycobacterium smegmatis as expression system. These enzymes showed different pH activity profiles. PLC-C was found to be the most active of the four recombinant PLCs under acidic conditions. All the enzymes tested induced cytotoxic effects on mouse macrophage RAW 264.7 cell lines, via direct or indirect enzymatic hydrolysis of cell membrane phospholipids. These results open new prospects for characterising biochemical and structural features of Mycobacterium tuberculosis PLCs, which might lead to the identification of novel anti-tuberculosis drug targets. All mycobacterial phospholipase Cs can now be studied in order to determine their role in the virulence and pathogenicity of bacteria of this kind. 2010 Elsevier B.V. All rights reserved.

Design and construction of a DNA origami drug delivery system based on MPT64 antibody aptamer for tuberculosis treatment.

PubMed

Ranjbar, Reza; Hafezi-Moghadam, Mohammad Sadegh

2016-02-01

With all of the developments on infectious diseases, tuberculosis (TB) remains a cause of death among people. One of the most promising assembly techniques in nano-technology is "scaffolded DNA origami" to design and construct a nano-scale drug delivery system. Because of the global health problems of tuberculosis, the development of potent new anti-tuberculosis drug delivery system without cross-resistance with known anti-mycobacterial agents is urgently needed. The aim of this study was to design a nano-scale drug delivery system for TB treatment using the DNA origami method. In this study, we presented an experimental research on a DNA drug delivery system for treating Tuberculosis. TEM images were visualized with an FEI Tecnai T12 BioTWIN at 120 kV. The model was designed by caDNAno software and computational prediction of the 3D solution shape and its flexibility was calculated with a CanDo server. Synthesizing the product was imaged using transmission electron microscopy after negative-staining by uranyl formate. We constructed a multilayer 3D DNA nanostructure system by designing square lattice geometry with the scaffolded-DNA-origami method. With changes in the lock and key sequences, we recommend that this system be used for other infectious diseases to target the pathogenic bacteria.

Comparative study of diagnostic accuracy of established PCR assays and in-house developed sdaA PCR method for detection of Mycobacterium tuberculosis in symptomatic patients with pulmonary tuberculosis.

PubMed

Nimesh, Manoj; Joon, Deepali; Pathak, Anil Kumar; Saluja, Daman

2013-11-01

Indian contribution to global burden of tuberculosis is about 26%. In the present study we have developed an in-house PCR assay using primers for sdaA gene of Mycobacterium tuberculosis and evaluated against already established primers devR, IS6110, MPB64, rpoB primers for diagnosis of pulmonary tuberculosis. Using universal sample preparation (USP) method, DNA was extracted from sputum specimens of 412 symptomatic patients from Delhi, India. The DNA so extracted was used as template for PCR amplification using primers targeting sdaA, devR, IS6110, MPB64 and rpoB genes. Out of 412, 149 specimens were considered positive based on composite reference standard (CRS) criteria. The in-house designed sdaA PCR showed high specificity (96.5%), the high positive likelihood ratio (28), the high sensitivity (95.9%), and the very low negative likelihood ratio (0.04) in comparison to CRS. Based on our results, the sdaA PCR assay can be considered as one of the most reliable diagnostic tests in comparison to other PCR based detection methods. Copyright © 2013 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

One-tube loop-mediated isothermal amplification combined with restriction endonuclease digestion and ELISA for colorimetric detection of resistance to isoniazid, ethambutol and streptomycin in Mycobacterium tuberculosis isolates.

PubMed

Lee, Mei-Feng; Chen, Yen-Hsu; Hsu, Hui-Jine; Peng, Chien-Fang

2010-10-01

In this study, we designed a simple and rapid colorimetric detection method, a one-tube loop-mediated isothermal amplification (LAMP)-PCR-hybridization-restriction endonuclease-ELISA [one-tube LAMP-PCR-HY-RE-ELISA] system, to detect resistance to isoniazid, ethambutol and streptomycin in strains of Mycobacterium tuberculosis isolated from clinical specimens. The clinical performance of this method for detecting isoniazid-resistant, ethambutol-resistant and streptomycin-resistant isolates of M. tuberculosis showed 98.9%, 94.3% and 93.8%, respectively. This assay is rapid and convenient that can be performed within one working day. One-tube LAMP-PCR-HY-RE-ELISA system was designed based on hot spot point mutations in target drug-resistant genes, using LAMP-PCR, hybridization, digestion with restriction endonuclease and colorimetric method of ELISA. In this study, LAMP assay was used to amplify DNA from drug-resistant M. tuberculosis, and ELISA was used for colorimetrical determination. This assay will be a useful tool for rapid diagnosis of mutant codons in strains of M. tuberculosis for isoniazid at katG 315 and katG 463, ethambutol at embB 306 and embB 497, and streptomycin at rpsL 43. Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.

Selective Mycobacterium tuberculosis Shikimate Kinase Inhibitors as Potential Antibacterials

PubMed Central

Gordon, Sara; Simithy, Johayra; Goodwin, Douglas C; Calderón, Angela I

2015-01-01

Owing to the persistence of tuberculosis (TB) as well as the emergence of multidrug-resistant and extensively drug-resistant (XDR) forms of the disease, the development of new antitubercular drugs is crucial. Developing inhibitors of shikimate kinase (SK) in the shikimate pathway will provide a selective target for antitubercular agents. Many studies have used in silico technology to identify compounds that are anticipated to interact with and inhibit SK. To a much more limited extent, SK inhibition has been evaluated by in vitro methods with purified enzyme. Currently, there are no data on in vivo activity of Mycobacterium tuberculosis shikimate kinase (MtSK) inhibitors available in the literature. In this review, we present a summary of the progress of SK inhibitor discovery and evaluation with particular attention toward development of new antitubercular agents. PMID:25861218

SU-E-J-150: Four-Dimensional Cone-Beam CT Algorithm by Extraction of Physical and Motion Parameter of Mobile Targets Retrospective to Image Reconstruction with Motion Modeling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ali, I; Ahmad, S; Alsbou, N

Purpose: To develop 4D-cone-beam CT (CBCT) algorithm by motion modeling that extracts actual length, CT numbers level and motion amplitude of a mobile target retrospective to image reconstruction by motion modeling. Methods: The algorithm used three measurable parameters: apparent length and blurred CT number distribution of a mobile target obtained from CBCT images to determine actual length, CT-number value of the stationary target, and motion amplitude. The predictions of this algorithm were tested with mobile targets that with different well-known sizes made from tissue-equivalent gel which was inserted into a thorax phantom. The phantom moved sinusoidally in one-direction to simulatemore » respiratory motion using eight amplitudes ranging 0–20mm. Results: Using this 4D-CBCT algorithm, three unknown parameters were extracted that include: length of the target, CT number level, speed or motion amplitude for the mobile targets retrospective to image reconstruction. The motion algorithms solved for the three unknown parameters using measurable apparent length, CT number level and gradient for a well-defined mobile target obtained from CBCT images. The motion model agreed with measured apparent lengths which were dependent on the actual target length and motion amplitude. The gradient of the CT number distribution of the mobile target is dependent on the stationary CT number level, actual target length and motion amplitude. Motion frequency and phase did not affect the elongation and CT number distribution of the mobile target and could not be determined. Conclusion: A 4D-CBCT motion algorithm was developed to extract three parameters that include actual length, CT number level and motion amplitude or speed of mobile targets directly from reconstructed CBCT images without prior knowledge of the stationary target parameters. This algorithm provides alternative to 4D-CBCT without requirement to motion tracking and sorting of the images into different breathing phases which has potential applications in diagnostic CT imaging and radiotherapy.« less

A motion algorithm to extract physical and motion parameters of mobile targets from cone-beam computed tomographic images.

PubMed

Alsbou, Nesreen; Ahmad, Salahuddin; Ali, Imad

2016-05-17

A motion algorithm has been developed to extract length, CT number level and motion amplitude of a mobile target from cone-beam CT (CBCT) images. The algorithm uses three measurable parameters: Apparent length and blurred CT number distribution of a mobile target obtained from CBCT images to determine length, CT-number value of the stationary target, and motion amplitude. The predictions of this algorithm are tested with mobile targets having different well-known sizes that are made from tissue-equivalent gel which is inserted into a thorax phantom. The phantom moves sinusoidally in one-direction to simulate respiratory motion using eight amplitudes ranging 0-20 mm. Using this motion algorithm, three unknown parameters are extracted that include: Length of the target, CT number level, speed or motion amplitude for the mobile targets from CBCT images. The motion algorithm solves for the three unknown parameters using measured length, CT number level and gradient for a well-defined mobile target obtained from CBCT images. The motion model agrees with the measured lengths which are dependent on the target length and motion amplitude. The gradient of the CT number distribution of the mobile target is dependent on the stationary CT number level, the target length and motion amplitude. Motion frequency and phase do not affect the elongation and CT number distribution of the mobile target and could not be determined. A motion algorithm has been developed to extract three parameters that include length, CT number level and motion amplitude or speed of mobile targets directly from reconstructed CBCT images without prior knowledge of the stationary target parameters. This algorithm provides alternative to 4D-CBCT without requirement of motion tracking and sorting of the images into different breathing phases. The motion model developed here works well for tumors that have simple shapes, high contrast relative to surrounding tissues and move nearly in regular motion pattern that can be approximated with a simple sinusoidal function. This algorithm has potential applications in diagnostic CT imaging and radiotherapy in terms of motion management.

Identification of the likely translational start of Mycobacterium tuberculosis GyrB.

PubMed

Karkare, Shantanu; Brown, Amanda C; Parish, Tanya; Maxwell, Anthony

2013-07-15

Bacterial DNA gyrase is a validated target for antibacterial chemotherapy. It consists of two subunits, GyrA and GyrB, which form an A₂B₂ complex in the active enzyme. Sequence alignment of Mycobacterium tuberculosis GyrB with other bacterial GyrBs predicts the presence of 40 potential additional amino acids at the GyrB N-terminus. There are discrepancies between the M. tuberculosis GyrB sequences retrieved from different databases, including sequences annotated with or without the additional 40 amino acids. This has resulted in differences in the GyrB sequence numbering that has led to the reporting of previously known fluoroquinolone-resistance mutations as novel mutations. We have expressed M. tuberculosis GyrB with and without the extra 40 amino acids in Escherichia coli and shown that both can be produced as soluble, active proteins. Supercoiling and other assays of the two proteins show no differences, suggesting that the additional 40 amino acids have no effect on the enzyme in vitro. RT-PCR analysis of M. tuberculosis mRNA shows that transcripts that could yield both the longer and shorter protein are present. However, promoter analysis showed that only the promoter elements leading to the shorter GyrB (lacking the additional 40 amino acids) had significant activity. We conclude that the most probable translational start codon for M. tuberculosis GyrB is GTG (Val) which results in translation of a protein of 674 amino acids (74 kDa).

Functional, thermodynamics, structural and biological studies of in silico-identified inhibitors of Mycobacterium tuberculosis enoyl-ACP(CoA) reductase enzyme

NASA Astrophysics Data System (ADS)

Martinelli, Leonardo K. B.; Rotta, Mariane; Villela, Anne D.; Rodrigues-Junior, Valnês S.; Abbadi, Bruno L.; Trindade, Rogério V.; Petersen, Guilherme O.; Danesi, Giuliano M.; Nery, Laura R.; Pauli, Ivani; Campos, Maria M.; Bonan, Carla D.; de Souza, Osmar Norberto; Basso, Luiz A.; Santos, Diogenes S.

2017-04-01

Novel chemotherapeutics agents are needed to kill Mycobacterium tuberculosis, the main causative agent of tuberculosis (TB). The M. tuberculosis 2-trans-enoyl-ACP(CoA) reductase enzyme (MtInhA) is the druggable bona fide target of isoniazid. New chemotypes were previously identified by two in silico approaches as potential ligands to MtInhA. The inhibition mode was determined by steady-state kinetics for seven compounds that inhibited MtInhA activity. Dissociation constant values at different temperatures were determined by protein fluorescence spectroscopy. van’t Hoff analyses of ligand binding to MtInhA:NADH provided the thermodynamic signatures of non-covalent interactions (ΔH°, ΔS°, ΔG°). Phenotypic screening showed that five compounds inhibited in vitro growth of M. tuberculosis H37Rv strain. Labio_16 and Labio_17 compounds also inhibited the in vitro growth of PE-003 multidrug-resistant strain. Cytotoxic effects on Hacat, Vero and RAW 264.7 cell lines were assessed for the latter two compounds. The Labio_16 was bacteriostatic and Labio_17 bactericidal in an M. tuberculosis-infected macrophage model. In Zebrafish model, Labio_16 showed no cardiotoxicity whereas Labio_17 showed dose-dependent cardiotoxicity. Accordingly, a model was built for the MtInhA:NADH:Labio_16 ternary complex. The results show that the Labio_16 compound is a direct inhibitor of MtInhA, and it may represent a hit for the development of chemotherapeutic agents to treat TB.

A molecular platform for the diagnosis of multidrug-resistant and pre-extensively drug-resistant tuberculosis based on single nucleotide polymorphism mutations present in Colombian isolates of Mycobacterium tuberculosis.

PubMed

Martínez, Luz Maira Wintaco; Castro, Gloria Puerto; Guerrero, Martha Inírida

2016-02-01

Developing a fast, inexpensive, and specific test that reflects the mutations present in Mycobacterium tuberculosis isolates according to geographic region is the main challenge for drug-resistant tuberculosis (TB) control. The objective of this study was to develop a molecular platform to make a rapid diagnosis of multidrug-resistant (MDR) and extensively drug-resistant TB based on single nucleotide polymorphism (SNP) mutations present in therpoB, katG, inhA,ahpC, and gyrA genes from Colombian M. tuberculosis isolates. The amplification and sequencing of each target gene was performed. Capture oligonucleotides, which were tested before being used with isolates to assess the performance, were designed for wild type and mutated codons, and the platform was standardised based on the reverse hybridisation principle. This method was tested on DNA samples extracted from clinical isolates from 160 Colombian patients who were previously phenotypically and genotypically characterised as having susceptible or MDR M. tuberculosis. For our method, the kappa index of the sequencing results was 0,966, 0,825, 0,766, 0,740, and 0,625 forrpoB, katG, inhA,ahpC, and gyrA, respectively. Sensitivity and specificity were ranked between 90-100% compared with those of phenotypic drug susceptibility testing. Our assay helps to pave the way for implementation locally and for specifically adapted methods that can simultaneously detect drug resistance mutations to first and second-line drugs within a few hours.

Phloretin Exerts Anti-Tuberculosis Activity and Suppresses Lung Inflammation.

PubMed

Jeon, Dasom; Jeong, Min-Cheol; Jnawali, Hum Nath; Kwak, Chulhee; Ryoo, Sungwon; Jung, In Duk; Kim, Yangmee

2017-01-22

An increase in the prevalence of the drug-resistant Mycobacteria tuberculosis necessitates developing new types of anti-tuberculosis drugs. Here, we found that phloretin, a naturally-occurring flavonoid, has anti-mycobacterial effects on H37Rv, multi-drug-, and extensively drug-resistant clinical isolates, with minimum inhibitory concentrations of 182 and 364 μM, respectively. Since Mycobacteria cause lung inflammation that contributes to tuberculosis pathogenesis, anti-inflammatory effects of phloretin in interferon-γ-stimulated MRC-5 human lung fibroblasts and lipopolysaccharide (LPS)-stimulated dendritic cells were investigated. The release of interleukin (IL)-1β, IL-12, and tumor necrosis factor (TNF)-α was inhibited by phloretin. The mRNA levels of IL-1β, IL-6, IL-12, TNF-α, and matrix metalloproteinase-1, as well as p38 mitogen-activated protein kinase and extracellular signal-regulated kinase phosphorylation, were suppressed. A mouse in vivo study of LPS-stimulated lung inflammation showed that phloretin effectively suppressed the levels of TNF-α, IL-1β, and IL-6 in lung tissue with low cytotoxicity. Phloretin was found to bind M. tuberculosis β-ketoacyl acyl carrier protein synthase III (mtKASIII) with high affinity (7.221 × 10⁷ M -1 ); a binding model showed hydrogen bonding of A-ring 2'-hydroxy and B-ring 4-hydroxy groups of phloretin with Asn261 and Cys122 of mtKASIII, implying that mtKASIII can be a potential target protein. Therefore, phloretin can be a useful dietary natural product with anti-tuberculosis benefits.

Potential market for novel tuberculosis diagnostics: worth the investment?

PubMed

Kik, Sandra V; Denkinger, Claudia M; Jefferson, Carole; Ginnard, Janet; Pai, Madhukar

2015-04-01

The potential available market (PAM) for new diagnostics for tuberculosis that meet the specifications of the high-priority target product profiles (TPPs) is currently unknown. We estimated the PAM in 2020 in 4 high-burden countries (South Africa, Brazil, China, and India) for tests that meet the specifications outlined in the TPPs. The yearly PAM was estimated for the most likely application of each TPP. In 2020 the PAM for all 4 countries together was estimated to be (1) 12M tests/year with a value of 48M-71M USD for a sputum smear-replacement test; (2) 16M tests/year with a value of 65M-97M USD for a biomarker test; (3) 18M tests/year with a value of 18M-35M USD for a triage test; (4) 12M tests/year with a value of 59M-2238M USD for a tuberculosis detection plus drug susceptibility test (DST) all-in-one or 1.5M tests/year for a DST that follows a positive tuberculosis detection test with a corresponding value of 75M-121M for both tuberculosis detection and DST. Although there is a considerable potential market for novel tuberculosis diagnostics that fit the specification of the TPPs in the 4 high-burden countries, the actual market for an individual product remains uncertain. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

A rare complication of pulmonary tuberculosis: a case report.

PubMed

Kumarihamy, Kulatunga Wijekoon Mudiyanselage Pramitha Prabhashini; Ralapanawa, Dissanayake Mudiyanselage Priyantha Udaya Kumara; Jayalath, Widana Arachchilage Thilak Ananda

2015-02-10

Pulmonary tuberculosis remains an important public health problem globally and one of the most prevalent infectious diseases in Sri Lanka. It can cause a wide variety of complications but hematological manifestations are rare. According to our literature survey, this is the first reported case of the disease associated with deep vein thrombosis in Sri Lanka. A 37 year old Sri Lankan Sinhalese female presented with fever of one month's duration with productive cough and two weeks painless left lower limb swelling. Chest X-ray showed bilateral inflammatory shadows with a cavitatory lesion on the right apical region. A computed tomographic pulmonary angiography scan excluded pulmonary embolism. She had rising mycoplasma antibody titre (four fold). Acute deep vein thrombosis of the left lower limb was confirmed by venous duplex. Pulmonary tuberculosis was confirmed with positive culture for Mycobacterium tuberculosis. She was treated with clarythromycin, enoxaparin, warfarin and anti tuberculus drugs. It was difficult to maintain her International Normalizing Ratio in the therapeutic range due to drug interactions and poor compliance. At five months of presentation she died of massive pulmonary embolism. Our case emphasizes that patients with severe pulmonary tuberculosis are at risk of developing thromboembolism and superadded infections. It should be noted that even though starting anti tuberculosis drugs improved haemostatic disturbances, achieving the target International Normalizing Ratio was difficult due to drug interactions. Therefore these patients should be closely followed up to prevent complications and death from pulmonary embolism.

Pharmacokinetics and Dosing of Levofloxacin in Children Treated for Active or Latent Multidrug-Resistant Tuberculosis, Federated States of Micronesia and Republic of the Marshall Islands

PubMed Central

Mase, Sundari R.; Jereb, John A.; Gonzalez, Daniel; Martin, Fatma; Daley, Charles L.; Fred, Dorina; Loeffler, Ann; Menon, Lakshmy; Morris, Sapna Bamrah; Brostrom, Richard; Chorba, Terence; Peloquin, Charles A.

2016-01-01

Background In the Federated States of Micronesia (FSM) and then the Republic of the Marshall Islands (RMI), levofloxacin pharmacokinetics (PK) were studied in children receiving directly observed once-daily regimens (10 mg/kg, age >5 years; 15–20 mg/kg, age ≤5 years) for either multidrug-resistant tuberculosis (MDR TB) disease or latent infection after MDR TB exposure, to inform future dosing strategies. Methods Blood samples were collected at 0 (RMI only), 1, 2, and 6 hours (50 children, aged 6 months to 15 years) after oral levofloxacin at >6 weeks of treatment. Clinical characteristics and levofloxacin Cmax, elimination half-life (t1/2), and area under the curve from 0 to 24 hours (AUC0–24 hours * µg/mL) were correlated to determine optimal dosage and to examine associations. Population PK and target attainment were modeled. With results from FSM, dosages were increased in RMI toward the target maximal drug concentration (Cmax) for Mycobacterium tuberculosis, 8–12 µg/ml. Results Cmax correlated linearly with per-weight dosage. Neither Cmax nor t1/2 was associated with gender, age, body mass index, concurrent medications, or pre-dose meals. At levofloxacin dosage of 15–20 mg/kg, Cmax ≥ 8 µg/ml was observed, and modeling corroborated a high target attainment across the ratio of the area under the free-concentration-versus-time curve to minimum inhibitory concentration (fAUCss,0–24/MIC) values. Conclusions Levofloxacin dosage should be 15–20 mg/kg for Cmax ≥ 8 µg/ml and a high target attainment across fAUCss,0–24/MIC values in children ≥2 years of age. PMID:26658531

Genomic analysis of globally diverse Mycobacterium tuberculosis strains provides insights into the emergence and spread of multidrug resistance.

PubMed

Manson, Abigail L; Cohen, Keira A; Abeel, Thomas; Desjardins, Christopher A; Armstrong, Derek T; Barry, Clifton E; Brand, Jeannette; Chapman, Sinéad B; Cho, Sang-Nae; Gabrielian, Andrei; Gomez, James; Jodals, Andreea M; Joloba, Moses; Jureen, Pontus; Lee, Jong Seok; Malinga, Lesibana; Maiga, Mamoudou; Nordenberg, Dale; Noroc, Ecaterina; Romancenco, Elena; Salazar, Alex; Ssengooba, Willy; Velayati, A A; Winglee, Kathryn; Zalutskaya, Aksana; Via, Laura E; Cassell, Gail H; Dorman, Susan E; Ellner, Jerrold; Farnia, Parissa; Galagan, James E; Rosenthal, Alex; Crudu, Valeriu; Homorodean, Daniela; Hsueh, Po-Ren; Narayanan, Sujatha; Pym, Alexander S; Skrahina, Alena; Swaminathan, Soumya; Van der Walt, Martie; Alland, David; Bishai, William R; Cohen, Ted; Hoffner, Sven; Birren, Bruce W; Earl, Ashlee M

2017-03-01

Multidrug-resistant tuberculosis (MDR-TB), caused by drug-resistant strains of Mycobacterium tuberculosis, is an increasingly serious problem worldwide. Here we examined a data set of whole-genome sequences from 5,310 M. tuberculosis isolates from five continents. Despite the great diversity of these isolates with respect to geographical point of isolation, genetic background and drug resistance, the patterns for the emergence of drug resistance were conserved globally. We have identified harbinger mutations that often precede multidrug resistance. In particular, the katG mutation encoding p.Ser315Thr, which confers resistance to isoniazid, overwhelmingly arose before mutations that conferred rifampicin resistance across all of the lineages, geographical regions and time periods. Therefore, molecular diagnostics that include markers for rifampicin resistance alone will be insufficient to identify pre-MDR strains. Incorporating knowledge of polymorphisms that occur before the emergence of multidrug resistance, particularly katG p.Ser315Thr, into molecular diagnostics should enable targeted treatment of patients with pre-MDR-TB to prevent further development of MDR-TB.

In silico design of Mycobacterium tuberculosis epitope ensemble vaccines.

PubMed

Shah, Preksha; Mistry, Jaymisha; Reche, Pedro A; Gatherer, Derek; Flower, Darren R

2018-05-01

Effective control of Mycobacterium tuberculosis is a global necessity. In 2015, tuberculosis (TB) caused more deaths than HIV. Considering the increasing prevalence of multi-drug resistant forms of M. tuberculosis, the need for effective TB vaccines becomes imperative. Currently, the only licensed TB vaccine is Bacillus Calmette-Guérin (BCG). Yet, BCG has many drawbacks limiting its efficacy and applicability. We applied advanced computational procedures to derive a universal TB vaccine and one targeting East Africa. Our approach selects an optimal set of highly conserved, experimentally validated epitopes, with high projected population coverage (PPC). Through rigorous data analysis, five different potential vaccine combinations were selected each with PPC above 80% for East Africa and above 90% for the World. Two potential vaccines only contained CD8+ epitopes, while the others included both CD4+ and CD8+ epitopes. Our prime vaccine candidate was a putative seven-epitope ensemble comprising: SRGWSLIKSVRLGNA, KPRIITLTMNPALDI, AAHKGLMNIALAISA, FPAGGSTGSL, MLLAVTVSL, QSSFYSDW and KMRCGAPRY, with a 97.4% global PPC and a 92.7% East African PPC. Copyright © 2018 Elsevier Ltd. All rights reserved.

Mycobacterium tuberculosis Exploits a Molecular Off Switch of the Immune System for Intracellular Survival.

PubMed

von Both, Ulrich; Berk, Maurice; Agapow, Paul-Michael; Wright, Joseph D; Git, Anna; Hamilton, Melissa Shea; Goldgof, Greg; Siddiqui, Nazneen; Bellos, Evangelos; Wright, Victoria J; Coin, Lachlan J; Newton, Sandra M; Levin, Michael

2018-01-12

Mycobacterium tuberculosis (M. tuberculosis) survives and multiplies inside human macrophages by subversion of immune mechanisms. Although these immune evasion strategies are well characterised functionally, the underlying molecular mechanisms are poorly understood. Here we show that during infection of human whole blood with M. tuberculosis, host gene transcriptional suppression, rather than activation, is the predominant response. Spatial, temporal and functional characterisation of repressed genes revealed their involvement in pathogen sensing and phagocytosis, degradation within the phagolysosome and antigen processing and presentation. To identify mechanisms underlying suppression of multiple immune genes we undertook epigenetic analyses. We identified significantly differentially expressed microRNAs with known targets in suppressed genes. In addition, after searching regions upstream of the start of transcription of suppressed genes for common sequence motifs, we discovered novel enriched composite sequence patterns, which corresponded to Alu repeat elements, transposable elements known to have wide ranging influences on gene expression. Our findings suggest that to survive within infected cells, mycobacteria exploit a complex immune "molecular off switch" controlled by both microRNAs and Alu regulatory elements.

Exosomes derived from tumor cells genetically modified to express Mycobacterium tuberculosis antigen: a novel vaccine for cancer therapy.

PubMed

Koyama, Yoshiyuki; Ito, Tomoko; Hasegawa, Aya; Eriguchi, Masazumi; Inaba, Toshio; Ushigusa, Takahiro; Sugiura, Kikuya

2016-11-01

To examine the potential of exosomes derived from the tumor cells, which had been genetically modified to express a Mycobacterium tuberculosis antigen, as a cancer vaccine aimed at overcoming the weak immunogenicity of tumor antigens. We transfected B16 melanoma cells with a plasmid encoding the M. tuberculosis antigen, early secretory antigenic target-6 (ESAT-6). The secreted exosomes bearing both tumor-associated antigens and the pathogenic antigen (or their epitopes) were collected. When the exosomes were injected into foot pads of mice, they significantly (p < 0.05) evoked cellular immunity against both ESAT-6, and B16 tumor cells. Intra-tumoral injection of the exosomes significantly suppressed (p < 0.001) tumor growth in syngeneic B16 tumor-bearing mice, while the exosomes derived from the non-transfected B16 cells showed no effect on tumor growth, although both exosomes should have similar tumor antigens. Exosomes bearing both tumor antigens and the M. tuberculosis antigen (or their epitopes) have a high potential as a candidate for cancer vaccine to overcome the immune escape by tumor cells.

Tuberculosis control, and the where and why of artificial intelligence

PubMed Central

Falzon, Dennis; Thomas, Bruce V.; Temesgen, Zelalem; Sadasivan, Lal; Raviglione, Mario

2017-01-01

Countries aiming to reduce their tuberculosis (TB) burden by 2035 to the levels envisaged by the World Health Organization End TB Strategy need to innovate, with approaches such as digital health (electronic and mobile health) in support of patient care, surveillance, programme management, training and communication. Alongside the large-scale roll-out required for such interventions to make a significant impact, products must stay abreast of advancing technology over time. The integration of artificial intelligence into new software promises to make processes more effective and efficient, endowing them with a potential hitherto unimaginable. Users can benefit from artificial intelligence-enabled pattern recognition software for tasks ranging from reading radiographs to adverse event monitoring, sifting through vast datasets to personalise a patient's care plan or to customise training materials. Many experts forecast the imminent transformation of the delivery of healthcare services. We discuss how artificial intelligence and machine learning could revolutionise the management of TB. PMID:28656130

Tuberculosis control, and the where and why of artificial intelligence.

PubMed

Doshi, Riddhi; Falzon, Dennis; Thomas, Bruce V; Temesgen, Zelalem; Sadasivan, Lal; Migliori, Giovanni Battista; Raviglione, Mario

2017-04-01

Countries aiming to reduce their tuberculosis (TB) burden by 2035 to the levels envisaged by the World Health Organization End TB Strategy need to innovate, with approaches such as digital health (electronic and mobile health) in support of patient care, surveillance, programme management, training and communication. Alongside the large-scale roll-out required for such interventions to make a significant impact, products must stay abreast of advancing technology over time. The integration of artificial intelligence into new software promises to make processes more effective and efficient, endowing them with a potential hitherto unimaginable. Users can benefit from artificial intelligence-enabled pattern recognition software for tasks ranging from reading radiographs to adverse event monitoring, sifting through vast datasets to personalise a patient's care plan or to customise training materials. Many experts forecast the imminent transformation of the delivery of healthcare services. We discuss how artificial intelligence and machine learning could revolutionise the management of TB.

Real-Time Measurement of Host Bioenergetics During Mycobacterium Tuberculosis Infection

DTIC Science & Technology

2015-05-01

antimycobacterial drugs on Mtb bioenergetics. We focused on Clofazimine (CFZ, targets Complex I), Bedaquiline (BDQ/TMC207, targets Complex V) and Q203 (targets... Complex III). Firstly we investigated the effect of CFZ and BDQ on the OCR profiles of Mtb mc2 6230 (Figure 3). These experiments were done in...addition with of CFZ. The decrease in OCR is consistent with ETC complex inhibition. BDQ caused a very surprising concentration-depended increase

Investigation of the mycobacterial enzyme HsaD as a potential novel target for anti-tubercular agents using a fragment-based drug design approach.

PubMed

Ryan, Ali; Polycarpou, Elena; Lack, Nathan A; Evangelopoulos, Dimitrios; Sieg, Christian; Halman, Alice; Bhakta, Sanjib; Eleftheriadou, Olga; McHugh, Timothy D; Keany, Sebastian; Lowe, Edward D; Ballet, Romain; Abuhammad, Areej; Jacobs, William R; Ciulli, Alessio; Sim, Edith

2017-07-01

With the emergence of extensively drug-resistant tuberculosis, there is a need for new anti-tubercular drugs that work through novel mechanisms of action. The meta cleavage product hydrolase, HsaD, has been demonstrated to be critical for the survival of Mycobacterium tuberculosis in macrophages and is encoded in an operon involved in cholesterol catabolism, which is identical in M. tuberculosis and M. bovis BCG. We generated a mutant strain of M. bovis BCG with a deletion of hsaD and tested its growth on cholesterol. Using a fragment based approach, over 1000 compounds were screened by a combination of differential scanning fluorimetry, NMR spectroscopy and enzymatic assay with pure recombinant HsaD to identify potential inhibitors. We used enzymological and structural studies to investigate derivatives of the inhibitors identified and to test their effects on growth of M. bovis BCG and M. tuberculosis. The hsaD deleted strain was unable to grow on cholesterol as sole carbon source but did grow on glucose. Of seven chemically distinct 'hits' from the library, two chemical classes of fragments were found to bind in the vicinity of the active site of HsaD by X-ray crystallography. The compounds also inhibited growth of M. tuberculosis on cholesterol. The most potent inhibitor of HsaD was also found to be the best inhibitor of mycobacterial growth on cholesterol-supplemented minimal medium. We propose that HsaD is a novel therapeutic target, which should be fully exploited in order to design and discover new anti-tubercular drugs. This article is part of a themed section on Drug Metabolism and Antibiotic Resistance in Micro-organisms. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.14/issuetoc. © 2017 The British Pharmacological Society.

Mycobacterium tuberculosis multistage antigens confer comprehensive protection against pre- and post-exposure infections by driving Th1-type T cell immunity

PubMed Central

Fan, Xionglin; Yu, Qi; Jing, Yukai; Wang, Weihua; Li, Li; Zhou, Zijie

2016-01-01

There is an urgent need for a vaccine against tuberculosis (TB) that is more effective than the current sole licensed option. However, target antigens of Mycobacterium tuberculosis with the vaccine potential remain elusive. Five immunodominant antigens with characteristic expressions at the stages of primary infection (Ag85A), the regulation of nutrition and metabolism when transferring from rapid growth to latency (PhoY2 and Rv3407), latency (Rv2626c), and reactivation (RpfB) were selected to construct the fusion polyprotein WH121, which has better immunogenicity and protection than each multistage antigen. DMT adjuvanted WH121 vaccinated C57BL/6 mice could confer persistent and significant protection against the respiratory challenge with 80 CFU of virulent M. tuberculosis H37Rv at 9 and 18 weeks after immunization, as the BCG vaccine did. Moreover, WH121/DMT could boost the BCG primed mice against post-exposure infection, and more significantly inhibit the growth of M. tuberculosis in the spleen than BCG repeat vaccination. The protection elicited by WH121/DMT is attributed to the WH121-specific Th1-type biased immune responses, characterized by increased antigen-specific IgG2a/IgG1 ratio and high levels of IFN-γ secreted by the splenocytes of vaccinated mice. In particular, high levels of IFN-γ+ TEM cells in the spleen are an effective biomarker for the vaccine-induced early protection, and the persistent protection mainly depends on the increasing IL-2+IFN-γ+CD4+ and CD8+ T cells, especially IL-2+ TCM cells. These findings demonstrate that multistage-specific antigens might be promising targets for the next generation TB vaccine, and a combination of these antigens such as WH121/DMT is required for further preclinical evaluation. PMID:27566581

Rv2744c Is a PspA Ortholog That Regulates Lipid Droplet Homeostasis and Nonreplicating Persistence in Mycobacterium tuberculosis

PubMed Central

Armstrong, Richard M.; Adams, Katherine L.; Zilisch, Joseph E.; Bretl, Daniel J.; Sato, Hiromi; Anderson, David M.

2016-01-01

ABSTRACT Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), remains a significant cause of morbidity and mortality worldwide, despite the availability of a live attenuated vaccine and anti-TB antibiotics. The vast majority of individuals infected with M. tuberculosis develop an asymptomatic latent infection in which the bacterium survives within host-generated granulomatous lesions in a physiologically altered metabolic state of nonreplicating persistence. The granuloma represents an adverse environment, as M. tuberculosis is exposed to various stressors capable of disrupting the essential constituents of the bacterium. In Gram-negative and Gram-positive bacteria, resistance to cell envelope stressors that perturb the plasma membrane is mediated in part by proteins comprising the phage shock protein (Psp) system. PspA is an important component of the Psp system; in the presence of envelope stress, PspA localizes to the inner face of the plasma membrane, homo-oligomerizes to form a large scaffold-like complex, and helps maintain plasma membrane integrity to prevent a loss of proton motive force. M. tuberculosis and other members of the Mycobacterium genus are thought to encode a minimal functional unit of the Psp system, including an ortholog of PspA. Here, we show that Rv2744c possesses structural and physical characteristics that are consistent with its designation as a PspA family member. However, although Rv2744c is upregulated under conditions of cell envelope stress, loss of Rv2744c does not alter resistance to cell envelope stressors. Furthermore, Rv2744c localizes to the surface of lipid droplets in Mycobacterium spp. and regulates lipid droplet number, size, and M. tuberculosis persistence during anaerobically induced dormancy. Collectively, our results indicate that Rv2744c is a bona fide ortholog of PspA that may function in a novel role to regulate lipid droplet homeostasis and nonreplicating persistence (NRP) in M. tuberculosis. IMPORTANCE Mycobacterium tuberculosis is the causative agent of tuberculosis, a disease associated with significant morbidity and mortality worldwide. M. tuberculosis is capable of establishing lifelong asymptomatic infections in susceptible individuals and reactivating during periods of immune suppression to cause active disease. The determinants that are important for persistent infection of M. tuberculosis or for reactivation of this organism from latency are poorly understood. In this study, we describe our initial characterizations of Rv2744c, an ortholog of phage shock protein A (PspA) that regulates the homeostasis of lipid bodies and nonreplicating persistence in M. tuberculosis. This function of PspA in M. tuberculosis is novel and suggests that PspA may represent a unique bacterial target upon which to base therapeutic interventions against this organism. PMID:27002134

Long-Chain Fatty Acyl Coenzyme A Ligase FadD2 Mediates Intrinsic Pyrazinamide Resistance in Mycobacterium tuberculosis

PubMed Central

Rosen, Brandon C.; Dillon, Nicholas A.; Peterson, Nicholas D.; Minato, Yusuke

2016-01-01

ABSTRACT Pyrazinamide (PZA) is a first-line tuberculosis (TB) drug that has been in clinical use for 60 years yet still has an unresolved mechanism of action. Based upon the observation that the minimum concentration of PZA required to inhibit the growth of Mycobacterium tuberculosis is approximately 1,000-fold higher than that of other first-line drugs, we hypothesized that M. tuberculosis expresses factors that mediate intrinsic resistance to PZA. To identify genes associated with intrinsic PZA resistance, a library of transposon-mutagenized Mycobacterium bovis BCG strains was screened for strains showing hypersusceptibility to the active form of PZA, pyrazinoic acid (POA). Disruption of the long-chain fatty acyl coenzyme A (CoA) ligase FadD2 enhanced POA susceptibility by 16-fold on agar medium, and the wild-type level of susceptibility was restored upon expression of fadD2 from an integrating mycobacterial vector. Consistent with the recent observation that POA perturbs mycobacterial CoA metabolism, the fadD2 mutant strain was more vulnerable to POA-mediated CoA depletion than the wild-type strain. Ectopic expression of the M. tuberculosis pyrazinamidase PncA, necessary for conversion of PZA to POA, in the fadD2 transposon insertion mutant conferred at least a 16-fold increase in PZA susceptibility under active growth conditions in liquid culture at neutral pH. Importantly, deletion of fadD2 in M. tuberculosis strain H37Rv also resulted in enhanced susceptibility to POA. These results indicate that FadD2 is associated with intrinsic PZA and POA resistance and provide a proof of concept for the target-based potentiation of PZA activity in M. tuberculosis. PMID:27855077

Structure-based screening and molecular dynamics simulations offer novel natural compounds as potential inhibitors of Mycobacterium tuberculosis isocitrate lyase.

PubMed

Shukla, Rohit; Shukla, Harish; Sonkar, Amit; Pandey, Tripti; Tripathi, Timir

2018-06-01

Mycobacterium tuberculosis is the etiological agent of tuberculosis in humans and is responsible for more than two million deaths annually. M. tuberculosis isocitrate lyase (MtbICL) catalyzes the first step in the glyoxylate cycle, plays a pivotal role in the persistence of M. tuberculosis, which acts as a potential target for an anti-tubercular drug. To identify the potential anti-tuberculosis compound, we conducted a structure-based virtual screening of natural compounds from the ZINC database (n = 1,67,748) against the MtbICL structure. The ligands were docked against MtbICL in three sequential docking modes that resulted in 340 ligands having better docking score. These compounds were evaluated for Lipinski and ADMET prediction, and 27 compounds were found to fit well with re-docking studies. After refinement by molecular docking and drug-likeness analyses, three potential inhibitors (ZINC1306071, ZINC2111081, and ZINC2134917) were identified. These three ligands and the reference compounds were further subjected to molecular dynamics simulation and binding energy analyses to compare the dynamic structure of protein after ligand binding and the stability of the MtbICL and bound complexes. The binding free energy analyses were calculated to validate and capture the intermolecular interactions. The results suggested that the three compounds had a negative binding energy with -96.462, -143.549, and -122.526 kJ mol -1 for compounds with IDs ZINC1306071, ZINC2111081, and ZINC2134917, respectively. These lead compounds displayed substantial pharmacological and structural properties to be drug candidates. We concluded that ZINC2111081 has a great potential to inhibit MtbICL and would add to the drug discovery process against tuberculosis.

Comparative Analyses of Nonpathogenic, Opportunistic, and Totally Pathogenic Mycobacteria Reveal Genomic and Biochemical Variabilities and Highlight the Survival Attributes of Mycobacterium tuberculosis

PubMed Central

Singh, Yadvir; Kohli, Sakshi; Ahmad, Javeed; Ehtesham, Nasreen Z.; Tyagi, Anil K.

2014-01-01

ABSTRACT Mycobacterial evolution involves various processes, such as genome reduction, gene cooption, and critical gene acquisition. Our comparative genome size analysis of 44 mycobacterial genomes revealed that the nonpathogenic (NP) genomes were bigger than those of opportunistic (OP) or totally pathogenic (TP) mycobacteria, with the TP genomes being smaller yet variable in size—their genomic plasticity reflected their ability to evolve and survive under various environmental conditions. From the 44 mycobacterial species, 13 species, representing TP, OP, and NP, were selected for genomic-relatedness analyses. Analysis of homologous protein-coding genes shared between Mycobacterium indicus pranii (NP), Mycobacterium intracellulare ATCC 13950 (OP), and Mycobacterium tuberculosis H37Rv (TP) revealed that 4,995 (i.e., ~95%) M. indicaus pranii proteins have homology with M. intracellulare, whereas the homologies among M. indicus pranii, M. intracellulare ATCC 13950, and M. tuberculosis H37Rv were significantly lower. A total of 4,153 (~79%) M. indicus pranii proteins and 4,093 (~79%) M. intracellulare ATCC 13950 proteins exhibited homology with the M. tuberculosis H37Rv proteome, while 3,301 (~82%) and 3,295 (~82%) M. tuberculosis H37Rv proteins showed homology with M. indicus pranii and M. intracellulare ATCC 13950 proteomes, respectively. Comparative metabolic pathway analyses of TP/OP/NP mycobacteria showed enzymatic plasticity between M. indicus pranii (NP) and M. intracellulare ATCC 13950 (OP), Mycobacterium avium 104 (OP), and M. tuberculosis H37Rv (TP). Mycobacterium tuberculosis seems to have acquired novel alternate pathways with possible roles in metabolism, host-pathogen interactions, virulence, and intracellular survival, and by implication some of these could be potential drug targets. PMID:25370496

Towards an easy-to-use tuberculosis diagnosis through exhaled breath analysis: a liquid fluorimeter with an excitation at 265 nm

NASA Astrophysics Data System (ADS)

Hue, J.; Dupoy, M.; Vignoud, S.; Ricaud, J. L.; Tran-Thi, T.; Karpe, S.; Novelli-Rousseau, A.; Mallard, F.

2013-03-01

The struggle against tuberculosis is one of the World Health Organization priorities. Identifying in a short time, patients with active tuberculosis, would bring a tremendous improvement to the current situation. Recovering from this infectious and deadly disease (2 million of death per year) is possible with a correct diagnosis to give an appropriate treatment. Unfortunately, most common tuberculosis diagnoses have few drawbacks: - skin tests: not reliable at 100% and need an incubation of 2 days before the diagnosis, - blood tests: costly and sophisticated technology, - chest X-ray: the first step before the sputum tests used for a bacterial culture with a final diagnosis given within 2 weeks. A tuberculosis test based on exhaled breath analysis is a prospective and noninvasive solution, cheap and easy to use and to transport. This test lies on a fluoregenic detection of niacin, a well-known mycobacterium tuberculosis specific metabolite. In this paper, it is assumed that the selected probe is specific to niacin and that exhaled breath does not contain any interfering species. To address this problem, a fluorimeter is developed with a cheap and cooled CCD ( 2k$) as a sensor, to easily determine the suitable "fluorescent zone". In comparing aqueous solutions with and without niacin, 250 pM of niacin have been detected. With a commercial fluorimeter (Fluorolog from Horiba), only 200 nM of niacin are detected. The present detection remains 10 times above the estimated targeted value for a tuberculosis test. The excitation source is a LED, which typically emits 20 °W at 265 nm through an optical fiber. The emission signal is detected around 545 nm. A typical light exposure lasts 700 seconds. Analysis of biomarkers with a liquid fluorimeter is generic and promising as health diagnosis.

Enriching the annotation of Mycobacterium tuberculosis H37Rv proteome using remote homology detection approaches: insights into structure and function.

PubMed

Ramakrishnan, Gayatri; Ochoa-Montaño, Bernardo; Raghavender, Upadhyayula S; Mudgal, Richa; Joshi, Adwait G; Chandra, Nagasuma R; Sowdhamini, Ramanathan; Blundell, Tom L; Srinivasan, Narayanaswamy

2015-01-01

The availability of the genome sequence of Mycobacterium tuberculosis H37Rv has encouraged determination of large numbers of protein structures and detailed definition of the biological information encoded therein; yet, the functions of many proteins in M. tuberculosis remain unknown. The emergence of multidrug resistant strains makes it a priority to exploit recent advances in homology recognition and structure prediction to re-analyse its gene products. Here we report the structural and functional characterization of gene products encoded in the M. tuberculosis genome, with the help of sensitive profile-based remote homology search and fold recognition algorithms resulting in an enhanced annotation of the proteome where 95% of the M. tuberculosis proteins were identified wholly or partly with information on structure or function. New information includes association of 244 proteins with 205 domain families and a separate set of new association of folds to 64 proteins. Extending structural information across uncharacterized protein families represented in the M. tuberculosis proteome, by determining superfamily relationships between families of known and unknown structures, has contributed to an enhancement in the knowledge of structural content. In retrospect, such superfamily relationships have facilitated recognition of probable structure and/or function for several uncharacterized protein families, eventually aiding recognition of probable functions for homologous proteins corresponding to such families. Gene products unique to mycobacteria for which no functions could be identified are 183. Of these 18 were determined to be M. tuberculosis specific. Such pathogen-specific proteins are speculated to harbour virulence factors required for pathogenesis. A re-annotated proteome of M. tuberculosis, with greater completeness of annotated proteins and domain assigned regions, provides a valuable basis for experimental endeavours designed to obtain a better understanding of pathogenesis and to accelerate the process of drug target discovery. Copyright © 2014 Elsevier Ltd. All rights reserved.

[Tuberculosis: steady dynamics between past and present to imagine the future].

PubMed

Cabello C, Felipe

2011-07-01

Progress in understanding the biological processes that allow Mycobacterium tuberculosis to be a successful parasite have accelerated in the last twenty years. This progress has been stimulated by the return of tuberculosis (TB) as an important disease in industrialized countries, by its increase in emergent nations in the tail of population increases and poverty and by the spread of multiple drug resistant (MDR) and extensively drug resistant (XDR) M. tuberculosis as a result of treatment failures. Progress on M. tuberculosis biology has also been fueled by advances in microbiology and molecular biology, including molecular genetics, genomics, proteomics and in vitro and in vivo models of infection. The study of latency or dormancy, a phenomenon central to understanding the persistence of M. tuberculosis and the development of TB in individuals, its spread in human populations and the emergence of antibiotic-resistant/tolerant organisms, has been preferred targets for investigators in this area. In this manner, factors that trigger M. tuberculosis latency (e. g, hypoxia, nutrient starvation, NO exposure) have been characterized and the metabolic shifts to host lipid utilization, tolerance to antimicrobials and resistance to host immune mechanisms involved in latency have been determined. Similarly, genetic changes and the resulting antimicrobial mechanisms mediating the MDR and XDR states have been characterized and potential new vaccines that avoid reactivation from latency and infection are being developed. Despite this progress, and given the fact that effective anti tuberculosis therapy was developed and first introduced clinically at the end of the 1940s, there are now more cases of latent and active TB worldwide than ever before. This reinforces the concept of TB as a bacterial disease with strong social and economical! determinants which are presently stimulating increased transmission in many human groups, undermining diagnostics, treatment and prevention. It suggests that in a scenario of global economical crisis the struggle against TB will be weakened, unless efforts are included to alleviate poverty, decrease economic inequality, improve public health and allow democracy and political organization.

The dynamic of tuberculosis case finding in the era of the public–private mix strategy for tuberculosis control in Central Java, Indonesia

PubMed Central

Reviono, Reviono; Setianingsih, Wahyu; Damayanti, Kusmadewi Eka; Ekasari, Ratna

2017-01-01

ABSTRACT Background: The public–private mix (PPM) strategy has strengthened tuberculosis care and control in many countries. Indonesia, a country with a high tuberculosis burden, has a low tuberculosis case detection rate (CDR), despite PPM implementation in 2003. The PPM in Indonesia involves primary healthcare centers, hospitals, and specialized chest clinics. The long-term impact of the strategy is unknown. Objective: We aimed to explore the case detection achievements of the tuberculosis program since PPM implementation in Central Java in 2003. Methods: This retrospective cohort study covered the period 1 January 2000 to 31 December 2014. The data from tuberculosis patients treated in all health facilities in Central Java implementing directly observed treatment short-course, recorded via a standardized form, were analyzed after being validated by the Office of Health of Central Java Province. We evaluated the CDR, case notification rate, and total number of cases, using linear regression to analyze the temporal trends of those indicators in the phases of PPM implementation. Results: The CDR increased during the initial phase (2000–2005), decreased during the mid-phase (2006–2009), and increased slightly during the late phase (2010–2014), ranging from 13 to 61.72. These trends were observed despite a steady increase in the number of participating healthcare facilities. The regression analysis showed that the CDR of referral institutions contributed the most to the total CDR of Central Java Province. Many of the smear-negative tuberculosis cases recorded at primary healthcare centers may have been smear positive; this probable misclassification could have been partially avoided if more specific and sensitive diagnostic tools were available. Conclusions: The CDR remains below the national target (70%). Early awareness of a negative trend in certain program indicators is important to ensure program sustainability. Careful observation of the indicator pattern will secure the long-term success of the program. PMID:28766465

The dynamic of tuberculosis case finding in the era of the public-private mix strategy for tuberculosis control in Central Java, Indonesia.

PubMed

Reviono, Reviono; Setianingsih, Wahyu; Damayanti, Kusmadewi Eka; Ekasari, Ratna

2017-01-01

The public-private mix (PPM) strategy has strengthened tuberculosis care and control in many countries. Indonesia, a country with a high tuberculosis burden, has a low tuberculosis case detection rate (CDR), despite PPM implementation in 2003. The PPM in Indonesia involves primary healthcare centers, hospitals, and specialized chest clinics. The long-term impact of the strategy is unknown.  We aimed to explore the case detection achievements of the tuberculosis program since PPM implementation in Central Java in 2003. This retrospective cohort study covered the period 1 January 2000 to 31 December 2014. The data from tuberculosis patients treated in all health facilities in Central Java implementing directly observed treatment short-course, recorded via a standardized form, were analyzed after being validated by the Office of Health of Central Java Province. We evaluated the CDR, case notification rate, and total number of cases, using linear regression to analyze the temporal trends of those indicators in the phases of PPM implementation. The CDR increased during the initial phase (2000-2005), decreased during the mid-phase (2006-2009), and increased slightly during the late phase (2010-2014), ranging from 13 to 61.72. These trends were observed despite a steady increase in the number of participating healthcare facilities. The regression analysis showed that the CDR of referral institutions contributed the most to the total CDR of Central Java Province. Many of the smear-negative tuberculosis cases recorded at primary healthcare centers may have been smear positive; this probable misclassification could have been partially avoided if more specific and sensitive diagnostic tools were available. The CDR remains below the national target (70%). Early awareness of a negative trend in certain program indicators is important to ensure program sustainability. Careful observation of the indicator pattern will secure the long-term success of the program.

Target-Based Screen Against a Periplasmic Serine Protease That Regulates Intrabacterial pH Homeostasis in Mycobacterium tuberculosis

PubMed Central

2015-01-01

Mycobacterium tuberculosis (Mtb) maintains its intrabacterial pH (pHIB) near neutrality in the acidic environment of phagosomes within activated macrophages. A previously reported genetic screen revealed that Mtb loses this ability when the mycobacterial acid resistance protease (marP) gene is disrupted. In the present study, a high throughput screen (HTS) of compounds against the protease domain of MarP identified benzoxazinones as inhibitors of MarP. A potent benzoxazinone, BO43 (6-chloro-2-(2′-methylphenyl)-4H-1,3-benzoxazin-4-one), acylated MarP and lowered Mtb’s pHIB and survival during incubation at pH 4.5. BO43 had similar effects on MarP-deficient Mtb, suggesting the existence of additional target(s). Reaction of an alkynyl-benzoxazinone, BO43T, with Mycobacterium bovis variant bacille Calmette-Guérin (BCG) followed by click chemistry with azido-biotin identified both the MarP homologue and the high temperature requirement A1 (HtrA1) homologue, an essential protein. Thus, the chemical probe identified through a target-based screen not only reacted with its intended target in the intact cells but also implicated an additional enzyme that had eluded a genetic screen biased against essential genes. PMID:25457457

Drug Discovery & Development: State-of-the-Art and Future Directions” on the topic of “Targets”

PubMed Central

Cook, Gregory M.; Hards, Kiel; Dunn, Elyse; Heikal, Adam; Nakatani, Yoshio; Greening, Chris; Crick, Dean C.; Fontes, Fabio L.; Pethe, Kevin; Hasenoehrl, Erik; Berney, Michael

2017-01-01

Chapter summary The emergence and spread of drug-resistant pathogens, and our inability to develop new antimicrobials to combat resistance, has inspired scientists to seek out new targets for drug development. The Mycobacterium tuberculosis complex is a group of obligately aerobic bacteria that have specialized for inhabiting a wide range of intracellular and extracellular environments. Two fundamental features in this adaptation are the flexible utilization of energy sources and continued metabolism in the absence of growth. M. tuberculosis is an obligately aerobic heterotroph that depends on oxidative phosphorylation (OXPHOS) for growth and survival. However, several studies are redefining the metabolic breadth of the genus. Alternative electron donors and acceptors may provide the maintenance energy for the pathogen to maintain viability in hypoxic, nonreplicating states relevant to latent infection. This hidden metabolic flexibility may ultimately decrease the efficacy of drugs targeted against primary dehydrogenases and terminal oxidases. However, it may also open up opportunities to develop novel antimycobacterials targeting persister cells. In this review, we discuss the progress in understanding the role of energetic targets in mycobacterial physiology and pathogenesis, and the opportunities for drug discovery. PMID:28597820

The Mycobacterium tuberculosis cell-surface glycoprotein apa as a potential adhesin to colonize target cells via the innate immune system pulmonary C-type lectin surfactant protein A.

PubMed

Ragas, Aude; Roussel, Lucie; Puzo, Germain; Rivière, Michel

2007-02-23

Tuberculosis is still a major health problem, and understanding the mechanism by which Mycobacterium tuberculosis (Mtb) invades and colonizes its host target cells remains an important issue for the control of infection. The innate immune system C-type lectins (C-TLs), including the human pulmonary surfactant protein A (PSP-A), have been recently identified as determinant players in the early recognition of the invading pathogen and in mounting the host defense response. Although the antigenic lipoglycan mannosylated lipoarabinomannan is currently considered to be the major C-TL target on the mycobacterial surface, the recognition by some C-TLs of the only mycobacterial species composing the "Mtb complex" indicates that mannosylated lipoarabinomannan cannot account alone for this specificity. Thus, we searched for the mycobacterial molecules targeted by human PSP-A, focusing our attention on the Mtb surface glycoproteins. We developed an original functional proteomic approach based on a lectin blot assay using crude human bronchoalveolar lavage fluid as a source of physiological PSP-A. Combined with selective cell-surface protein extraction and mass spectrometry peptide mapping, this strategy allowed us to identify the Apa (alanine- and proline-rich antigenic) glycoprotein as new potential target for PSP-A. This result was supported by direct binding of PSP-A to purified Apa. Moreover, EDTA addition or deglycosylation of purified Apa samples completely abolished the interaction, demonstrating that the interaction is calcium- and mannose-dependent, as expected. Finally, we provide convincing evidence that Apa, formerly considered as mainly secreted, is associated with the cell wall for a sufficiently long time to aid in the attachment of PSP-A. Because, to date, Apa seems to be restricted to the Mtb complex strains, we propose that it may account for the selective recognition of those strains by PSP-A and other immune system C-TLs containing homologous functional domains.

Interventions to increase tuberculosis case detection at primary healthcare or community-level services

PubMed Central

Mhimbira, Francis A; Cuevas, Luis E.; Dacombe, Russell; Mkopi, Abdallah; Sinclair, David

2017-01-01

Background Pulmonary tuberculosis is usually diagnosed when symptomatic individuals seek care at healthcare facilities, and healthcare workers have a minimal role in promoting the health-seeking behaviour. However, some policy specialists believe the healthcare system could be more active in tuberculosis diagnosis to increase tuberculosis case detection. Objectives To evaluate the effectiveness of different strategies to increase tuberculosis case detection through improving access (geographical, financial, educational) to tuberculosis diagnosis at primary healthcare or community-level services. Search methods We searched the following databases for relevant studies up to 19 December 2016: the Cochrane Infectious Disease Group Specialized Register; the Cochrane Central Register of Controlled Trials (CENTRAL), published in the Cochrane Library, Issue 12, 2016; MEDLINE; Embase; Science Citation Index Expanded, Social Sciences Citation Index; BIOSIS Previews; and Scopus. We also searched the World Health Organization International Clinical Trials Registry Platform (WHO ICTRP), ClinicalTrials.gov, and the metaRegister of Controlled Trials (mRCT) for ongoing trials. Selection criteria Randomized and non-randomized controlled studies comparing any intervention that aims to improve access to a tuberculosis diagnosis, with no intervention or an alternative intervention. Data collection and analysis Two review authors independently assessed trials for eligibility and risk of bias, and extracted data. We compared interventions using risk ratios (RR) and 95% confidence intervals (CI). We assessed the certainty of the evidence using the GRADE approach. Main results We included nine cluster-randomized trials, one individual randomized trial, and seven non-randomized controlled studies. Nine studies were conducted in sub-Saharan Africa (Ethiopia, Nigeria, South Africa, Zambia, and Zimbabwe), six in Asia (Bangladesh, Cambodia, India, Nepal, and Pakistan), and two in South America (Brazil and Colombia); which are all high tuberculosis prevalence areas. Tuberculosis outreach screening, using house-to-house visits, sometimes combined with printed information about going to clinic, may increase tuberculosis case detection (RR 1.24, 95% CI 0.86 to 1.79; 4 trials, 6,458,591 participants in 297 clusters, low-certainty evidence); and probably increases case detection in areas with tuberculosis prevalence of 5% or more (RR 1.52, 95% CI 1.10 to 2.09; 3 trials, 155,918 participants, moderate-certainty evidence; prespecified stratified analysis). These interventions may lower the early default (prior to starting treatment) or default during treatment (RR 0.67, 95% CI 0.47 to 0.96; 3 trials, 849 participants, low-certainty evidence). However, this intervention may have may have little or no effect on treatment success (RR 1.07, 95% CI 1.00 to 1.15; 3 trials, 849 participants, low-certainty evidence), and we do not know if there is an effect on treatment failure or mortality. One study investigated long-term prevalence in the community, but with no clear effect due to imprecision and differences in care between the two groups (RR 1.14, 95% CI 0.65 to 2.00; 1 trial, 556,836 participants, very low-certainty evidence). Four studies examined health promotion activities to encourage people to attend for screening, including mass media strategies and more locally organized activities. There was some increase, but this could have been related to temporal trends, with no corresponding increase in case notifications, and no evidence of an effect on long-term tuberculosis prevalence. Two studies examined the effects of two to six nurse practitioner educational sessions in tuberculosis diagnosis, with no clear effect on tuberculosis cases detected. One trial compared mobile clinics every five days with house-to-house screening every six months, and showed an increase in tuberculosis cases. There was also insufficient evidence to determine if sustained improvements in case detection impact on long-term tuberculosis prevalence; this was evaluated in one study, which indicated little or no effect after four years of either contact tracing, extensive health promotion activities, or both (RR 1.31, 95% CI 0.75 to 2.30; 1 study, 405,788 participants in 12 clusters, very low-certainty evidence). Authors' conclusions The available evidence demonstrates that when used in appropriate settings, active case-finding approaches may result in increase in tuberculosis case detection in the short term. The effect of active case finding on treatment outcome needs to be further evaluated in sufficiently powered studies. Interventions to increase the number of tuberculosis cases being diagnosed This review summarized trials evaluating the effects of interventions aiming to increase the diagnosis of tuberculosis and reduce the number of undiagnosed tuberculosis cases in communities. After searching for relevant trials up to 19 December 2016, we included 17 studies conducted in sub-Saharan Africa (nine studies), Asia (six studies), and South America (two studies). Why does tuberculosis go undiagnosed and how might programmes improve this? Tuberculosis is a chronic infectious disease that affects over 10 million people worldwide, with an estimated four million tuberculosis patients remaining undiagnosed each year. Interventions such as outreach tuberculosis screening with or without health promotion that actively screen for tuberculosis among individuals presenting with symptoms of tuberculosis, may increase detection of microbiologically confirmed tuberculosis cases. These interventions may improve treatment outcomes by increasing the number of tuberculosis patients who are cured and complete treatment. However, we do not know if these interventions reduce either tuberculosis treatment failure, or tuberculosis-associated death or long-term tuberculosis burden in moderate- and high-tuberculosis settings. What the research says House-to-house screening for active tuberculosis, and organizing tuberculosis diagnostic clinics nearer to where people live and work, may increase tuberculosis case detection in settings where the prevalence of undiagnosed disease is high (low-certainty evidence). These people may have higher levels of treatment success and lower levels of default from treatment (low-certainty evidence). There was insufficient evidence to determine if health promotion activities alone increase tuberculosis case detection (very low-certainty evidence). There was also insufficient evidence to determine if sustained improvements in case detection impact on long-term tuberculosis prevalence, as the only study to evaluate this found no effect after four years (very low-certainty evidence). PMID:29182800

Active Site Flexibility of Mycobacterium tuberculosis Isocitrate Lyase in Dimer Form.

PubMed

Lee, Yie-Vern; Choi, Sy Bing; Wahab, Habibah A; Choong, Yee Siew

2017-09-25

Tuberculosis (TB) still remains a global threat due to the emergence of a drug-resistant strain. Instead of focusing on the drug target of active stage TB, we are highlighting the isocitrate lyase (ICL) at the dormant stage TB. ICL is one of the persistent factors for Mycobacterium tuberculosis (MTB) to survive during the dormant phase. In addition, the absence of ICL in human has made ICL a potential drug target for TB therapy. However, the dynamic details of ICL which could give insights to the ICL-ligand interaction have yet to be solved. Therefore, a series of ICL dimer dynamics studies through molecular dynamics simulation were performed in this work. The ICL active site entrance gate closure is contributed to by hydrogen bonding and electrostatic interactions with the C-terminal. Analysis suggested that the open-closed behavior of the ICL active site entrance depends on the type of ligand present in the active site. We also observed four residues (Ser91, Asp108, Asp153, and Cys191) which could possibly be the nucleophiles for nucleophilic attack on the cleavage of isocitrate at the C 2 -C 3 bond. We hope that the elucidation of ICL dynamics can benefit future works such as lead identification or antibody design against ICL for TB therapeutics.

Toward Mycobacterium tuberculosis DXR inhibitor design: homology modeling and molecular dynamics simulations

NASA Astrophysics Data System (ADS)

Singh, Nidhi; Avery, Mitchell A.; McCurdy, Christopher R.

2007-09-01

Mycobacterium tuberculosis 1-deoxy- d-xylulose-5-phosphate reductoisomerase ( MtDXR) is a potential target for antitubercular chemotherapy. In the absence of its crystallographic structure, our aim was to develop a structural model of MtDXR. This will allow us to gain early insight into the structure and function of the enzyme and its likely binding to ligands and cofactors and thus, facilitate structure-based inhibitor design. To achieve this goal, initial models of MtDXR were generated using MODELER. The best quality model was refined using a series of minimizations and molecular dynamics simulations. A protein-ligand complex was also developed from the initial homology model of the target protein by including information about the known ligand as spatial restraints and optimizing the mutual interactions between the ligand and the binding site. The final model was evaluated on the basis of its ability to explain several site-directed mutagenesis data. Furthermore, a comparison of the homology model with the X-ray structure published in the final stages of the project shows excellent agreement and validates the approach. The knowledge gained from the current study should prove useful in the design and development of inhibitors as potential novel therapeutic agents against tuberculosis by either de novo drug design or virtual screening of large chemical databases.

A review of the use of ethionamide and prothionamide in childhood tuberculosis.

PubMed

Thee, S; Garcia-Prats, A J; Donald, P R; Hesseling, A C; Schaaf, H S

2016-03-01

Ethionamide (ETH) and prothionamide (PTH), both thioamides, have proven efficacy in clinical studies and form important components for multidrug-resistant tuberculosis treatment regimens and for treatment of tuberculous meningitis in adults and children. ETH and PTH are pro-drugs that, following enzymatic activation by mycobacterial EthA inhibit InhA, a target shared with isoniazid (INH), and subsequently inhibit mycolic acid synthesis of Mycobacterium tuberculosis. Co-resistance to INH and ETH is conferred by mutations in the mycobacterial inhA promoter region; mutations in the ethA gene often underlie ETH and PTH monoresistance. An oral daily dose of ETH or PTH of 15-20 mg/kg with a maximum daily dose of 1000 mg is recommended in children to achieve adult-equivalent serum concentrations shown to be efficacious in adults, although information on optimal pharmacodynamic targets is still lacking. Gastrointestinal disturbances, and hypothyroidism during long-term therapy, are frequent adverse effects observed in adults and children, but are rarely life-threatening and seldom necessitate cessation of ETH therapy. More thorough investigation of the therapeutic effects and toxicity of ETH and PTH is needed in childhood TB while child-friendly formulations are needed to appropriately dose children. Copyright © 2015 Elsevier Ltd. All rights reserved.

Structural investigation of inhibitor designs targeting 3-dehydroquinate dehydratase from the shikimate pathway of Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dias, Marcio V.B.; Snee, William C.; Bromfield, Karen M.

The shikimate pathway is essential in Mycobacterium tuberculosis and its absence from humans makes the enzymes of this pathway potential drug targets. In the present paper, we provide structural insights into ligand and inhibitor binding to 3-dehydroquinate dehydratase (dehydroquinase) from M. tuberculosis (MtDHQase), the third enzyme of the shikimate pathway. The enzyme has been crystallized in complex with its reaction product, 3-dehydroshikimate, and with six different competitive inhibitors. The inhibitor 2,3-anhydroquinate mimics the flattened enol/enolate reaction intermediate and serves as an anchor molecule for four of the inhibitors investigated. MtDHQase also forms a complex with citrazinic acid, a planar analoguemore » of the reaction product. The structure of MtDHQase in complex with a 2,3-anhydroquinate moiety attached to a biaryl group shows that this group extends to an active-site subpocket inducing significant structural rearrangement. The flexible extensions of inhibitors designed to form {pi}-stacking interactions with the catalytic Tyr{sup 24} have been investigated. The high-resolution crystal structures of the MtDHQase complexes provide structural evidence for the role of the loop residues 19-24 in MtDHQase ligand binding and catalytic mechanism and provide a rationale for the design and efficacy of inhibitors.« less

Use of NMR Metabolomics to Analyze the Targets of D-cycloserine in Mycobacteria: Role of D-Alanine Racemase

PubMed Central

Halouska, Steven; Chacon, Ofelia; Fenton, Robert J.; Zinniel, Denise K.; Barletta, Raul G.; Powers, Robert

2008-01-01

D-cycloserine (DCS) is only used with multi-drug resistant strains of tuberculosis because of serious side-effects. DCS is known to inhibit cell wall biosynthesis, but the in vivo lethal target is still unknown. We have applied NMR-based metabolomics combined with principal component analysis to monitor the in vivo affect of DCS on M. smegmatis. Our analysis suggests DCS functions by inhibiting multiple protein targets. PMID:17979227

First Insights into the Phylogenetic Diversity of Mycobacterium tuberculosis in Nepal

PubMed Central

Malla, Bijaya; Stucki, David; Borrell, Sonia; Feldmann, Julia; Maharjan, Bhagwan; Shrestha, Bhawana

2012-01-01

Background Tuberculosis (TB) is a major public health problem in Nepal. Strain variation in Mycobacterium tuberculosis may influence the outcome of TB infection and disease. To date, the phylogenetic diversity of M. tuberculosis in Nepal is unknown. Methods and Findings We analyzed 261 M. tuberculosis isolates recovered from pulmonary TB patients recruited between August 2009 and August 2010 in Nepal. M. tuberculosis lineages were determined by single nucleotide polymorphisms (SNP) typing and spoligotyping. Drug resistance was determined by sequencing the hot spot regions of the relevant target genes. Overall, 164 (62.8%) TB patients were new, and 97 (37.2%) were previously treated. Any drug resistance was detected in 50 (19.2%) isolates, and 16 (6.1%) were multidrug-resistant. The most frequent M. tuberculosis lineage was Lineage 3 (CAS/Delhi) with 106 isolates (40.6%), followed by Lineage 2 (East-Asian lineage, includes Beijing genotype) with 84 isolates (32.2%), Lineage 4 (Euro-American lineage) with 41 (15.7%) isolates, and Lineage 1 (Indo-Oceanic lineage) with 30 isolates (11.5%). Based on spoligotyping, we found 45 different spoligotyping patterns that were previously described. The Beijing (83 isolates, 31.8%) and CAS spoligotype (52, 19.9%) were the dominant spoligotypes. A total of 36 (13.8%) isolates could not be assigned to any known spoligotyping pattern. Lineage 2 was associated with female sex (adjusted odds ratio [aOR] 2.58, 95% confidence interval [95% CI] 1.42–4.67, p = 0.002), and any drug resistance (aOR 2.79; 95% CI 1.43–5.45; p = 0.002). We found no evidence for an association of Lineage 2 with age or BCG vaccination status. Conclusions We found a large genetic diversity of M. tuberculosis in Nepal with representation of all four major lineages. Lineages 3 and 2 were dominating. Lineage 2 was associated with clinical characteristics. This study fills an important gap on the map of the M. tuberculosis genetic diversity in the Asian region. PMID:23300635

Future vaccination strategies against tuberculosis: thinking outside the box.

PubMed

Kaufmann, Stefan H E

2010-10-29

With almost a dozen vaccine candidates in clinical trials, tuberculosis (TB) research and development is finally reaping the first fruits of its labors. Vaccine candidates in clinical trials may prevent TB disease reactivation by efficiently containing the pathogen Mycobacterium tuberculosis (Mtb). Future research should target vaccines that achieve sterile eradication of Mtb or even prevent stable infection. These are ambitious goals that can be reached only by highly cooperative engagement of basic immunologists, vaccinologists, and clinical researchers--or in other words, by translation from basic immunology to vaccine research and development, as well as reverse translation of insights from clinical trials back to hypothesis-driven research in the basic laboratory. Here, we review current and future strategies toward the rational design of novel vaccines against TB, as well as the progress made thus far, and the hurdles that need to be overcome in the near and distant future. Copyright © 2010 Elsevier Inc. All rights reserved.

Lassomycin, a ribosomally synthesized cyclic peptide, kills Mycobacterium tuberculosis by targeting the ATP-dependent protease ClpC1P1P2

PubMed Central

Gavrish, Ekaterina; Sit, Clarissa S.; Cao, Shugeng; Kandror, Olga; Spoering, Amy; Peoples, Aaron; Ling, Losee; Fetterman, Ashley; Hughes, Dallas; Bissell, Anthony; Torrey, Heather; Akopian, Tatos; Mueller, Andreas; Epstein, Slava; Goldberg, Alfred; Clardy, Jon; Lewis, Kim

2014-01-01

Summary Languishing antibiotic discovery and flourishing antibiotic resistance have prompted development of alternative untapped sources for antibiotic discovery, including previously uncultured bacteria. Here, we screen extracts from uncultured species against M. tuberculosis and identify lassomycin, an antibiotic that exhibits potent bactericidal activity against both growing and dormant mycobacteria, including drug-resistant forms of M. tuberculosis, but little activity against other bacteria or mammalian cells. Lassomycin is a highly basic, ribosomally-encoded cyclic peptide with an unusual structural fold that only partially resembles that of other lasso peptides. We show that lassomycin binds to a highly acidic region of the ClpC1 ATPase complex and markedly stimulates its ATPase activity without stimulating ClpP1P2 catalyzed protein breakdown, which is essential for viability of mycobacteria. This mechanism, uncoupling ATPase from proteolytic activity, accounts for lassomycin's bacteriocidal activity. PMID:24684906

Bacterial cytoskeleton and implications for new antibiotic targets.

PubMed

Wang, Huan; Xie, Longxiang; Luo, Hongping; Xie, Jianping

2016-01-01

Traditionally eukaryotes exclusive cytoskeleton has been found in bacteria and other prokaryotes. FtsZ, MreB and CreS are bacterial counterpart of eukaryotic tubulin, actin filaments and intermediate filaments, respectively. FtsZ can assemble to a Z-ring at the cell division site, regulate bacterial cell division; MreB can form helical structure, and involve in maintaining cell shape, regulating chromosome segregation; CreS, found in Caulobacter crescentus (C. crescentus), can form curve or helical filaments in intracellular membrane. CreS is crucial for cell morphology maintenance. There are also some prokaryotic unique cytoskeleton components playing crucial roles in cell division, chromosome segregation and cell morphology. The cytoskeleton components of Mycobacterium tuberculosis (M. tuberculosis), together with their dynamics during exposure to antibiotics are summarized in this article to provide insights into the unique organization of this formidable pathogen and druggable targets for new antibiotics.

R&D in Vaccines Targeting Neglected Diseases: An Exploratory Case Study Considering Funding for Preventive Tuberculosis Vaccine Development from 2007 to 2014

PubMed Central

Costa Barbosa Bessa, Theolis; Santos de Aragão, Erika; Medeiros Guimarães, Jane Mary

2017-01-01

Based on an exploratory case study regarding the types of institutions funding the research and development to obtain new tuberculosis vaccines, this article intends to provoke discussion regarding the provision of new vaccines targeting neglected disease. Although our findings and discussion are mainly relevant to the case presented here, some aspects are more generally applicable, especially regarding the dynamics of development in vaccines to prevent neglected diseases. Taking into account the dynamics of innovation currently seen at work in the vaccine sector, a highly concentrated market dominated by few multinational pharmaceutical companies, we feel that global PDP models can play an important role throughout the vaccine development cycle. In addition, the authors call attention to issues surrounding the coordination of actors and resources in the research, development, manufacturing, and distribution processes of vaccine products arising from PDP involvement. PMID:28133608

Non-Steroidal Anti-inflammatory Drugs As Host-Directed Therapy for Tuberculosis: A Systematic Review

PubMed Central

Kroesen, Vera M.; Gröschel, Matthias I.; Martinson, Neil; Zumla, Alimuddin; Maeurer, Markus; van der Werf, Tjip S.; Vilaplana, Cristina

2017-01-01

Lengthy, antimicrobial therapy targeting the pathogen is the mainstay of conventional tuberculosis treatment, complicated by emerging drug resistances. Host-directed therapies, including non-steroidal anti-inflammatory drugs (NSAIDs), in contrast, target host factors to mitigate disease severity. In the present Systematic Review, we investigate whether NSAIDs display any effects as therapy of TB and discuss possible mechanisms of action of NSAIDs as adjunctive therapy of TB. Ten studies, seven preclinical studies in mice and three clinical trials, were included and systematically reviewed. Our results point toward a beneficial effect of NSAIDs as adjunct to current TB therapy regimens, mediated by decreased lung pathology balancing host-immune reaction. The determination of the best timing for their administration in order to obtain the potential beneficial effects needs further investigation. Even if the preclinical evidence requires clinical evaluation, NSAIDs might represent a potential safe, simple, and cheap improvement in therapy of TB. PMID:28713389

Tuberculosis of hip in children: A retrospective analysis

PubMed Central

Moon, Myung-Sang; Kim, Sung-Soo; Lee, Sung-Rak; Moon, Young-Wan; Moon, Jeong-Lim; Moon, Seog-In

2012-01-01

Background: Tuberculosis (TB) of hip constitutes nearly 15% of all cases of osteoarticular tuberculosis. We report a retrospective study carried out on 43 children with hip TB. Materials and Methods: Forty-three children of TB hip treated between 1971 and 2000 were analysed. Twenty-four children of the early series were treated with streptomycin (S), isoniazid (H) and PAS (Pa) for 18 months (3HPaS, 15 HPa), while 19 children in the later series were treated with isoniazid (H), rifampicin (R) and ethambutol (E) or pyrazinamide (Z) for 12 months [(12 RHE(Z)]. Five out of 18 children with radiologically normal appearing type hip TB were treated with chemotherapy alone and 38 children were subjected to surgery; simple synovectomy alone in 31 hips, joint debridement in six hips, and proximal femoral varisation osteotomy in one. After surgery hips were immobilized in cast for one to three months according to the severity of the disease and patients pain tolerance, and then were mobilized under leg traction in bed gradually till pain subsided completely. Results: TB of hip healed with minimum sequelae in all children. In 18 Type one hip TB, normal hip (synovial form) anatomy was maintained, and in 25 patients with advanced lesions some defect in the femoral head and acetabulum was noticed, though painless good hip motion was maintained. Excellent to good results were obtained in 31 children (73.1%), fair in eight (18.6%), and poor in four (9.3%). In four patients with poor results, there was some residual morphological defect in the hip. None developed ankylosis of hip. Conclusion: We achieved good outcome with minimum sequelae in this series. The management goal should be aimed not only to heal the disease but also to maintain a painless mobile hip and anatomical cephalocotyloid relationship until maturity, and retard the development of secondary osteoarthritis. PMID:22448058

76 FR 39885 - Risk-Based Targeting of Foreign Flagged Mobile Offshore Drilling Units (MODUs)

Federal Register 2010, 2011, 2012, 2013, 2014

2011-07-07

... Foreign Flagged Mobile Offshore Drilling Units (MODUs) AGENCY: Coast Guard, DHS. ACTION: Notice of... 11-06, Risk-Based Targeting of Foreign Flagged Mobile Offshore Drilling Units (MODUs). This policy... applicable regulations, every foreign-flagged mobile offshore drilling unit (MODU) must undergo a Coast Guard...

Insights into Integrated Lead Generation and Target Identification in Malaria and Tuberculosis Drug Discovery

PubMed Central

2017-01-01

Conspectus New, safe and effective drugs are urgently needed to treat and control malaria and tuberculosis, which affect millions of people annually. However, financial return on investment in the poor settings where these diseases are mostly prevalent is very minimal to support market-driven drug discovery and development. Moreover, the imminent loss of therapeutic lifespan of existing therapies due to evolution and spread of drug resistance further compounds the urgency to identify novel effective drugs. However, the advent of new public–private partnerships focused on tropical diseases and the recent release of large data sets by pharmaceutical companies on antimalarial and antituberculosis compounds derived from phenotypic whole cell high throughput screening have spurred renewed interest and opened new frontiers in malaria and tuberculosis drug discovery. This Account recaps the existing challenges facing antimalarial and antituberculosis drug discovery, including limitations associated with experimental animal models as well as biological complexities intrinsic to the causative pathogens. We enlist various highlights from a body of work within our research group aimed at identifying and characterizing new chemical leads, and navigating these challenges to contribute toward the global drug discovery and development pipeline in malaria and tuberculosis. We describe a catalogue of in-house efforts toward deriving safe and efficacious preclinical drug development candidates via cell-based medicinal chemistry optimization of phenotypic whole-cell medium and high throughput screening hits sourced from various small molecule chemical libraries. We also provide an appraisal of target-based screening, as invoked in our laboratory for mechanistic evaluation of the hits generated, with particular focus on the enzymes within the de novo pyrimidine biosynthetic and hemoglobin degradation pathways, the latter constituting a heme detoxification process and an associated cysteine protease-mediated hydrolysis of hemoglobin. We further expound on the recombinant enzyme assays, heme fractionation experiments, and genomic and chemoproteomic methods that we employed to identify Plasmodium falciparum falcipain 2 (PfFP2), hemozoin formation, phosphatidylinositol 4-kinase (PfPI4K) and Mycobacterium tuberculosis cytochrome bc1 complex as the targets of the antimalarial chalcones, pyrido[1,2-a]benzimidazoles, aminopyridines, and antimycobacterial pyrrolo[3,4-c]pyridine-1,3(2H)-diones, respectively. In conclusion, we argue for the expansion of chemical space through exploitation of privileged natural product scaffolds and diversity-oriented synthesis, as well as the broadening of druggable spaces by exploiting available protein crystal structures, -omics data, and bioinformatics infrastructure to explore hitherto untargeted spaces like lipid metabolism and protein kinases in P. falciparum. Finally, we audit the merits of both target-based and whole-cell phenotypic screening in steering antimalarial and antituberculosis chemical matter toward populating drug discovery pipelines with new lead molecules. PMID:28636311

A nucleic acid strand displacement system for the multiplexed detection of tuberculosis-specific mRNA using quantum dots

NASA Astrophysics Data System (ADS)

Gliddon, H. D.; Howes, P. D.; Kaforou, M.; Levin, M.; Stevens, M. M.

2016-05-01

The development of rapid, robust and high performance point-of-care diagnostics relies on the advancement and combination of various areas of research. We have developed an assay for the detection of multiple mRNA molecules that combines DNA nanotechnology with fluorescent nanomaterials. The core switching mechanism is toehold-mediated strand displacement. We have used fluorescent quantum dots (QDs) as signal transducers in this assay, as they bring many benefits including bright fluorescence and multiplexing abilities. The resulting assay is capable of multiplexed detection of long RNA targets against a high concentration of background non-target RNA, with high sensitivity and specificity and limits of detection in the nanomolar range using only a standard laboratory plate reader. We demonstrate the utility of our QD-based system for the detection of two genes selected from a microarray-derived tuberculosis-specific gene expression signature. Levels of up- and downregulated gene transcripts comprising this signature can be combined to give a disease risk score, making the signature more amenable for use as a diagnostic marker. Our QD-based approach to detect these transcripts could pave the way for novel diagnostic assays for tuberculosis.The development of rapid, robust and high performance point-of-care diagnostics relies on the advancement and combination of various areas of research. We have developed an assay for the detection of multiple mRNA molecules that combines DNA nanotechnology with fluorescent nanomaterials. The core switching mechanism is toehold-mediated strand displacement. We have used fluorescent quantum dots (QDs) as signal transducers in this assay, as they bring many benefits including bright fluorescence and multiplexing abilities. The resulting assay is capable of multiplexed detection of long RNA targets against a high concentration of background non-target RNA, with high sensitivity and specificity and limits of detection in the nanomolar range using only a standard laboratory plate reader. We demonstrate the utility of our QD-based system for the detection of two genes selected from a microarray-derived tuberculosis-specific gene expression signature. Levels of up- and downregulated gene transcripts comprising this signature can be combined to give a disease risk score, making the signature more amenable for use as a diagnostic marker. Our QD-based approach to detect these transcripts could pave the way for novel diagnostic assays for tuberculosis. Electronic supplementary information (ESI) available: Base pair mismatch tuning of CProbes. Binding capacity of the QDs. Theoretical limit of detection (LOD) for the monoplex systems. Kinetics of strand displacement. Kinetics of QProbe-CProbe binding. LOD and saturation point calculations. See DOI: 10.1039/c6nr00484a

Tuberculosis therapeutics: Engineering of nanomedicinal systems for local delivery of targeted drug cocktails

NASA Astrophysics Data System (ADS)

D'Addio, Suzanne M.

In this thesis, a multifunctional nanocarrier drug delivery system was investigated and optimized to improve tuberculosis therapy by promoting the intracellular delivery of high payloads of antibiotics. To meet the needs of a patient population which continues to grow by close to 10 million people a year, innovative therapeutics must be formulated by robust and scalable processes. We use Flash NanoPrecipitation for the continuous precipitation of nanocarriers by block copolymer directed assembly, which enables the development of nanocarriers with tunable properties. Stable nanocarriers of Rifampicin and a hydrophobic Rifampicin prodrug have efficacy against tuberculosis in vitro that is equivalent to the soluble Rifampicin. To overcome poor in vivo efficacy of the recently discovered antitubercular drug SQ641, we co-encapsulate SQ641 and Cyclosporine A in a stable aqueous nanocarrier suspension, which enables drug administration and also enhances intracellular accumulation and antitubercular efficacy relative to SQ641 in solution. Since the mannose receptor is involved in the phagocytosis of tuberculosis bacilli, we modify the surface of nanocarriers with mannoside residues to target specific intracellular accumulation in macrophages. The surface density of mannoside terminated polyethylene glycol chains was controlled between 0 and 75% and in vitro cellular association reveals a 9% surface density is optimal for internalization mediated by the mannose receptor. We explore the preparation of large, porous aerosol carrier particles of with tunable deposition characteristics by spray freeze drying with ultrasonic atomization for direct dosing to the lungs. Nanocarriers are loaded at 3 - 50 wt% in mannitol particles with constant size, limited nanocarrier aggregation, and 63% dose delivered to the lungs, as determined by in vitro cascade impaction. There has been a lag in the development of new technologies to facilitate development and commercialization of therapeutic nanocarrier formulations. We present three translational technologies. (1) The intrinsic dissolution rates of drug nanocarriers are determined using a novel assay, based on high surface area lipid sink particles and magnetic separations, to improve in vitro/in vivo correlations. (2) The nanocarrier interaction with whole serum and the polymer surface conformation are correlated to in vivo clearance and general rules are proposed for the design of nanocarriers produced by Flash NanoPrecipitation with extended circulation times for targeted delivery. (3) In Hydrogen Bonding Coacervate Precipitation, polyethylene glycol coated nanocarriers are controllably flocculated with the addition of polyacids to enable rapid filtration and drying. In summary, this research outlines approaches to the customization of nanocarrier drug delivery systems to specifically improve outcomes in tuberculosis therapy. New assays and processing techniques for transitioning formulations from bench research to the clinic are developed. The methods are flexible and can be applied to target various diseases, coupled with rational design of nanocarrier payloads, surface functionality, and dosing route.

Genetic Determinants of Drug Resistance in Mycobacterium tuberculosis and Their Diagnostic Value.

PubMed

Farhat, Maha R; Sultana, Razvan; Iartchouk, Oleg; Bozeman, Sam; Galagan, James; Sisk, Peter; Stolte, Christian; Nebenzahl-Guimaraes, Hanna; Jacobson, Karen; Sloutsky, Alexander; Kaur, Devinder; Posey, James; Kreiswirth, Barry N; Kurepina, Natalia; Rigouts, Leen; Streicher, Elizabeth M; Victor, Tommie C; Warren, Robin M; van Soolingen, Dick; Murray, Megan

2016-09-01

The development of molecular diagnostics that detect both the presence of Mycobacterium tuberculosis in clinical samples and drug resistance-conferring mutations promises to revolutionize patient care and interrupt transmission by ensuring early diagnosis. However, these tools require the identification of genetic determinants of resistance to the full range of antituberculosis drugs. To determine the optimal molecular approach needed, we sought to create a comprehensive catalog of resistance mutations and assess their sensitivity and specificity in diagnosing drug resistance. We developed and validated molecular inversion probes for DNA capture and deep sequencing of 28 drug-resistance loci in M. tuberculosis. We used the probes for targeted sequencing of a geographically diverse set of 1,397 clinical M. tuberculosis isolates with known drug resistance phenotypes. We identified a minimal set of mutations to predict resistance to first- and second-line antituberculosis drugs and validated our predictions in an independent dataset. We constructed and piloted a web-based database that provides public access to the sequence data and prediction tool. The predicted resistance to rifampicin and isoniazid exceeded 90% sensitivity and specificity but was lower for other drugs. The number of mutations needed to diagnose resistance is large, and for the 13 drugs studied it was 238 across 18 genetic loci. These data suggest that a comprehensive M. tuberculosis drug resistance diagnostic will need to allow for a high dimension of mutation detection. They also support the hypothesis that currently unknown genetic determinants, potentially discoverable by whole-genome sequencing, encode resistance to second-line tuberculosis drugs.

Genetic Determinants of Drug Resistance in Mycobacterium tuberculosis and Their Diagnostic Value

PubMed Central

Sultana, Razvan; Iartchouk, Oleg; Bozeman, Sam; Galagan, James; Sisk, Peter; Stolte, Christian; Nebenzahl-Guimaraes, Hanna; Jacobson, Karen; Sloutsky, Alexander; Kaur, Devinder; Posey, James; Kreiswirth, Barry N.; Kurepina, Natalia; Rigouts, Leen; Streicher, Elizabeth M.; Victor, Tommie C.; Warren, Robin M.; van Soolingen, Dick; Murray, Megan

2016-01-01

Rationale: The development of molecular diagnostics that detect both the presence of Mycobacterium tuberculosis in clinical samples and drug resistance–conferring mutations promises to revolutionize patient care and interrupt transmission by ensuring early diagnosis. However, these tools require the identification of genetic determinants of resistance to the full range of antituberculosis drugs. Objectives: To determine the optimal molecular approach needed, we sought to create a comprehensive catalog of resistance mutations and assess their sensitivity and specificity in diagnosing drug resistance. Methods: We developed and validated molecular inversion probes for DNA capture and deep sequencing of 28 drug-resistance loci in M. tuberculosis. We used the probes for targeted sequencing of a geographically diverse set of 1,397 clinical M. tuberculosis isolates with known drug resistance phenotypes. We identified a minimal set of mutations to predict resistance to first- and second-line antituberculosis drugs and validated our predictions in an independent dataset. We constructed and piloted a web-based database that provides public access to the sequence data and prediction tool. Measurements and Main Results: The predicted resistance to rifampicin and isoniazid exceeded 90% sensitivity and specificity but was lower for other drugs. The number of mutations needed to diagnose resistance is large, and for the 13 drugs studied it was 238 across 18 genetic loci. Conclusions: These data suggest that a comprehensive M. tuberculosis drug resistance diagnostic will need to allow for a high dimension of mutation detection. They also support the hypothesis that currently unknown genetic determinants, potentially discoverable by whole-genome sequencing, encode resistance to second-line tuberculosis drugs. PMID:26910495

Mycobacterium tuberculosis Controls MicroRNA-99b (miR-99b) Expression in Infected Murine Dendritic Cells to Modulate Host Immunity*

PubMed Central

Singh, Yogesh; Kaul, Vandana; Mehra, Alka; Chatterjee, Samit; Tousif, Sultan; Dwivedi, Ved Prakash; Suar, Mrutyunjay; Van Kaer, Luc; Bishai, William R.; Das, Gobardhan

2013-01-01

Mycobacterium tuberculosis resides and replicates within host phagocytes by modulating host microbicidal responses. In addition, it suppresses the production of host protective cytokines to prevent activation of and antigen presentation by M. tuberculosis-infected cells, causing dysregulation of host protective adaptive immune responses. Many cytokines are regulated by microRNAs (miRNAs), a newly discovered class of small noncoding RNAs, which have been implicated in modulating host immune responses in many bacterial and viral diseases. Here, we show that miRNA-99b (miR-99b), an orphan miRNA, plays a key role in the pathogenesis of M. tuberculosis infection. We found that miR-99b expression was highly up-regulated in M. tuberculosis strain H37Rv-infected dendritic cells (DCs) and macrophages. Blockade of miR-99b expression by antagomirs resulted in significantly reduced bacterial growth in DCs. Interestingly, knockdown of miR-99b in DCs significantly up-regulated proinflammatory cytokines such as IL-6, IL-12, and IL-1β. Furthermore, mRNA and membrane-bound protein data indicated that inhibition of miR-99b augments TNF-α and TNFRSF-4 production. Thus, miR-99b targets TNF-α and TNFRSF-4 receptor genes. Treatment of anti-miR-99b-transfected DCs with anti-TNF-α antibody resulted in increased bacterial burden. Thus, our findings unveil a novel host evasion mechanism adopted by M. tuberculosis via miR-99b, which may open up new avenues for designing miRNA-based vaccines and therapies. PMID:23233675

Tuberculosis Prevention in the Private Sector: Using Claims-Based Methods to Identify and Evaluate Latent Tuberculosis Infection Treatment With Isoniazid Among the Commercially Insured.

PubMed

Stockbridge, Erica L; Miller, Thaddeus L; Carlson, Erin K; Ho, Christine

Targeted identification and treatment of people with latent tuberculosis infection (LTBI) are key components of the US tuberculosis elimination strategy. Because of recent policy changes, some LTBI treatment may shift from public health departments to the private sector. To (1) develop methodology to estimate initiation and completion of treatment with isoniazid for LTBI using claims data, and (2) estimate treatment completion rates for isoniazid regimens from commercial insurance claims. Medical and pharmacy claims data representing insurance-paid services rendered and prescriptions filled between January 2011 and March 2015 were analyzed. Four million commercially insured individuals 0 to 64 years of age. Six-month and 9-month treatment completion rates for isoniazid LTBI regimens. There was an annual isoniazid LTBI treatment initiation rate of 12.5/100 000 insured persons. Of 1074 unique courses of treatment with isoniazid for which treatment completion could be assessed, almost half (46.3%; confidence interval, 43.3-49.3) completed 6 or more months of therapy. Of those, approximately half (48.9%; confidence interval, 44.5-53.3) completed 9 months or more. Claims data can be used to identify and evaluate LTBI treatment with isoniazid occurring in the commercial sector. Completion rates were in the range of those found in public health settings. These findings suggest that the commercial sector may be a valuable adjunct to more traditional venues for tuberculosis prevention. In addition, these newly developed claims-based methods offer a means to gain important insights and open new avenues to monitor, evaluate, and coordinate tuberculosis prevention.

A small-molecule allosteric inhibitor of Mycobacterium tuberculosis tryptophan synthase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wellington, Samantha; Nag, Partha P.; Michalska, Karolina

New antibiotics with novel targets are greatly needed. Bacteria have numerous essential functions, but only a small fraction of such processes—primarily those involved in macromolecular synthesis—are inhibited by current drugs. Targeting metabolic enzymes has been the focus of recent interest, but effective inhibitors have been difficult to identify. We describe a synthetic azetidine derivative, BRD4592, that kills Mycobacterium tuberculosis (Mtb) through allosteric inhibition of tryptophan synthase (TrpAB), a previously untargeted, highly allosterically regulated enzyme. BRD4592 binds at the TrpAB a–b-subunit interface and affects multiple steps in the enzyme’s overall reaction, resulting in inhibition not easily overcome by changes in metabolicmore » environment. We show that TrpAB is required for the survival of Mtb and Mycobacterium marinum in vivo and that this requirement may be independent of an adaptive immune response. This work highlights the effectiveness of allosteric inhibition for targeting proteins that are naturally highly dynamic and that are essential in vivo, despite their apparent dispensability under in vitro conditions, and suggests a framework for the discovery of a next generation of allosteric inhibitors.« less

A small-molecule allosteric inhibitor of Mycobacterium tuberculosis tryptophan synthase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wellington, Samantha; Nag, Partha P.; Michalska, Karolina

New antibiotics with novel targets are greatly needed. Bacteria have numerous essential functions, but only a small fraction of such processes—primarily those involved in macromolecular synthesis—are inhibited by current drugs. Targeting metabolic enzymes has been the focus of recent interest, but effective inhibitors have been difficult to identify. We describe a synthetic azetidine derivative, BRD4592, that kills Mycobacterium tuberculosis (Mtb) through allosteric inhibition of tryptophan synthase (TrpAB), a previously untargeted, highly allosterically regulated enzyme. BRD4592 binds at the TrpAB α–β-subunit interface and affects multiple steps in the enzyme's overall reaction, resulting in inhibition not easily overcome by changes in metabolicmore » environment. We show that TrpAB is required for the survival of Mtb and Mycobacterium marinum in vivo and that this requirement may be independent of an adaptive immune response. This work highlights the effectiveness of allosteric inhibition for targeting proteins that are naturally highly dynamic and that are essential in vivo, despite their apparent dispensability under in vitro conditions, and suggests a framework for the discovery of a next generation of allosteric inhibitors.« less

Insights on how the Mycobacterium tuberculosis heme uptake pathway can be used as a drug target

PubMed Central

Owens, Cedric P; Chim, Nicholas; Goulding, Celia W

2013-01-01

Mycobacterium tuberculosis (Mtb) acquires non-heme iron through salicylate-derived siderophores termed mycobactins whereas heme iron is obtained through a cascade of heme uptake proteins. Three proteins are proposed to mediate Mtb heme iron uptake, a secreted heme transporter (Rv0203), and MmpL3 and MmpL11, which are potential transmembrane heme transfer proteins. Furthermore, MhuD, a cytoplasmic heme-degrading enzyme, has been identified. Rv0203, MmpL3 and MmpL11 are mycobacteria-specific proteins, making them excellent drug targets. Importantly, MmpL3, a necessary protein, has also been implicated in trehalose monomycolate export. Recent drug-discovery efforts revealed that MmpL3 is the target of several compounds with antimycobacterial activity. Inhibition of the Mtb heme uptake pathway has yet to be explored. We propose that inhibitor design could focus on heme analogs, with the goal of blocking specific steps of this pathway. In addition, heme uptake could be hijacked as a method of importing drugs into the mycobacterial cytosol. PMID:23919550

Novel approach based on one-tube nested PCR and a lateral flow strip for highly sensitive diagnosis of tuberculous meningitis

PubMed Central

Sun, Yajuan; Chen, Jiajun; Li, Jia; Xu, Yawei; Jin, Hui; Xu, Na; Yin, Rui

2017-01-01

Rapid and sensitive detection of Mycobacterium tuberculosis (M. Tb) in cerebrospinal fluid is crucial in the diagnosis of tuberculous meningitis (TBM), but conventional diagnostic technologies have limited sensitivity and specificity or are time-consuming. In this work, a novel, highly sensitive molecular diagnostic method, one-tube nested PCR-lateral flow strip test (OTNPCR-LFST), was developed for detecting M. tuberculosis. This one-tube nested PCR maintains the sensitivity of conventional two-step nested PCR and reduces both the chance of cross-contamination and the time required for analysis. The PCR product was detected by a lateral flow strip assay, which provided a basis for migration of the test to a point-of-care (POC) microfluidic format. The developed assay had an improved sensitivity compared with traditional PCR, and the limit of detection was up to 1 fg DNA isolated from M. tuberculosis. The assay was also specific for M. tuberculosis, and no cross-reactions were found in other non-target bacteria. The application of this technique to clinical samples was successfully evaluated, and OTNPCR-LFST showed 89% overall sensitivity and 100% specificity for TBM patients. This one-tube nested PCR-lateral flow strip assay is useful for detecting M. tuberculosis in TBM due to its rapidity, high sensitivity and simple manipulation. PMID:29084241

The Tuberculosis Drug Discovery and Development Pipeline and Emerging Drug Targets

PubMed Central

Mdluli, Khisimuzi; Kaneko, Takushi; Upton, Anna

2015-01-01

The recent accelerated approval for use in extensively drug-resistant and multidrug-resistant-tuberculosis (MDR-TB) of two first-in-class TB drugs, bedaquiline and delamanid, has reinvigorated the TB drug discovery and development field. However, although several promising clinical development programs are ongoing to evaluate new TB drugs and regimens, the number of novel series represented is few. The global early-development pipeline is also woefully thin. To have a chance of achieving the goal of better, shorter, safer TB drug regimens with utility against drug-sensitive and drug-resistant disease, a robust and diverse global TB drug discovery pipeline is key, including innovative approaches that make use of recently acquired knowledge on the biology of TB. Fortunately, drug discovery for TB has resurged in recent years, generating compounds with varying potential for progression into developable leads. In parallel, advances have been made in understanding TB pathogenesis. It is now possible to apply the lessons learned from recent TB hit generation efforts and newly validated TB drug targets to generate the next wave of TB drug leads. Use of currently underexploited sources of chemical matter and lead-optimization strategies may also improve the efficiency of future TB drug discovery. Novel TB drug regimens with shorter treatment durations must target all subpopulations of Mycobacterium tuberculosis existing in an infection, including those responsible for the protracted TB treatment duration. This review summarizes the current TB drug development pipeline and proposes strategies for generating improved hits and leads in the discovery phase that could help achieve this goal. PMID:25635061

Clinical evaluation of a mobile digital specimen radiography system for intraoperative specimen verification.

PubMed

Wang, Yingbing; Ebuoma, Lilian; Saksena, Mansi; Liu, Bob; Specht, Michelle; Rafferty, Elizabeth

2014-08-01

Use of mobile digital specimen radiography systems expedites intraoperative verification of excised breast specimens. The purpose of this study was to evaluate the performance of a such a system for verifying targets. A retrospective review included 100 consecutive pairs of breast specimen radiographs. Specimens were imaged in the operating room with a mobile digital specimen radiography system and then with a conventional digital mammography system in the radiology department. Two expert reviewers independently scored each image for image quality on a 3-point scale and confidence in target visualization on a 5-point scale. A target was considered confidently verified only if both reviewers declared the target to be confidently detected. The 100 specimens contained a total of 174 targets, including 85 clips (49%), 53 calcifications (30%), 35 masses (20%), and one architectural distortion (1%). Although a significantly higher percentage of mobile digital specimen radiographs were considered poor quality by at least one reviewer (25%) compared with conventional digital mammograms (1%), 169 targets (97%), were confidently verified with mobile specimen radiography; 172 targets (98%) were verified with conventional digital mammography. Three faint masses were not confidently verified with mobile specimen radiography, and conventional digital mammography was needed for confirmation. One faint mass and one architectural distortion were not confidently verified with either method. Mobile digital specimen radiography allows high diagnostic confidence for verification of target excision in breast specimens across target types, despite lower image quality. Substituting this modality for conventional digital mammography can eliminate delays associated with specimen transport, potentially decreasing surgical duration and increasing operating room throughput.

Sustained release formulation of an anti-tuberculosis drug based on para-amino salicylic acid-zinc layered hydroxide nanocomposite.

PubMed

Saifullah, Bullo; Hussein, Mohd Zobir; Hussein-Al-Ali, Samer Hasan; Arulselvan, Palanisamy; Fakurazi, Sharida

2013-04-20

Tuberculosis (TB), is caused by the bacteria, Mycobacterium tuberculosis and its a threat to humans since centuries. Depending on the type of TB, its treatment can last for 6-24 months which is a major cause for patients non-compliance and treatment failure. Many adverse effects are associated with the currently available TB medicines, and there has been no new anti-tuberculosis drug on the market for more than 50 year, as the drug development is very lengthy and budget consuming process.Development of the biocompatible nano drug delivery systems with the ability to minimize the side effects of the drugs, protection of the drug from enzymatic degradation. And most importantly the drug delivery systems which can deliver the drug at target site would increase the therapeutic efficacy. Nanovehicles with their tendency to release the drug in a sustained manner would result in the bioavalibilty of the drugs in the body for a longer period of time and this would reduce the dosing frequency in drug administration. The biocompatible nanovehicles with the properties like sustained release of drug of the target site, protection of the drug from physio-chemical degradation, reduction in dosing frequency, and prolong bioavailability of drug in the body would result in the shortening of the treatment duration. All of these factors would improve the patient compliance with chemotherapy of TB. An anti-tuberculosis drug, 4-amino salicylic acid (4-ASA) was successfully intercalated into the interlamellae of zinc layered hydroxide (ZLH) via direct reaction with zinc oxide suspension. The X-ray diffraction patterns and FTIR analyses indicate that the molecule was successfully intercalated into the ZLH interlayer space with an average basal spacing of 24 Å. Furthermore, TGA and DTG results show that the drug 4-ASA is stabilized in the interlayers by electrostatic interaction. The release of 4-ASA from the nanocomposite was found to be in a sustained manner. The nanocomposite treated with normal 3T3 cells shows it reduces cell viability in a dose- and time-dependent manner. Sustained release formulation of the nanocomposite, 4-ASA intercalated into zinc layered hydroxides, with its ease of preparation, sustained release of the active and less-toxic to the cell is a step forward for a more patient-friendly chemotherapy of Tuberculosis.

Sustained release formulation of an anti-tuberculosis drug based on para-amino salicylic acid-zinc layered hydroxide nanocomposite

PubMed Central

2013-01-01

Background Tuberculosis (TB), is caused by the bacteria, Mycobacterium tuberculosis and its a threat to humans since centuries. Depending on the type of TB, its treatment can last for 6–24 months which is a major cause for patients non-compliance and treatment failure. Many adverse effects are associated with the currently available TB medicines, and there has been no new anti-tuberculosis drug on the market for more than 50 year, as the drug development is very lengthy and budget consuming process. Development of the biocompatible nano drug delivery systems with the ability to minimize the side effects of the drugs, protection of the drug from enzymatic degradation. And most importantly the drug delivery systems which can deliver the drug at target site would increase the therapeutic efficacy. Nanovehicles with their tendency to release the drug in a sustained manner would result in the bioavalibilty of the drugs in the body for a longer period of time and this would reduce the dosing frequency in drug administration. The biocompatible nanovehicles with the properties like sustained release of drug of the target site, protection of the drug from physio-chemical degradation, reduction in dosing frequency, and prolong bioavailability of drug in the body would result in the shortening of the treatment duration. All of these factors would improve the patient compliance with chemotherapy of TB. Result An anti-tuberculosis drug, 4-amino salicylic acid (4-ASA) was successfully intercalated into the interlamellae of zinc layered hydroxide (ZLH) via direct reaction with zinc oxide suspension. The X-ray diffraction patterns and FTIR analyses indicate that the molecule was successfully intercalated into the ZLH interlayer space with an average basal spacing of 24 Å. Furthermore, TGA and DTG results show that the drug 4-ASA is stabilized in the interlayers by electrostatic interaction. The release of 4-ASA from the nanocomposite was found to be in a sustained manner. The nanocomposite treated with normal 3T3 cells shows it reduces cell viability in a dose- and time-dependent manner. Conclusions Sustained release formulation of the nanocomposite, 4-ASA intercalated into zinc layered hydroxides, with its ease of preparation, sustained release of the active and less-toxic to the cell is a step forward for a more patient-friendly chemotherapy of Tuberculosis. PMID:23601852

Impact of Targeted Tuberculosis Vaccination Among a Mining Population in South Africa: A Model-Based Study.

PubMed

Shrestha, Sourya; Chihota, Violet; White, Richard G; Grant, Alison D; Churchyard, Gavin J; Dowdy, David W

2017-12-15

Optimizing the use of new tools, such as vaccines, may play a crucial role in reaching global targets for tuberculosis (TB) control. Some of the most promising candidate vaccines target adults, although high-coverage mass vaccinations may be logistically more challenging among this population than among children. Vaccine-delivery strategies that target high-risk groups or settings might yield proportionally greater impact than do those that target the general population. We developed an individual-based TB transmission model representing a hypothetical population consisting of people who worked in South African gold mines or lived in associated labor-sending communities. We simulated the implementation of a postinfection adult vaccine with 60% efficacy and a mean effect duration of 10 years. We then compared the impact of a mine-targeted vaccination strategy, in which miners were vaccinated while in the mines, with that of a community-targeted strategy, in which random individuals within the labor-sending communities were vaccinated. Mine-targeted vaccination averted an estimated 0.37 TB cases per vaccine dose compared with 0.25 for community-targeted vaccination, for a relative efficacy of 1.46 (95% range, 1.13-1.91). The added benefit of mine-targeted vaccination primarily reflected the disproportionate demographic burden of TB among the population of adult males as a whole. As novel vaccines for TB are developed, venue-based vaccine delivery that targets high-risk demographic groups may improve both vaccine feasibility and the impact on transmission. © The Author(s) 2017. Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Factors influencing polymerase chain reaction outcomes in patients with clinically suspected ocular tuberculosis.

PubMed

Balne, Praveen Kumar; Modi, Rohit Ramesh; Choudhury, Nuzhat; Mohan, Neha; Barik, Manas Ranjan; Padhi, Tapas Ranjan; Sharma, Savitri; Panigrahi, Satya Ranjan; Basu, Soumyava

2014-03-25

Polymerase chain reaction (PCR) assay can be a useful method for definitive diagnosis in paucibacillary infections such as ocular tuberculosis (TB). In this study, we have evaluated factors affecting PCR outcomes in patients with clinically suspected ocular TB. Patients with clinically suspected ocular TB were investigated by PCR of aqueous or vitreous samples. Three control groups were also tested: group 1 included culture-proven non-tuberculous endophthalmitis, group 2 culture-negative non-tuberculous endophthalmitis, and group 3 patients undergoing surgery for uncomplicated cataract. PCR targeted one or more of following targets: IS6110, MPB64, and protein b genes of Mycobacterium tuberculosis complex. Multiple regression analysis (5% level of significance) was done to evaluate the associations between positive PCR outcome and laterality of disease, tuberculin skin test (TST)/interferon-gamma release assay (IGRA), chest radiography, and type of sample (aqueous or vitreous). The main outcome measures were positive PCR by one or more gene targets, and factors influencing positive PCR outcomes. All 114 samples were tested for MPB64, 110 for protein b, and 88 for IS6110. MPB64 was positive in 70.2% (n = 80) of tested samples, protein b in 40.0% (n = 44), and IS6110 in only 9.1% (n = 8). DNA sequencing of amplicons from four randomly chosen PCR reactions showed homology for M. tuberculosis complex. Of the 80 PCR-positive patients, 71 completed a full course of antitubercular therapy, of which 65 patients (91.5%) had complete resolution of inflammation at final follow-up. Among controls, 12.5% (3 out of 24) in group 1 and 18.7% (6 out of 32) in group 2 also tested positive by PCR. No PCR-positive outcome was observed in control group 3 (n = 25). Multiple regression analysis revealed significant association of positive PCR outcome with bilateral presentation, but not with a positive TST/IGRA, chest radiography, or type of sample (aqueous/vitreous) used. Careful selection of gene targets can yield high PCR positivity in clinically suspected ocular TB. Bilateral disease presentation but not any evidence of latent systemic TB influences PCR outcomes. False-positive results may be seen in ocular inflammation unrelated to ocular TB.

Essential but Not Vulnerable: Indazole Sulfonamides Targeting Inosine Monophosphate Dehydrogenase as Potential Leads against Mycobacterium tuberculosis.

PubMed

Park, Yumi; Pacitto, Angela; Bayliss, Tracy; Cleghorn, Laura A T; Wang, Zhe; Hartman, Travis; Arora, Kriti; Ioerger, Thomas R; Sacchettini, Jim; Rizzi, Menico; Donini, Stefano; Blundell, Tom L; Ascher, David B; Rhee, Kyu; Breda, Ardala; Zhou, Nian; Dartois, Veronique; Jonnala, Surendranadha Reddy; Via, Laura E; Mizrahi, Valerie; Epemolu, Ola; Stojanovski, Laste; Simeons, Fred; Osuna-Cabello, Maria; Ellis, Lucy; MacKenzie, Claire J; Smith, Alasdair R C; Davis, Susan H; Murugesan, Dinakaran; Buchanan, Kirsteen I; Turner, Penelope A; Huggett, Margaret; Zuccotto, Fabio; Rebollo-Lopez, Maria Jose; Lafuente-Monasterio, Maria Jose; Sanz, Olalla; Diaz, Gracia Santos; Lelièvre, Joël; Ballell, Lluis; Selenski, Carolyn; Axtman, Matthew; Ghidelli-Disse, Sonja; Pflaumer, Hannah; Bösche, Markus; Drewes, Gerard; Freiberg, Gail M; Kurnick, Matthew D; Srikumaran, Myron; Kempf, Dale J; Green, Simon R; Ray, Peter C; Read, Kevin; Wyatt, Paul; Barry, Clifton E; Boshoff, Helena I

2017-01-13

A potent, noncytotoxic indazole sulfonamide was identified by high-throughput screening of >100,000 synthetic compounds for activity against Mycobacterium tuberculosis (Mtb). This noncytotoxic compound did not directly inhibit cell wall biogenesis but triggered a slow lysis of Mtb cells as measured by release of intracellular green fluorescent protein (GFP). Isolation of resistant mutants followed by whole-genome sequencing showed an unusual gene amplification of a 40 gene region spanning from Rv3371 to Rv3411c and in one case a potential promoter mutation upstream of guaB2 (Rv3411c) encoding inosine monophosphate dehydrogenase (IMPDH). Subsequent biochemical validation confirmed direct inhibition of IMPDH by an uncompetitive mode of inhibition, and growth inhibition could be rescued by supplementation with guanine, a bypass mechanism for the IMPDH pathway. Beads containing immobilized indazole sulfonamides specifically interacted with IMPDH in cell lysates. X-ray crystallography of the IMPDH-IMP-inhibitor complex revealed that the primary interactions of these compounds with IMPDH were direct pi-pi interactions with the IMP substrate. Advanced lead compounds in this series with acceptable pharmacokinetic properties failed to show efficacy in acute or chronic murine models of tuberculosis (TB). Time-kill experiments in vitro suggest that sustained exposure to drug concentrations above the minimum inhibitory concentration (MIC) for 24 h were required for a cidal effect, levels that have been difficult to achieve in vivo. Direct measurement of guanine levels in resected lung tissue from tuberculosis-infected animals and patients revealed 0.5-2 mM concentrations in caseum and normal lung tissue. The high lesional levels of guanine and the slow lytic, growth-rate-dependent effect of IMPDH inhibition pose challenges to developing drugs against this target for use in treating TB.

Essential but not vulnerable: indazole sulfonamides targeting inosine monophosphate dehydrogenase as potential leads against Mycobacterium tuberculosis

PubMed Central

Park, Yumi; Pacitto, Angela; Bayliss, Tracy; Cleghorn, Laura A. T.; Wang, Zhe; Hartman, Travis; Arora, Kriti; Ioerger, Thomas R.; Sacchettini, Jim; Rizzi, Menico; Donini, Stefano; Blundell, Tom L.; Ascher, David B.; Rhee, Kyu; Breda, Ardala; Zhou, Nian; Dartois, Veronique; Jonnala, Surendranadha Reddy; Via, Laura E.; Mizrahi, Valerie; Epemolu, Ola; Stojanovski, Laste; Simeons, Fred; Osuna-Cabello, Maria; Ellis, Lucy; MacKenzie, Claire J.; Smith, Alasdair R. C.; Davis, Susan H.; Murugesan, Dinakaran; Buchanan, Kirsteen I.; Turner, Penelope A.; Huggett, Margaret; Zuccotto, Fabio; Rebollo-Lopez, Maria Jose; Lafuente-Monasterio, Maria Jose; Sanz, Olalla; Santos Diaz, Gracia; Lelièvre, Joël; Ballell, Lluis; Selenski, Carolyn; Axtman, Matthew; Ghidelli-Disse, Sonja; Pflaumer, Hannah; Bösche, Markus; Drewes, Gerard; Freiberg, Gail M.; Kurnick, Matthew D.; Srikumaran, Myron; Kempf, Dale J.; Green, Simon R.; Ray, Peter C.; Read, Kevin; Wyatt, Paul; Barry, Clifton E; Boshoff, Helena I.

2018-01-01

A potent, non-cytotoxic indazole sulfonamide was identified by high-throughput screening of >100,000 synthetic compounds for activity against Mycobacterium tuberculosis (Mtb). This non-cytotoxic compound did not directly inhibit cell wall biogenesis but triggered a slow lysis of Mtb cells as measured by release of intracellular green fluorescent protein (GFP). Isolation of resistant mutants followed by whole-genome sequencing showed an unusual gene amplification of a 40 gene region spanning Rv3371 to Rv3411c and in one case a potential promoter mutation upstream of guaB2 (Rv3411c) encoding inosine monophosphate dehydrogenase (IMPDH). Subsequent biochemical validation confirmed direct inhibition of IMPDH by an uncompetitive mode of inhibition and growth inhibition could be rescued by supplementation with guanine, a bypass mechanism for the IMPDH pathway. Beads containing immobilized indazole sulfonamides specifically interacted with IMPDH in cell lysates. X-ray crystallography of the IMPDH-IMP-inhibitor complex revealed that the primary interactions of these compounds with IMPDH were direct pi-pi interactions with the IMP substrate. Advanced lead compounds in this series with acceptable pharmacokinetic properties failed to show efficacy in acute or chronic murine models of tuberculosis (TB). Time-kill experiments in vitro suggest that sustained exposure to drug concentrations above MIC for 24 hours were required for a cidal effect, levels that have been difficult to achieve in vivo. Direct measurement of guanine levels in resected lung tissue from tuberculosis infected animals and patients revealed 0.5–2 mM concentrations in caseum and normal lung tissue. The high lesional levels of guanine and the slow lytic, growth-rate dependent, effect of IMPDH inhibition pose challenges to developing drugs against this target for use in treating TB. PMID:27704782

Drug Concentration Thresholds Predictive of Therapy Failure and Death in Children With Tuberculosis: Bread Crumb Trails in Random Forests

PubMed Central

Swaminathan, Soumya; Pasipanodya, Jotam G.; Ramachandran, Geetha; Hemanth Kumar, A. K.; Srivastava, Shashikant; Deshpande, Devyani; Nuermberger, Eric; Gumbo, Tawanda

2016-01-01

Background. The role of drug concentrations in clinical outcomes in children with tuberculosis is unclear. Target concentrations for dose optimization are unknown. Methods. Plasma drug concentrations measured in Indian children with tuberculosis were modeled using compartmental pharmacokinetic analyses. The children were followed until end of therapy to ascertain therapy failure or death. An ensemble of artificial intelligence algorithms, including random forests, was used to identify predictors of clinical outcome from among 30 clinical, laboratory, and pharmacokinetic variables. Results. Among the 143 children with known outcomes, there was high between-child variability of isoniazid, rifampin, and pyrazinamide concentrations: 110 (77%) completed therapy, 24 (17%) failed therapy, and 9 (6%) died. The main predictors of therapy failure or death were a pyrazinamide peak concentration <38.10 mg/L and rifampin peak concentration <3.01 mg/L. The relative risk of these poor outcomes below these peak concentration thresholds was 3.64 (95% confidence interval [CI], 2.28–5.83). Isoniazid had concentration-dependent antagonism with rifampin and pyrazinamide, with an adjusted odds ratio for therapy failure of 3.00 (95% CI, 2.08–4.33) in antagonism concentration range. In regard to death alone as an outcome, the same drug concentrations, plus z scores (indicators of malnutrition), and age <3 years, were highly ranked predictors. In children <3 years old, isoniazid 0- to 24-hour area under the concentration-time curve <11.95 mg/L × hour and/or rifampin peak <3.10 mg/L were the best predictors of therapy failure, with relative risk of 3.43 (95% CI, .99–11.82). Conclusions. We have identified new antibiotic target concentrations, which are potential biomarkers associated with treatment failure and death in children with tuberculosis. PMID:27742636

Implementing a successful tuberculosis programme within primary care services in a conflict area using the stop TB strategy: Afghanistan case study

PubMed Central

2014-01-01

Introduction Afghanistan has faced health consequences of war including those due to displacement of populations, breakdown of health and social services, and increased risks of disease transmission for over three decades. Yet it was able to restructure its National Tuberculosis Control Programme (NTP), integrate tuberculosis treatment into primary health care and achieve most of its targets by the year 2011. What were the processes that enabled the programme to achieve its targets? More importantly, what were the underpinning factors that made this success possible? We addressed these important questions through a case study. Case description We adopted a processes and outcomes framework for this study, which began with examining the change in key programme indicators, followed by backwards tracing of the processes and underlying factors, responsible for this change. Methods included review of the published and grey literature along with in-depth interviews of 15 key informants involved with the care of tuberculosis patients in Afghanistan. Discussion and evaluation TB incidence and mortality per 100,000 decreased from 325 and 92 to 189 and 39 respectively, while case notification and treatment success improved during the decade under study. Efficient programme structures were enabled through high political commitment from the Government, strong leadership from the programme, effective partnership and coordination among stakeholders, and adequate technical and financial support from the development partners. Conclusions The NTP Afghanistan is an example that public health programmes can be effectively implemented in fragile states. High political commitment and strong local leadership are essential factors for such programmes. To ensure long-term effectiveness of the NTP, the international support should be withdrawn in a phased manner, coupled with a sequential increase in resources allocated to the NTP by the Government of Afghanistan. PMID:24507446

Dihydrolipoamide dehydrogenase-Lpd (Rv0462)-specific T cell recall responses are higher in healthy household contacts of TB: a novel immunodominant antigen from M. tuberculosis.

PubMed

Devasundaram, Santhi; Raja, Alamelu

2017-07-01

The partial effectiveness against pulmonary tuberculosis (PTB), displayed by the existing tuberculosis (TB) vaccine, bacillus Calmette-Guérin (BCG), highlights the need for novel vaccines to replace or improve BCG. In TB immunology, antigen-specific cellular immune response is frequently considered indispensable. Latency-associated antigens are intriguing as targets for TB vaccine development. The mycobacterial protein, dihydrolipoamide dehydrogenase (Lpd; Rv0462), the third enzyme of the pyruvate dehydrogenase (PDH) complex, facilitates Mycobacterium tuberculosis to resist host reactive nitrogen intermediates. Multicolor flow cytometry analysis of whole-blood cultures showed higher Lpd-specific Th1 recall response (IFN-γ, TNF-α, and IL-2; P = 0.0006) and memory CD4 + and CD8 + T cells (CCR7 + CD45RA - and CCR7 - CD45RA - ) in healthy household contacts (HHC) of TB ( P < 0.0001), which is comparable with or higher than the standard antigens, ESAT-6 and CFP-10. The frequency of Lpd-specific multifunctional T cells was higher in HHC compared with PTB patients. However, there is no significant statistical correlation. Regulatory T cell (T reg ) analysis of HHCs and active TB patients demonstrated very low Lpd-specific CD4 + T regs relative to ESAT-6 and CFP-10. Our study demonstrates that the Lpd antigen induces a strong cellular immune response in healthy mycobacteria-infected individuals. In consideration of this population having demonstrated immunologic protection against active TB disease development, our data are encouraging about the possible use of Lpd as a target for further TB subunit vaccine development. © Society for Leukocyte Biology.

Rapid screening of rpoB and katG mutations in Mycobacterium tuberculosis isolates by high-resolution melting curve analysis.

PubMed

Haeili, M; Fooladi, A I; Bostanabad, S Z; Sarokhalil, D D; Siavoshi, F; Feizabadi, M M

2014-01-01

Early detection of multidrug-resistant tuberculosis (MDR-TB) is essential to prevent its transmission in the community and initiate effective anti-TB treatment regimen. High-resolution melting curve (HRM) analysis was evaluated for rapid detection of resistance conferring mutations in rpoB and katG genes. We screened 95 Mycobacterium tuberculosis clinical isolates including 20 rifampin resistant (RIF-R), 21 isoniazid resistant (INH-R) and 54 fully susceptible (S) isolates determined by proportion method of drug susceptibility testing. Nineteen M. tuberculosis isolates with known drug susceptibility genotypes were used as references for the assay validation. The nucleotide sequences of the target regions rpoB and katG genes were determined to investigate the frequency and type of mutations and to confirm HRM results. HRM analysis of a 129-bp fragment of rpoB allowed correct identification of 19 of the 20 phenotypically RIF-R and all RIF-S isolates. All INH-S isolates generated wild-type HRM curves and 18 out of 21 INH-R isolates harboured any mutation in 109-bp fragment of katG exhibited mutant type HRM curves. However, 1 RIF-R and 3 INH-R isolates were falsely identified as susceptible which were confirmed for having no mutation in their target regions by sequencing. The main mutations involved in RIF and INH resistance were found at codons rpoB531 (60% of RIF-R isolates) and katG315 (85.7% of INH-R isolates), respectively. HRM was found to be a reliable, rapid and low cost method to characterise drug susceptibility of clinical TB isolates in resource-limited settings.

Viral booster vaccines improve Mycobacterium bovis BCG-induced protection against bovine tuberculosis.

PubMed

Vordermeier, H Martin; Villarreal-Ramos, Bernardo; Cockle, Paul J; McAulay, Martin; Rhodes, Shelley G; Thacker, Tyler; Gilbert, Sarah C; McShane, Helen; Hill, Adrian V S; Xing, Zhou; Hewinson, R Glyn

2009-08-01

Previous work with small-animal laboratory models of tuberculosis has shown that vaccination strategies based on heterologous prime-boost protocols using Mycobacterium bovis bacillus Calmette-Guérin (BCG) to prime and modified vaccinia virus Ankara strain (MVA85A) or recombinant attenuated adenoviruses (Ad85A) expressing the mycobacterial antigen Ag85A to boost may increase the protective efficacy of BCG. Here we report the first efficacy data on using these vaccines in cattle, a natural target species of tuberculous infection. Protection was determined by measuring development of disease as an end point after M. bovis challenge. Either Ad85A or MVA85A boosting resulted in protection superior to that given by BCG alone: boosting BCG with MVA85A or Ad85A induced significant reduction in pathology in four/eight parameters assessed, while BCG vaccination alone did so in only one parameter studied. Protection was particularly evident in the lungs of vaccinated animals (median lung scores for naïve and BCG-, BCG/MVA85A-, and BCG/Ad85A-vaccinated animals were 10.5, 5, 2.5, and 0, respectively). The bacterial loads in lymph node tissues were also reduced after viral boosting of BCG-vaccinated calves compared to those in BCG-only-vaccinated animals. Analysis of vaccine-induced immunity identified memory responses measured by cultured enzyme-linked immunospot assay as well as in vitro interleukin-17 production as predictors of vaccination success, as both responses, measured before challenge, correlated positively with the degree of protection. Therefore, this study provides evidence of improved protection against tuberculosis by viral booster vaccination in a natural target species and has prioritized potential correlates of vaccine efficacy for further evaluation. These findings also have implications for human tuberculosis vaccine development.

Structure of the Mycobacterium tuberculosis D-Alanine:D-Alanine Ligase, a Target of the Antituberculosis Drug D-Cycloserine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bruning, John B.; Murillo, Ana C.; Chacon, Ofelia

D-Alanine:D-alanine ligase (EC 6.3.2.4; Ddl) catalyzes the ATP-driven ligation of two D-alanine (D-Ala) molecules to form the D-alanyl:D-alanine dipeptide. This molecule is a key building block in peptidoglycan biosynthesis, making Ddl an attractive target for drug development. D-Cycloserine (DCS), an analog of D-Ala and a prototype Ddl inhibitor, has shown promise for the treatment of tuberculosis. Here, we report the crystal structure of Mycobacterium tuberculosis Ddl at a resolution of 2.1 {angstrom}. This structure indicates that Ddl is a dimer and consists of three discrete domains; the ligand binding cavity is at the intersection of all three domains and conjoinedmore » by several loop regions. The M. tuberculosis apo Ddl structure shows a novel conformation that has not yet been observed in Ddl enzymes from other species. The nucleotide and D-alanine binding pockets are flexible, requiring significant structural rearrangement of the bordering regions for entry and binding of both ATP and D-Ala molecules. Solution affinity and kinetic studies showed that DCS interacts with Ddl in a manner similar to that observed for D-Ala. Each ligand binds to two binding sites that have significant differences in affinity, with the first binding site exhibiting high affinity. DCS inhibits the enzyme, with a 50% inhibitory concentration (IC{sub 50}) of 0.37 mM under standard assay conditions, implicating a preferential and weak inhibition at the second, lower-affinity binding site. Moreover, DCS binding is tighter at higher ATP concentrations. The crystal structure illustrates potential drugable sites that may result in the development of more-effective Ddl inhibitors.« less

Brucella melitensis and Mycobacterium tuberculosis depict overlapping gene expression patterns induced in infected THP-1 macrophages.

PubMed

Masoudian, M; Derakhshandeh, A; Ghahramani Seno, M M

2015-01-01

Pathogens infecting mammalian cells have developed various strategies to suppress and evade their hosts' defensive mechanisms. In this line, the intracellular bacteria that are able to survive and propagate within their host cells must have developed strategies to avert their host's killing attitude. Studying the interface of host-pathogen confrontation can provide valuable information for defining therapeutic approaches. Brucellosis, caused by the Brucella strains, is a zoonotic bacterial disease that affects thousands of humans and animals around the world inflicting discomfort and huge economic losses. Similar to many other intracellular dwelling bacteria, infections caused by Brucella are difficult to treat, and hence any attempt at identifying new and common therapeutic targets would prove beneficial for the purpose of curing infections caused by the intracellular bacteria. In THP-1 macrophage infected with Brucella melitensis we studied the expression levels of four host's genes, i.e. EMP2, ST8SIA4, HCP5 and FRMD5 known to be involved in pathogenesis of Mycobacterium tuberculosis. Our data showed that at this molecular level, except for FRMD5 that was downregulated, the other three genes were upregulated by B. melitensis. Brucella melitensis and M. tuberculosis go through similar intracellular processes and interestingly two of the investigated genes, i.e. EMP2 and ST4SIA8 were upregulated in THP-1 cell infected with B. melitensis similar to that reported for THP-1 cells infected with M. tuberculosis. At the host-pathogen interaction interface, this study depicts overlapping changes for different bacteria with common survival strategies; a fact that implies designing therapeutic approaches based on common targets may be possible.

The dynamics of pulmonary tuberculosis in Colima, Mexico (1999-2002).

PubMed

Chowell, Gerardo; Diaz-Dueñas, Porfirio; Chowell, Diego

2005-01-01

Tuberculosis is a public health problem in Mexico. From 1999 to 2002, we assessed retrospectively the epidemiological, clinical, and treatment characteristics of pulmonary tuberculosis in the hospitals of the Mexican Institute of Public Health in the state of Colima (Mexico). We included 184 cases diagnosed with pulmonary tuberculosis. A database containing demographic, epidemiological, and clinical information was constructed and analyzed. We estimate a median patient delay of 83 d and a mean treatment delay of 2.3 d. Of 14 cases suspected for multiresistance and microbiologically assayed, 5 were found to carry a multi-drug-resistant strain. We also found a significant association between a short patient delay and the presence of hemoptysis (p = 0.002) or dyspnea (p<0.001). 86 patients (46.8%) were sputum smear microscopy negative at the end of treatment and 40 (21.7%) completed treatment giving an overall success rate of 68.5%, which compares unfavorably with the World Health Organization target success rate of 85%. Five (2.7%) patients failed treatment, 10 (5.4%) died, 39 (21.2%) interrupted treatment, and 4 (2.2%) transferred to another reporting unit. A 2002 strategic change in drug distribution seemed to prove successful.

Potential Inhibitors for Isocitrate Lyase of Mycobacterium tuberculosis and Non-M. tuberculosis: A Summary

PubMed Central

Lee, Yie-Vern; Wahab, Habibah A.

2015-01-01

Isocitrate lyase (ICL) is the first enzyme involved in glyoxylate cycle. Many plants and microorganisms are relying on glyoxylate cycle enzymes to survive upon downregulation of tricarboxylic acid cycle (TCA cycle), especially Mycobacterium tuberculosis (MTB). In fact, ICL is a potential drug target for MTB in dormancy. With the urge for new antitubercular drug to overcome tuberculosis treat such as multidrug resistant strain and HIV-coinfection, the pace of drug discovery has to be increased. There are many approaches to discovering potential inhibitor for MTB ICL and we hereby review the updated list of them. The potential inhibitors can be either a natural compound or synthetic compound. Moreover, these compounds are not necessary to be discovered only from MTB ICL, as it can also be discovered by a non-MTB ICL. Our review is categorized into four sections, namely, (a) MTB ICL with natural compounds; (b) MTB ICL with synthetic compounds; (c) non-MTB ICL with natural compounds; and (d) non-MTB ICL with synthetic compounds. Each of the approaches is capable of overcoming different challenges of inhibitor discovery. We hope that this paper will benefit the discovery of better inhibitor for ICL. PMID:25649791

Identifying components for programmatic latent tuberculosis infection control in the European Union

PubMed Central

Sandgren, Andreas; Vonk Noordegraaf-Schouten, Jannigje M; Oordt-Speets, Anouk M; van Kessel, Gerarda B; de Vlas, Sake J; van der Werf, Marieke J

2016-01-01

Individuals with latent tuberculosis infection (LTBI) are the reservoir of Mycobacterium tuberculosis in a population and as long as this reservoir exists, elimination of tuberculosis (TB) will not be feasible. In 2013, the European Centre for Disease Prevention and Control (ECDC) started an assessment of benefits and risks of introducing programmatic LTBI control, with the aim of providing guidance on how to incorporate LTBI control into national TB strategies in European Union/European Economic Area (EU/EEA) Member States and candidate countries. In a first step, experts from the Member States, candidate countries, and international and national organisations were consulted on the components of programmatic LTBI control that should be considered and evaluated in literature reviews, mathematical models and cost-effectiveness studies. This was done through a questionnaire and two interactive discussion rounds. The main components identified were identification and targeting of risk groups, determinants of LTBI and progression to active TB, optimal diagnostic tests for LTBI, effective preventive treatment regimens, and to explore the potential for combining LTBI control with other health programmes. Political commitment, a solid healthcare infrastructure, and favourable economic situation in specific countries were identified as essential to facilitate the implementation of programmatic LTBI control. PMID:27589214

The antibiotic cyclomarin blocks arginine-phosphate-induced millisecond dynamics in the N-terminal domain of ClpC1 from Mycobacterium tuberculosis.

PubMed

Weinhäupl, Katharina; Brennich, Martha; Kazmaier, Uli; Lelievre, Joel; Ballell, Lluis; Goldberg, Alfred; Schanda, Paul; Fraga, Hugo

2018-06-01

Mycobacterium tuberculosis can remain dormant in the host, an ability that explains the failure of many current tuberculosis treatments. Recently, the natural products cyclomarin, ecumicin, and lassomycin have been shown to efficiently kill Mycobacterium tuberculosis persisters. Their target is the N-terminal domain of the hexameric AAA+ ATPase ClpC1, which recognizes, unfolds, and translocates protein substrates, such as proteins containing phosphorylated arginine residues, to the ClpP1P2 protease for degradation. Surprisingly, these antibiotics do not inhibit ClpC1 ATPase activity, and how they cause cell death is still unclear. Here, using NMR and small-angle X-ray scattering, we demonstrate that arginine-phosphate binding to the ClpC1 N-terminal domain induces millisecond dynamics. We show that these dynamics are caused by conformational changes and do not result from unfolding or oligomerization of this domain. Cyclomarin binding to this domain specifically blocked these N-terminal dynamics. On the basis of these results, we propose a mechanism of action involving cyclomarin-induced restriction of ClpC1 dynamics, which modulates the chaperone enzymatic activity leading eventually to cell death. © 2018 Weinhäupl et al.

Rv1458c: a new diagnostic marker for identification of Mycobacterium tuberculosis complex in a novel duplex PCR assay.

PubMed

Shrivastava, Kamal; Garima, Kushal; Narang, Anshika; Bhattacharyya, Kausik; Vishnoi, Ekta; Singh, Roshan Kumar; Chaudhry, Anil; Prasad, Rajendra; Bose, Mridula; Varma-Basil, Mandira

2017-03-01

We explored the efficiency of Rv1458c, the gene encoding a putative ABC drug transporter specific for the Mycobacterium tuberculosis complex (MTBC), as a diagnostic marker. A 190 bp region of Rv1458c and a 300 bp region of hsp65 were targeted in a novel duplex PCR assay and the results were compared with those for PCR restriction analysis(PRA) using the restriction enzymes NruI and BamHI. Species identification of a subset of the isolates (n=50) was confirmed by sequencing. Clinical isolates of M. tuberculosis (n=426) obtained from clinically suspected patients of pulmonary tuberculosis and mycobacterial (n=13) and non-mycobacterial (n=8) reference strains were included in the study. The duplex PCR assay correctly identified 320/426 isolates as MTBC and 106/426 isolates as non-tuberculous mycobacteria(NTM). The test was 100 % specific and sensitive when compared with NruI/BamHI PCR restriction analysis and highlighted the use of Rv1458c as a diagnostic marker for MTBC. The duplex PCR assay could be developed for use as a screening test to identify MTBC in clinical specimens in peripheral laboratories with limited resources.

The Role of ESX-1 in Mycobacterium tuberculosis Pathogenesis.

PubMed

Wong, Ka-Wing

2017-05-01

In this article, we have described several cellular pathological effects caused by the Mycobacterium tuberculosis ESX-1. The effects include induction of necrosis, NOD2 signaling, type I interferon production, and autophagy. We then attempted to suggest that these pathological effects are mediated by the cytosolic access of M. tuberculosis -derived materials as a result of the phagosome-disrupting activity of the major ESX-1 substrate ESAT-6. Such activity of ESAT-6 is most likely due to its pore-forming activity at the membrane. The amyloidogenic characteristic of ESAT-6 is reviewed here as a potential mechanism of membrane pore formation. In addition to ESAT-6, the ESX-1 substrate EspB interferes with membrane-mediated innate immune mechanisms such as efferocytosis and autophagy, most likely through its ability to bind phospholipids. Overall, the M. tuberculosis ESX-1 secretion system appears to be a specialized system for the deployment of host membrane-targeting proteins, whose primary function is to interrupt key steps in innate immune mechanisms against pathogens. Inhibitors that block the ESX-1 system or block host factors critical for ESX-1 toxicity have been identified and should represent attractive potential new antituberculosis drugs.

Incidence of active tuberculosis in individuals with latent tuberculosis infection in rural China: follow-up results of a population-based, multicentre, prospective cohort study.

PubMed

Gao, Lei; Li, Xiangwei; Liu, Jianmin; Wang, Xinhua; Lu, Wei; Bai, Liqiong; Xin, Henan; Zhang, Haoran; Li, Hengjing; Zhang, Zongde; Ma, Yu; Li, Mufei; Feng, Boxuan; Du, Jiang; Sui, Hongtao; Zhao, Rong; Su, Haoxiang; Pan, Shouguo; Guan, Ling; Shen, Fei; He, Jian; Yang, Shumin; Si, Hongyan; Cheng, Xu; Xu, Zuhui; Tan, Yunhong; Chen, Tianzhu; Xu, Weiguo; Peng, Hong; Wang, Zhijian; Zhu, Tao; Chen, Xiaoyou; Zhou, Xinhua; Guan, Xueling; Jin, Qi

2017-10-01

The management of latent Mycobacterium tuberculosis infection is a new priority action for the WHO End Tuberculosis (TB) Strategy. However, national guidelines on latent tuberculosis infection testing and treatment have not yet been developed in China. Here, we present the results from the 2-year follow-up of a study that aimed to track the development of active disease in individuals with latent tuberculosis infection, identify priority populations for latent infection management, and explore the most suitable latent infection diagnostic approach. A population-based multicentre prospective study was done in four sites in rural China, between 2013 and 2015. The baseline survey in 2013 measured the prevalence of latent tuberculosis infection using QuantiFERON-TB Gold In-Tube (QFT) and tuberculin skin test (TST) in eligible participants. During the follow-up phase between 2014-15, we assessed individuals who had tuberculosis infection at baseline (QFT-positivity or TST tuberculin reaction size [induration] of ≥10 mm) for the development of active disease through active case finding. Eligible participants included in follow-up survey had a birth date before June 1, 2008 (5 years or older in 2013), and continuous residence at the study site for 6 months or longer in the past year. Participants with current active tuberculosis at baseline survey were excluded. Between Sept 1, 2013, and Aug 31, 2015, 7505 eligible participants (aged 5 years or older) were included in tuberculosis infection test positive cohorts (4455 were QFT positive, 6404 had TST induration ≥10 mm, and 3354 were positive for both tests) after baseline examination. During the 2-year follow-up period, 84 incident cases of active tuberculosis were diagnosed. Of participants who developed active tuberculosis, 75 were diagnosed with latent infection by QFT, 62 were diagnosed by TST, and 53 were diagnosed by both tests. An incidence rate of 0·87 (95% CI 0·68-1·07) per 100 person-years was observed for individuals who tested positive with QFT, 0·50 (0·38-0·63) per 100 person-years for those who tested positive with TST (p<0·0001), and 0·82 (0·60-1·04) per 100 person-years for those who tested positive with both tests. Male sex and a history of tuberculosis were significantly associated with increased risk of disease development with adjusted hazard ratios of 2·36 (95% CI 1·30-4·30) for male sex and 5·40 (3·34-8·71) for a history of tuberculosis. Our results suggest that high-risk populations in communities in rural China, such as individuals at a high risk of disease reactivation from previous tuberculosis, should be targeted for latent infection screening and treatment with an interferon-γ releasing assay rather than a TST. National Science and Technology Major Project of China, Program for Changjiang Scholars and Innovative Research Team in University of China, CAMS Innovation Fund for Medical Sciences, and Sanming Project of Medicine in Shenzhen. Copyright © 2017 Elsevier Ltd. All rights reserved.

Antituberculosis activity of the molecular libraries screening center network library.

PubMed

Maddry, Joseph A; Ananthan, Subramaniam; Goldman, Robert C; Hobrath, Judith V; Kwong, Cecil D; Maddox, Clinton; Rasmussen, Lynn; Reynolds, Robert C; Secrist, John A; Sosa, Melinda I; White, E Lucile; Zhang, Wei

2009-09-01

There is an urgent need for the discovery and development of new antitubercular agents that target novel biochemical pathways and treat drug-resistant forms of the disease. One approach to addressing this need is through high-throughput screening of drug-like small molecule libraries against the whole bacterium in order to identify a variety of new, active scaffolds that will stimulate additional biological research and drug discovery. Through the Molecular Libraries Screening Center Network, the NIAID Tuberculosis Antimicrobial Acquisition and Coordinating Facility tested a 215,110-compound library against Mycobacterium tuberculosis strain H37Rv. A medicinal chemistry survey of the results from the screening campaign is reported herein.

APC targeting enhances immunogenicity of a novel multistage Fc-fusion tuberculosis vaccine in mice.

PubMed

Soleimanpour, Saman; Farsiani, Hadi; Mosavat, Arman; Ghazvini, Kiarash; Eydgahi, Mohammad Reza Akbari; Sankian, Mojtaba; Sadeghian, Hamid; Meshkat, Zahra; Rezaee, Seyed Abdolrahim

2015-12-01

Numerous studies have demonstrated that targeting immunogens to FcγR on antigen-presenting cells (APCs) can selectively uptake and increase cellular immunity in vitro and in vivo. Therefore, the present study was conducted to evaluate immunogenicity of a novel multistage tuberculosis vaccine, a combination of an early and a dormant immunogenic protein, ESAT6 and HspX, fused to Fcγ2a fragment of mouse IgG2a to target all forms of tuberculosis. Codon-optimized genes consisting of ESAT6, a linker, and HspX fused either to mouse Fcγ2a (ESAT6:HspX:mFcγ2a) or 6× His-tag (ESAT6:HspX:His) were synthesized. The resulting proteins were then produced in Pichia pastoris. The fusion proteins were separately emulsified in dimethyldioctadecylammonium bromide(DDA)-trehalose-6,6-dibehenate(TDB) adjuvant, and their immunogenicity with and without bacille Calmette-Guérin (BCG) was assessed in C57BL/6 mice. Th1, Th2, Th17, and T-reg cytokine patterns were evaluated using the ELISA method. Both multistage vaccines induced very strong IL-12 and IFN-γ secretion from splenic cells; the Fc-tagged subunit vaccine induced a more effective Th1 immune response (IFN-γ, 910 pg/mL, and IL-12, 854 pg/mL) with a very low increase in IL-17 (∼0.1 pg/mL) and IL-4 (37 pg/mL) and a mild increase in TGF-β (543 pg/mL) compared to the BCG or ESAT6:HspX:His primed and boosted groups. The production of IFN-γ to ESAT6:HspX:Fcγ2a was very consistent and showed an increasing trend for IL-12 compared to the BCG or ESAT6:HspX:His primed and boosted groups. Fcγ2a used as a delivery vehicle supported the idea of selective uptake, inducing cross-presentation and forming a proper anti-tuberculosis response in context of Th1/Th2 and Th17/T-reg balances, which is important for protection and prevention of damage.

Point-of-care C-reactive protein-based tuberculosis screening for people living with HIV: a diagnostic accuracy study.

PubMed

Yoon, Christina; Semitala, Fred C; Atuhumuza, Elly; Katende, Jane; Mwebe, Sandra; Asege, Lucy; Armstrong, Derek T; Andama, Alfred O; Dowdy, David W; Davis, J Luke; Huang, Laurence; Kamya, Moses; Cattamanchi, Adithya

2017-12-01

Symptom-based screening for tuberculosis is recommended for all people living with HIV. This recommendation results in unnecessary Xpert MTB/RIF testing in many individuals living in tuberculosis-endemic areas and thus poor implementation of intensified case finding and tuberculosis preventive therapy. Novel approaches to tuberculosis screening are needed to help achieve global targets for tuberculosis elimination. We assessed the performance of C-reactive protein (CRP) measured with a point-of-care assay as a screening tool for active pulmonary tuberculosis. For this prospective study, we enrolled adults (aged ≥18 years) living with HIV with CD4 cell count less than or equal to 350 cells per μL who were initiating antiretroviral therapy (ART) from two HIV/AIDS clinics in Uganda. CRP concentrations were measured at study entry with a point-of-care assay using whole blood obtained by fingerprick (concentration ≥10 mg/L defined as screen positive for tuberculosis). Sputum samples were collected for Xpert MTB/RIF testing and culture. We calculated the sensitivity and specificity of point-of-care CRP and WHO symptom-based screening in reference to culture results. We repeated the sensitivity analysis with Xpert MTB/RIF as the reference standard. Between July 8, 2013, and Dec 15, 2015, 1237 HIV-infected adults were enrolled and underwent point-of-care CRP testing. 60 (5%) patients with incomplete or contaminated cultures were excluded from the analysis. Of the remaining 1177 patients (median CD4 count 165 cells per μL [IQR 75-271]), 163 (14%) had culture-confirmed tuberculosis. Point-of-care CRP testing had 89% sensitivity (145 of 163, 95% CI 83-93) and 72% specificity (731 of 1014, 95% CI 69-75) for culture-confirmed tuberculosis. Compared with WHO symptom-based screening, point-of-care CRP testing had lower sensitivity (difference -7%, 95% CI -12 to -2; p=0·002) but substantially higher specificity (difference 58%, 95% CI 55 to 61; p<0·0001). When Xpert MTB/RIF results were used as the reference standard, sensitivity of point-of-care CRP and WHO symptom-based screening were similar (94% [79 of 84] vs 99% [83 of 84], respectively; difference -5%, 95% CI -12 to 2; p=0·10). The performance characteristics of CRP support its use as a tuberculosis screening test for people living with HIV with CD4 count less than or equal to 350 cells per μL who are initiating ART. HIV/AIDS programmes should consider point-of-care CRP-based tuberculosis screening to improve the efficiency of intensified case finding and increase uptake of tuberculosis preventive therapy. National Institutes of Health; President's Emergency Plan for AIDS Relief; University of California, San Francisco, Nina Ireland Program for Lung Health. Copyright © 2017 Elsevier Ltd. All rights reserved.

Crowd Sourcing a New Paradigm for Interactome Driven Drug Target Identification in Mycobacterium tuberculosis

PubMed Central

Rohira, Harsha; Bhat, Ashwini G.; Passi, Anurag; Mukherjee, Keya; Choudhary, Kumari Sonal; Kumar, Vikas; Arora, Anshula; Munusamy, Prabhakaran; Subramanian, Ahalyaa; Venkatachalam, Aparna; S, Gayathri; Raj, Sweety; Chitra, Vijaya; Verma, Kaveri; Zaheer, Salman; J, Balaganesh; Gurusamy, Malarvizhi; Razeeth, Mohammed; Raja, Ilamathi; Thandapani, Madhumohan; Mevada, Vishal; Soni, Raviraj; Rana, Shruti; Ramanna, Girish Muthagadhalli; Raghavan, Swetha; Subramanya, Sunil N.; Kholia, Trupti; Patel, Rajesh; Bhavnani, Varsha; Chiranjeevi, Lakavath; Sengupta, Soumi; Singh, Pankaj Kumar; Atray, Naresh; Gandhi, Swati; Avasthi, Tiruvayipati Suma; Nisthar, Shefin; Anurag, Meenakshi; Sharma, Pratibha; Hasija, Yasha; Dash, Debasis; Sharma, Arun; Scaria, Vinod; Thomas, Zakir; Chandra, Nagasuma; Brahmachari, Samir K.; Bhardwaj, Anshu

2012-01-01

A decade since the availability of Mycobacterium tuberculosis (Mtb) genome sequence, no promising drug has seen the light of the day. This not only indicates the challenges in discovering new drugs but also suggests a gap in our current understanding of Mtb biology. We attempt to bridge this gap by carrying out extensive re-annotation and constructing a systems level protein interaction map of Mtb with an objective of finding novel drug target candidates. Towards this, we synergized crowd sourcing and social networking methods through an initiative ‘Connect to Decode’ (C2D) to generate the first and largest manually curated interactome of Mtb termed ‘interactome pathway’ (IPW), encompassing a total of 1434 proteins connected through 2575 functional relationships. Interactions leading to gene regulation, signal transduction, metabolism, structural complex formation have been catalogued. In the process, we have functionally annotated 87% of the Mtb genome in context of gene products. We further combine IPW with STRING based network to report central proteins, which may be assessed as potential drug targets for development of drugs with least possible side effects. The fact that five of the 17 predicted drug targets are already experimentally validated either genetically or biochemically lends credence to our unique approach. PMID:22808064

Rv2969c, essential for optimal growth in Mycobacterium tuberculosis, is a DsbA-like enzyme that interacts with VKOR-derived peptides and has atypical features of DsbA-like disulfide oxidases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Premkumar, Lakshmanane, E-mail: p.lakshmanane@imb.uq.edu.au; Heras, Begoña; Duprez, Wilko

The gene product of M. tuberculosis Rv2969c is shown to be a disulfide oxidase enzyme that has a canonical DsbA-like fold with novel structural and functional characteristics. The bacterial disulfide machinery is an attractive molecular target for developing new antibacterials because it is required for the production of multiple virulence factors. The archetypal disulfide oxidase proteins in Escherichia coli (Ec) are DsbA and DsbB, which together form a functional unit: DsbA introduces disulfides into folding proteins and DsbB reoxidizes DsbA to maintain it in the active form. In Mycobacterium tuberculosis (Mtb), no DsbB homologue is encoded but a functionally similarmore » but structurally divergent protein, MtbVKOR, has been identified. Here, the Mtb protein Rv2969c is investigated and it is shown that it is the DsbA-like partner protein of MtbVKOR. It is found that it has the characteristic redox features of a DsbA-like protein: a highly acidic catalytic cysteine, a highly oxidizing potential and a destabilizing active-site disulfide bond. Rv2969c also has peptide-oxidizing activity and recognizes peptide segments derived from the periplasmic loops of MtbVKOR. Unlike the archetypal EcDsbA enzyme, Rv2969c has little or no activity in disulfide-reducing and disulfide-isomerase assays. The crystal structure of Rv2969c reveals a canonical DsbA fold comprising a thioredoxin domain with an embedded helical domain. However, Rv2969c diverges considerably from other DsbAs, including having an additional C-terminal helix (H8) that may restrain the mobility of the catalytic helix H1. The enzyme is also characterized by a very shallow hydrophobic binding surface and a negative electrostatic surface potential surrounding the catalytic cysteine. The structure of Rv2969c was also used to model the structure of a paralogous DsbA-like domain of the Ser/Thr protein kinase PknE. Together, these results show that Rv2969c is a DsbA-like protein with unique properties and a limited substrate-binding specificity.« less

Diagnostic accuracy of nucleic acid amplification tests (NAATs) in urine for genitourinary tuberculosis: a systematic review and meta-analysis.

PubMed

Altez-Fernandez, Carlos; Ortiz, Victor; Mirzazadeh, Majid; Zegarra, Luis; Seas, Carlos; Ugarte-Gil, Cesar

2017-06-05

Genitourinary tuberculosis is the third most common form of extrapulmonary tuberculosis. Diagnosis is difficult because of unspecific clinical manifestations and low accuracy of conventional tests. Unfortunately, the delayed diagnosis impacts the urinary tract severely. Nucleic acid amplification tests yield fast results, and among these, new technologies can also detect drug resistance. There is lack of consensus regarding the use of these tests in genitourinary tuberculosis; we therefore aimed to assess the accuracy of nucleic acid amplification tests in the diagnosis of genitourinary tuberculosis and to evaluate the heterogeneity between studies. We did a systematic review and meta-analysis of research articles comparing the accuracy of a reference standard and a nucleic acid amplification test for diagnosis of urinary tract tuberculosis. We searched Medline, EMBASE, Web of Science, LILACS, Cochrane Library, and Scopus for articles published between Jan 1, 1990, and Apr 14, 2016. Two investigators identified eligible articles and extracted data for individual study sites. We analyzed data in groups with the same index test. Then, we generated pooled summary estimates (95% CIs) for sensitivity and specificity by use of random-effects meta-analysis when studies were not heterogeneous. We identified eleven relevant studies from ten articles, giving information on PCR, LCR and Xpert MTB/RIF tests. All PCR studies were "in-house" tests, with different gene targets and had several quality concerns therefore we did not proceed with a pooled analysis. Only one study used LCR. Xpert studies were of good quality and not heterogeneous, pooled sensitivity was 0·87 (0·66-0·96) and specificity was 0·91 (0·84-0·95). PCR studies were highly heterogeneous. Among Xpert MTB/RIF studies, specificity was favorable with an acceptable confidence interval, however new studies can update meta-analysis and get more precise estimates. Further high-quality studies are urgently needed to improve diagnosis of genitourinary tuberculosis. PROSPERO CRD42016039020.

Human Tuberculosis Caused by Mycobacterium bovis in the United States, 2006-2013.

PubMed

Scott, Colleen; Cavanaugh, Joseph S; Pratt, Robert; Silk, Benjamin J; LoBue, Philip; Moonan, Patrick K

2016-09-01

Using genotyping techniques that have differentiated Mycobacterium bovis from Mycobacterium tuberculosis since 2005, we review the epidemiology of human tuberculosis caused by M. bovis in the United States and validate previous findings nationally. All tuberculosis cases with a genotyped M. tuberculosis complex isolate reported during 2006-2013 in the United States were eligible for analysis. We used binomial regression to identify characteristics independently associated with M. bovis disease using adjusted prevalence ratios (aPRs) and corresponding 95% confidence intervals (CIs). During 2006-2013, the annual percentages of tuberculosis cases attributable to M. bovis remained consistent nationally (range, 1.3%-1.6%) among all tuberculosis cases (N = 59 273). Compared with adults 25-44 years of age, infants aged 0-4 years (aPR, 1.9 [95% CI, 1.4-2.8]) and children aged 5-14 years (aPR, 4.0 [95% CI, 3.1-5.3]) had higher prevalences of M. bovis disease. Patients who were foreign-born (aPR, 1.4 [95% CI, 1.2-1.7]), Hispanic (aPR, 3.9 [95% CI, 3.0-5.0]), female (aPR, 1.4 [95% CI, 1.3-1.6]), and resided in US-Mexico border counties (aPR, 2.0 [95% CI, 1.7-2.4]) also had higher M. bovis prevalences. Exclusively extrapulmonary disease (aPR, 3.7 [95% CI, 3.3-4.2]) or disease that was both pulmonary and extrapulmonary (aPR, 2.4 [95% CI, 2.1-2.9]) were associated with a higher prevalence of M. bovis disease. Children, foreign-born persons, Hispanics, and females are disproportionately affected by M. bovis, which was independently associated with extrapulmonary disease. Targeted prevention efforts aimed at Hispanic mothers and caregivers are warranted. Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Infection rate and tissue localization of murine IL-12p40-producing monocyte-derived CD103(+) lung dendritic cells during pulmonary tuberculosis.

PubMed

Leepiyasakulchai, Chaniya; Taher, Chato; Chuquimia, Olga D; Mazurek, Jolanta; Söderberg-Naucler, Cecilia; Fernández, Carmen; Sköld, Markus

2013-01-01

Non-hematopoietic cells, including lung epithelial cells, influence host immune responses. By co-culturing primary alveolar epithelial cells and monocytes from naïve donor mice, we show that alveolar epithelial cells support monocyte survival and differentiation in vitro, suggesting a role for non-hematopoietic cells in monocyte differentiation during the steady state in vivo. CD103(+) dendritic cells (αE-DC) are present at mucosal surfaces. Using a murine primary monocyte adoptive transfer model, we demonstrate that αE-DC in the lungs and pulmonary lymph nodes are monocyte-derived during pulmonary tuberculosis. The tissue localization may influence the functional potential of αE-DC that accumulate in Mycobacterium tuberculosis-infected lungs. Here, we confirm the localization of αE-DC in uninfected mice beneath the bronchial epithelial cell layer and near the vascular wall, and show that αE-DC have a similar distribution in the lungs during pulmonary tuberculosis and are detected in the bronchoalveolar lavage fluid from infected mice. Lung DC can be targeted by M. tuberculosis in vivo and play a role in bacterial dissemination to the draining lymph node. In contrast to other DC subsets, only a fraction of lung αE-DC are infected with the bacterium. We also show that virulent M. tuberculosis does not significantly alter cell surface expression levels of MHC class II on infected cells in vivo and that αE-DC contain the highest frequency of IL-12p40(+) cells among the myeloid cell subsets in infected lungs. Our results support a model in which inflammatory monocytes are recruited into the M. tuberculosis-infected lung tissue and, depending on which non-hematopoietic cells they interact with, differentiate along different paths to give rise to multiple monocyte-derived cells, including DC with a distinctive αE-DC phenotype.

Infection Rate and Tissue Localization of Murine IL-12p40-Producing Monocyte-Derived CD103+ Lung Dendritic Cells during Pulmonary Tuberculosis

PubMed Central

Leepiyasakulchai, Chaniya; Taher, Chato; Chuquimia, Olga D.; Mazurek, Jolanta; Söderberg-Naucler, Cecilia; Fernández, Carmen; Sköld, Markus

2013-01-01

Non-hematopoietic cells, including lung epithelial cells, influence host immune responses. By co-culturing primary alveolar epithelial cells and monocytes from naïve donor mice, we show that alveolar epithelial cells support monocyte survival and differentiation in vitro, suggesting a role for non-hematopoietic cells in monocyte differentiation during the steady state in vivo. CD103+ dendritic cells (αE-DC) are present at mucosal surfaces. Using a murine primary monocyte adoptive transfer model, we demonstrate that αE-DC in the lungs and pulmonary lymph nodes are monocyte-derived during pulmonary tuberculosis. The tissue localization may influence the functional potential of αE-DC that accumulate in Mycobacterium tuberculosis-infected lungs. Here, we confirm the localization of αE-DC in uninfected mice beneath the bronchial epithelial cell layer and near the vascular wall, and show that αE-DC have a similar distribution in the lungs during pulmonary tuberculosis and are detected in the bronchoalveolar lavage fluid from infected mice. Lung DC can be targeted by M. tuberculosis in vivo and play a role in bacterial dissemination to the draining lymph node. In contrast to other DC subsets, only a fraction of lung αE-DC are infected with the bacterium. We also show that virulent M. tuberculosis does not significantly alter cell surface expression levels of MHC class II on infected cells in vivo and that αE-DC contain the highest frequency of IL-12p40+ cells among the myeloid cell subsets in infected lungs. Our results support a model in which inflammatory monocytes are recruited into the M. tuberculosis-infected lung tissue and, depending on which non-hematopoietic cells they interact with, differentiate along different paths to give rise to multiple monocyte-derived cells, including DC with a distinctive αE-DC phenotype. PMID:23861965

QuantiFERON-TB Gold In-Tube as a Confirmatory Test for Tuberculin Skin Test in Tuberculosis Contact Tracing: A Noninferiority Clinical Trial.

PubMed

Muñoz, Laura; Santin, Miguel; Alcaide, Fernando; Ruíz-Serrano, Maria Jesús; Gijón, Paloma; Bermúdez, Elena; Domínguez-Castellano, Angel; Navarro, María Dolores; Ramírez, Encarnación; Pérez-Escolano, Elvira; López-Prieto, María Dolores; Gutiérrez-Rodriguez, José; Anibarro, Luis; Calviño, Laura; Trigo, Matilde; Cifuentes, Carmen; García-Gasalla, Mercedes; Payeras, Antoni; Gasch, Oriol; Espasa, Mateu; Agüero, Ramon; Ferrer, Diego; Casas, Xavier; González-Cuevas, Araceli; García-Zamalloa, Alberto; Bikuña, Edurne; Lecuona, María; Galindo, Rosa; Ramírez-Lapausa, Marta; Carrillo, Raquel

2018-01-18

Screening strategies based on interferon-γ release assays in tuberculosis contact tracing may reduce the need for preventive therapy without increasing subsequent active disease. We conducted an open-label, randomized trial to test the noninferiority of a 2-step strategy with the tuberculin skin test (TST) followed by QuantiFERON-TB Gold In-Tube (QFT-GIT) as a confirmatory test (the TST/QFT arm) to the standard TST-alone strategy (TST arm) for targeting preventive therapy in household contacts of patients with tuberculosis. Participants were followed for 24 months after randomization. The primary endpoint was the development of tuberculosis, with a noninferiority margin of 1.5 percentage points. A total of 871 contacts were randomized. Four contacts in the TST arm and 2 in the TST/QFT arm developed tuberculosis. In the modified intention-to-treat analysis, this accounted for 0.99% in the TST arm and 0.51% in the TST/QFT arm (-0.48% difference; 97.5% confidence interval [CI], -1.86% to 0.90%); in the per-protocol analysis, the corresponding rates were 1.67% and 0.82% in the TST and TST/QFT arms, respectively (-0.85% difference; 97.5% CI, -3.14% to 1.43%). Of the 792 contacts analyzed, 65.3% in the TST arm and 42.2% in the TST/QFT arm were diagnosed with tuberculosis infection (23.1% difference; 95% CI, 16.4% to 30.0%). In low-incidence settings, screening household contacts with the TST and using QFT-GIT as a confirmatory test is not inferior to TST-alone for preventing active tuberculosis, allowing a safe reduction of preventive treatments. NCT01223534. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Comparing Galactan Biosynthesis in Mycobacterium tuberculosis and Corynebacterium diphtheriae*

PubMed Central

Wesener, Darryl A.; Levengood, Matthew R.

2017-01-01

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. PMID:28039359

Rapid detection of rifampin resistance in Mycobacterium tuberculosis isolates from India and Mexico by a molecular beacon assay.

PubMed

Varma-Basil, Mandira; El-Hajj, Hiyam; Colangeli, Roberto; Hazbón, Manzour Hernando; Kumar, Sujeet; Bose, Mridula; Bobadilla-del-Valle, Miriam; García, Lourdes García; Hernández, Araceli; Kramer, Fred Russell; Osornio, Jose Sifuentes; Ponce-de-León, Alfredo; Alland, David

2004-12-01

We assessed the performance of a rapid, single-well, real-time PCR assay for the detection of rifampin-resistant Mycobacterium tuberculosis by using clinical isolates from north India and Mexico, regions with a high incidence of tuberculosis. The assay uses five differently colored molecular beacons to determine if a short region of the M. tuberculosis rpoB gene contains mutations that predict rifampin resistance in most isolates. Until now, the assay had not been sufficiently tested on samples from countries with a high incidence of tuberculosis. In the present study, the assay detected mutations in 16 out of 16 rifampin-resistant isolates from north India (100%) and in 55 of 64 rifampin-resistant isolates from Mexico (86%) compared to results with standard susceptibility testing. The assay did not detect mutations (a finding predictive of rifampin susceptibility) in 37 out of 37 rifampin-susceptible isolates from India (100%) and 125 out of 126 rifampin-susceptible isolates from Mexico (99%). DNA sequencing revealed that none of the nine rifampin-resistant isolates from Mexico, which were misidentified as rifampin susceptible by the molecular beacon assay, contained a mutation in the region targeted by the molecular beacons. The one rifampin-susceptible isolate from Mexico that appeared to be rifampin resistant by the molecular beacon assay contained an S531W mutation, which is usually associated with rifampin resistance. Of the rifampin-resistant isolates that were correctly identified in the molecular beacon assay, one contained a novel L530A mutation and another contained a novel deletion between codons 511 and 514. Overall, the molecular beacon assay appears to have sufficient sensitivity (89%) and specificity (99%) for use in countries with a high prevalence of tuberculosis.

Absorption, distribution and excretion of the anti-tuberculosis drug delamanid in rats: Extensive tissue distribution suggests potential therapeutic value for extrapulmonary tuberculosis.

PubMed

Shibata, Masakazu; Shimokawa, Yoshihiko; Sasahara, Katsunori; Yoda, Noriaki; Sasabe, Hiroyuki; Suzuki, Mitsunari; Umehara, Ken

2017-05-01

Delamanid (OPC-67683, Deltyba™, nitro-dihydro-imidazooxazoles derivative) is approved for the treatment of adult pulmonary multidrug-resistant tuberculosis. The absorption, distribution and excretion of delamanid-derived radioactivity were investigated after a single oral administration of 14 C-delamanid at 3 mg/kg to rats. In both male and female rats, radioactivity in blood and all tissues reached peak levels by 8 or 24 h post-dose, and thereafter decreased slowly. Radioactivity levels were 3- to 5-fold higher in lung tissue at time to maximum concentration compared with plasma. In addition, radioactivity was broadly distributed in various tissues, including the central nervous system, eyeball, placenta and fetus, indicating that 14 C-delamanid permeated the brain, retinal and placental blood barriers. By 168 h post-dose, radioactivity in almost all the tissues was higher than that in the plasma. Radioactivity was also transferred into the milk of lactating rats. Approximately 6% and 92% of radioactivity was excreted in the urine and feces, respectively, indicating that the absorbed radioactivity was primarily excreted via the biliary route. No significant differences in the absorption, distribution and excretion of 14 C-delamanid were observed between male and female rats. The pharmacokinetic results suggested that delamanid was broadly distributed to the lungs and various tissues for a prolonged duration of time at concentrations expected to effectively target tuberculosis bacteria. These data indicate that delamanid, in addition to its previously demonstrated efficacy in pulmonary tuberculosis, might be an effective therapeutic approach to treating extrapulmonary tuberculosis. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Transmission of Mycobacterium Tuberculosis in Households and the Community: A Systematic Review and Meta-Analysis.

PubMed

Martinez, Leonardo; Shen, Ye; Mupere, Ezekiel; Kizza, Allan; Hill, Philip C; Whalen, Christopher C

2017-06-15

The individual- and population-level impact of household tuberculosis exposure on transmission is unclear but may have implications for the effectiveness and implementation of control interventions. We systematically searched for and included studies in which latent tuberculosis infection was assessed in 2 groups: children exposed and unexposed to a household member with tuberculosis. We also extracted data on the smear and culture status of index cases, the age and bacillus Calmette-Guérin vaccination status of contacts, and study design characteristics. Of 6,176 citations identified from our search strategy, 26 studies (13,999 children with household exposure to tuberculosis and 174,097 children without) from 1929-2015 met inclusion criteria. Exposed children were 3.79 (95% confidence interval (CI): 3.01, 4.78) times more likely to be infected than were their community counterparts. Metaregression demonstrated higher infection among children aged 0-4 years of age compared with children aged 10-14 years (ratio of odds ratios = 2.24, 95% CI: 1.43, 3.51) and among smear-positive versus smear-negative index cases (ratio of odds ratios = 5.45, 95% CI: 3.43, 8.64). At the population level, we estimated that a small proportion (<20%) of transmission was attributable to household exposure. Our results suggest that targeting tuberculosis prevention efforts to household contacts is highly effective. However, a large proportion of transmission at the population level may occur outside the household. © The Author 2017. Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Direct detection of Mycobacterium tuberculosis complex in bovine and bubaline tissues through nested-PCR.

PubMed

Araújo, Cristina P; Osório, Ana Luiza A R; Jorge, Klaudia S G; Ramos, Carlos A N; Souza Filho, Antonio F; Vidal, Carlos E S; Vargas, Agueda P C; Roxo, Eliana; Rocha, Adalgiza S; Suffys, Philip N; Fonseca, Antônio A; Silva, Marcio R; Barbosa Neto, José D; Cerqueira, Valíria D; Araújo, Flábio R

2014-01-01

Post-mortem bacterial culture and specific biochemical tests are currently performed to characterize the etiologic agent of bovine tuberculosis. Cultures take up to 90 days to develop. A diagnosis by molecular tests such as PCR can provide fast and reliable results while significantly decreasing the time of confirmation. In the present study, a nested-PCR system, targeting rv2807, with conventional PCR followed by real-time PCR, was developed to detect Mycobacterium tuberculosis complex (MTC) organisms directly from bovine and bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other Actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. Regarding the analytical sensitivity, DNA of the M. bovis AN5 strain was detected up to 1.5 pg by nested-PCR, whereas DNA of M. tuberculosis H37Rv strain was detected up to 6.1 pg. The nested-PCR system showed 100% analytical specificity for MTC when tested with DNA of reference strains of non-tuberculous mycobacteria and closely-related Actinomycetales. A clinical sensitivity level of 76.7% was detected with tissues samples positive for MTC by means of the culture and conventional PCR. A clinical specificity of 100% was detected with DNA from tissue samples of cattle with negative results in the comparative intradermal tuberculin test. These cattle exhibited no visible lesions and were negative in the culture for MTC. The use of the nested-PCR assay to detect M. tuberculosis complex in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.

Direct detection of Mycobacterium tuberculosis complex in bovine and bubaline tissues through nested-PCR

PubMed Central

Araújo, Cristina P.; Osório, Ana Luiza A.R.; Jorge, Klaudia S.G.; Ramos, Carlos A.N.; Souza Filho, Antonio F.; Vidal, Carlos E.S.; Vargas, Agueda P.C.; Roxo, Eliana; Rocha, Adalgiza S.; Suffys, Philip N.; Fonseca, Antônio A.; Silva, Marcio R.; Barbosa Neto, José D.; Cerqueira, Valíria D.; Araújo, Flábio R.

2014-01-01

Post-mortem bacterial culture and specific biochemical tests are currently performed to characterize the etiologic agent of bovine tuberculosis. Cultures take up to 90 days to develop. A diagnosis by molecular tests such as PCR can provide fast and reliable results while significantly decreasing the time of confirmation. In the present study, a nested-PCR system, targeting rv2807, with conventional PCR followed by real-time PCR, was developed to detect Mycobacterium tuberculosis complex (MTC) organisms directly from bovine and bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other Actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. Regarding the analytical sensitivity, DNA of the M. bovis AN5 strain was detected up to 1.5 pg by nested-PCR, whereas DNA of M. tuberculosis H37Rv strain was detected up to 6.1 pg. The nested-PCR system showed 100% analytical specificity for MTC when tested with DNA of reference strains of non-tuberculous mycobacteria and closely-related Actinomycetales. A clinical sensitivity level of 76.7% was detected with tissues samples positive for MTC by means of the culture and conventional PCR. A clinical specificity of 100% was detected with DNA from tissue samples of cattle with negative results in the comparative intradermal tuberculin test. These cattle exhibited no visible lesions and were negative in the culture for MTC. The use of the nested-PCR assay to detect M. tuberculosis complex in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis. PMID:25242951

Spatial analysis of deaths from pulmonary tuberculosis in the city of São Luís, Brazil*

PubMed Central

Santos-Neto, Marcelino; Yamamura, Mellina; Garcia, Maria Concebida da Cunha; Popolin, Marcela Paschoal; Silveira, Tatiane Ramos dos Santos; Arcêncio, Ricardo Alexandre

2014-01-01

OBJECTIVE: To characterize deaths from pulmonary tuberculosis, according to sociodemographic and operational variables, in the city of São Luís, Brazil, and to describe their spatial distribution. METHODS: This was an exploratory ecological study based on secondary data from death certificates, obtained from the Brazilian Mortality Database, related to deaths from pulmonary tuberculosis. We included all deaths attributed to pulmonary tuberculosis that occurred in the urban area of São Luís between 2008 and 2012. We performed univariate and bivariate analyses of the sociodemographic and operational variables of the deaths investigated, as well as evaluating the spatial distribution of the events by kernel density estimation. RESULTS: During the study period, there were 193 deaths from pulmonary tuberculosis in São Luís. The median age of the affected individuals was 52 years. Of the 193 individuals who died, 142 (73.60%) were male, 133 (68.91%) were Mulatto, 102 (53.13%) were single, and 64 (33.16%) had completed middle school. There was a significant positive association between not having received medical care prior to death and an autopsy having been performed (p = 0.001). A thematic map by density of points showed that the spatial distribution of those deaths was heterogeneous and that the density was as high as 8.12 deaths/km2. CONCLUSIONS: The sociodemographic and operational characteristics of the deaths from pulmonary tuberculosis evaluated in this study, as well as the identification of priority areas for control and surveillance of the disease, could promote public health policies aimed at reducing health inequities, allowing the optimization of resources, as well as informing decisions regarding the selection of strategies and specific interventions targeting the most vulnerable populations. PMID:25410843

The 1.25 Å resolution structure of phosphoribosyl-ATP pyrophosphohydrolase from Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Javid-Majd, Farah; Yang, Dong; Ioerger, Thomas R.

2008-06-01

The crystal structure of M. tuberculosis phosphoribosyl-ATP pyrophosphohydrolase, the second enzyme in the histidine-biosynthetic pathway, is presented. The structural and inferred functional relationships between M. tuberculosis phosphoribosyl-ATP pyrophosphohydrolase and other members of the nucleoside-triphosphate pyrophosphatase-fold family are described. Phosphoribosyl-ATP pyrophosphohydrolase is the second enzyme in the histidine-biosynthetic pathway, irreversibly hydrolyzing phosphoribosyl-ATP to phosphoribosyl-AMP and pyrophosphate. It is encoded by the hisE gene, which is present as a separate gene in many bacteria and archaea but is fused to hisI in other bacteria, fungi and plants. Because of its essentiality for growth in vitro, HisE is a potential drug target formore » tuberculosis. The crystal structures of two native (uncomplexed) forms of HisE from Mycobacterium tuberculosis have been determined to resolutions of 1.25 and 1.79 Å. The structure of the apoenzyme reveals that the protein is composed of five α-helices with connecting loops and is a member of the α-helical nucleoside-triphosphate pyrophosphatase superfamily. The biological unit of the protein is a homodimer, with an active site on each subunit composed of residues exclusively from that subunit. A comparison with the Campylobacter jejuni dUTPase active site allowed the identification of putative metal- and substrate-binding sites in HisE, including four conserved glutamate and glutamine residues in the sequence that are consistent with a motif for pyrophosphohydrolase activity. However, significant differences between family members are observed in the loop region between α-helices H1 and H3. The crystal structure of M. tuberculosis HisE provides insights into possible mechanisms of substrate binding and the diversity of the nucleoside-triphosphate pyrophosphatase superfamily.« less

The application of anti-ESAT-6 monoclonal antibody fluorescent probe in ex vivo near-infrared fluorescence imaging in mice with pulmonary tuberculosis.

PubMed

Feng, Feng; Zhang, Haoling; Zhu, Zhaoqin; Li, Cong; Shi, Yuxin; Zhang, Zhiyong

2014-09-01

Here, we aimed to assess the feasibility of anti-ESAT-6 monoclonal antibody (mAb) coupling with IR783 and rhodamine fluorescent probe in the detection of ESAT-6 expression in tuberculosis tissue of mice using near-infrared fluorescence imaging. IR783 and rhodamine were conjugated to the anti-ESAT-6 mAb or IgG. Mice in the experimental group were injected with fluorescence-labeled mAb probe, and mice in the control group were injected with fluorescence-labeled non-specific IgG antibody. Twenty-four hours later, the lung tissue of mice was examined using ex vivo near-infrared fluorescence imaging. In addition, the contrast-to-noise ratio (CNR) was calculated by measuring the signal intensities of the pulmonary lesions, normal lung tissue and background noise. The frozen lung tissue section was examined under fluorescence microscopy and compared with hemoxylin and eosin (HE) staining. The ex vivo near-infrared fluorescence imaging showed that the fluorescence signal in the lung tuberculosis lesions in the experimental group was significantly enhanced, whereas there was only a weak fluorescence signal or even no fluorescence signal in the control group. CNR values were 64.40 ± 7.02 (n = 6) and 8.75 ± 3.87 (n = 6), respectively (t = 17.01, p < 0.001). The fluorescence accumulation distribution detected under fluorescence microscopy was consistent with HE staining of the tuberculosis region. In conclusion, anti-ESAT-6 mAb fluorescent probe could target and be applied in specific ex vivo imaging of mice tuberculosis, and may be of further use in tuberculosis in living mice. Copyright © 2013 John Wiley & Sons, Ltd.

Spatial analysis of deaths from pulmonary tuberculosis in the city of São Luís, Brazil.

PubMed

Santos-Neto, Marcelino; Yamamura, Mellina; Garcia, Maria Concebida da Cunha; Popolin, Marcela Paschoal; Silveira, Tatiane Ramos Dos Santos; Arcêncio, Ricardo Alexandre

2014-10-01

To characterize deaths from pulmonary tuberculosis, according to sociodemographic and operational variables, in the city of São Luís, Brazil, and to describe their spatial distribution. This was an exploratory ecological study based on secondary data from death certificates, obtained from the Brazilian Mortality Database, related to deaths from pulmonary tuberculosis. We included all deaths attributed to pulmonary tuberculosis that occurred in the urban area of São Luís between 2008 and 2012. We performed univariate and bivariate analyses of the sociodemographic and operational variables of the deaths investigated, as well as evaluating the spatial distribution of the events by kernel density estimation. During the study period, there were 193 deaths from pulmonary tuberculosis in São Luís. The median age of the affected individuals was 52 years. Of the 193 individuals who died, 142 (73.60%) were male, 133 (68.91%) were Mulatto, 102 (53.13%) were single, and 64 (33.16%) had completed middle school. There was a significant positive association between not having received medical care prior to death and an autopsy having been performed (p = 0.001). A thematic map by density of points showed that the spatial distribution of those deaths was heterogeneous and that the density was as high as 8.12 deaths/km2. The sociodemographic and operational characteristics of the deaths from pulmonary tuberculosis evaluated in this study, as well as the identification of priority areas for control and surveillance of the disease, could promote public health policies aimed at reducing health inequities, allowing the optimization of resources, as well as informing decisions regarding the selection of strategies and specific interventions targeting the most vulnerable populations.

Directed evolution approach to a structural genomics project: Rv2002 from Mycobacterium tuberculosis.

PubMed

Yang, Jin Kuk; Park, Min S; Waldo, Geoffrey S; Suh, Se Won

2003-01-21

One of the serious bottlenecks in structural genomics projects is overexpression of the target proteins in soluble form. We have applied the directed evolution technique and prepared soluble mutants of the Mycobacterium tuberculosis Rv2002 gene product, the wild type of which had been expressed as inclusion bodies in Escherichia coli. A triple mutant I6TV47MT69K (Rv2002-M3) was chosen for structural and functional characterizations. Enzymatic assays indicate that the Rv2002-M3 protein has a high catalytic activity as a NADH-dependent 3alpha, 20beta-hydroxysteroid dehydrogenase. We have determined the crystal structures of a binary complex with NAD(+) and a ternary complex with androsterone and NADH. The structure reveals that Asp-38 determines the cofactor specificity. The catalytic site includes the triad Ser-140Tyr-153Lys-157. Additionally, it has an unusual feature, Glu-142. Enzymatic assays of the E142A mutant of Rv2002-M3 indicate that Glu-142 reverses the effect of Lys-157 in influencing the pKa of Tyr-153. This study suggests that the Rv2002 gene product is a unique member of the SDR family and is likely to be involved in steroid metabolism in M. tuberculosis. Our work demonstrates the power of the directed evolution technique as a general way of overcoming the difficulties in overexpressing the target proteins in soluble form.

Targeted screening and treatment for latent tuberculosis infection using QuantiFERON-TB Gold is cost-effective in Mexico.

PubMed

Burgos, J L; Kahn, J G; Strathdee, S A; Valencia-Mendoza, A; Bautista-Arredondo, S; Laniado-Laborin, R; Castañeda, R; Deiss, R; Garfein, R S

2009-08-01

To assess the cost-effectiveness of screening for latent tuberculosis infection (LTBI) using a commercially available detection test and treating individuals at high risk for human immunodeficiency virus (HIV) infection in a middle-income country. We developed a Markov model to evaluate the cost per LTBI case detected, TB case averted and quality-adjusted life year (QALY) gained for a cohort of 1000 individuals at high risk for HIV infection over 20 years. Baseline model inputs for LTBI prevalence were obtained from published literature and cross-sectional data from tuberculosis (TB) screening using QuantiFERON-TB Gold In-Tube (QFT-GIT) testing among sex workers and illicit drug users at high risk for HIV recruited through street outreach in Tijuana, Mexico. Costs are reported in 2007 US dollars. Future costs and QALYs were discounted at 3% per year. Sensitivity analyses were performed to evaluate model robustness. Over 20 years, we estimate the program would prevent 78 cases of active TB and 55 TB-related deaths. The incremental cost per case of LTBI detected was US$730, cost per active TB averted was US$529 and cost per QALY gained was US$108. In settings of endemic TB and escalating HIV incidence, targeting LTBI screening and treatment among high-risk groups may be highly cost-effective.

A Target-Based Whole Cell Screen Approach To Identify Potential Inhibitors of Mycobacterium tuberculosis Signal Peptidase

PubMed Central

2016-01-01

The general secretion (Sec) pathway is a conserved essential pathway in bacteria and is the primary route of protein export across the cytoplasmic membrane. During protein export, the signal peptidase LepB catalyzes the cleavage of the signal peptide and subsequent release of mature proteins into the extracellular space. We developed a target-based whole cell assay to screen for potential inhibitors of LepB, the sole signal peptidase in Mycobacterium tuberculosis, using a strain engineered to underexpress LepB (LepB-UE). We screened 72,000 compounds against both the Lep-UE and wild-type (wt) strains. We identified the phenylhydrazone (PHY) series as having higher activity against the LepB-UE strain. We conducted a limited structure–activity relationship determination around a representative PHY compound with differential activity (MICs of 3.0 μM against the LepB-UE strain and 18 μM against the wt); several analogues were less potent against the LepB overexpressing strain. A number of chemical modifications around the hydrazone moiety resulted in improved potency. Inhibition of LepB activity was observed for a number of compounds in a biochemical assay using cell membrane fraction derived from M. tuberculosis. Compounds did not increase cell permeability, dissipate membrane potential, or inhibit an unrelated mycobacterial enzyme, suggesting a specific mode of action related to the LepB secretory mechanism. PMID:27642770

Proteome analysis of ofloxacin and moxifloxacin induced mycobacterium tuberculosis isolates by proteomic approach.

PubMed

Lata, Manju; Sharma, Divakar; Kumar, Bhavnesh; Deo, Nirmala; Tiwari, Pramod Kumar; Bisht, Deepa; Venkatesan, Krishnamurthy

2015-01-01

Ofloxacin (OFX) and moxifloxacin (MOX) are the most promising second line drugs for tuberculosis treatment. Although the primary mechanism of action of OFX and MOX is gyrase inhibition, other possible mechanisms cannot be ruled out. Being the functional moiety of cell, the proteins act as primary targets for developing drugs, diagnostics and therapeutics. In this study we have investigated the proteomic changes of Mycobacterium tuberculosis isolates induced by OFX and MOX by applying comparative proteomic approaches based on two-dinensional gel electrophoresis (2DE) along with matrix assisted laser desorption ionisation time of flight mass spectrometry (MALDI TOF/TOF-MS) and bioinformatic tools. The findings are likely to provide new understanding of OFX and MOX mechanisms that might be helpful in exploring new diagnostics and drug targets. Our study explored eleven proteins (Rv2889c, Rv2623, Rv0952, Rv1827, Rv1932, Rv0054, Rv1080c, Rv3418c, Rv3914, Rv1636 and Rv0009) that were overexpressed in the presence of drugs. Among them, Rv2623, Rv1827 and Rv1636 were identified as proteins with unknown function. InterProScan and molecular docking revealed that the conserved domain of hypothetical proteins interact with OFX and MOX which indicate a probable inhibition/modulation of the functioning of these proteins by both drugs, which might be overexpressed to overcome this effect.

Advances in nanotechnology-based carrier systems for targeted delivery of bioactive drug molecules with special emphasis on immunotherapy in drug resistant tuberculosis - a critical review.

PubMed

Singh, Jagdeep; Garg, Tarun; Rath, Goutam; Goyal, Amit K

2016-06-01

From the early sixteenth and seventeenth centuries to the present day of life, tuberculosis (TB) still is a global health threat with some new emergence of resistance. This type of emergence poses a vital challenge to control TB cases across the world. Mortality and morbidity rates are high due to this new face of TB. The newer nanotechnology-based drug-delivery approaches involving micro-metric and nano-metric carriers are much needed at this stage. These delivery systems would provide more advantages over conventional systems of treatment by producing enhanced therapeutic efficacy, uniform distribution of drug molecule to the target site, sustained and controlled release of drug molecules and lesser side effects. The main aim to develop these novel drug-delivery systems is to improve the patient compliance and reduce therapy time. This article reviews and elaborates the new concepts and drug-delivery approaches for the treatment of TB involving solid-lipid particulate drug-delivery systems (solid-lipid micro- and nanoparticles, nanostructured lipid carriers), vesicular drug-delivery systems (liposomes, niosomes and liposphere), emulsion-based drug-delivery systems (micro and nanoemulsion) and some other novel drug-delivery systems for the effective treatment of tuberculosis and role of immunomodulators as an adjuvant therapy for management of MDR-TB and XDR-TB.

Targeted screening and treatment for latent tuberculosis infection using QuantiFERON®-TB Gold is cost-effective in Mexico

PubMed Central

Burgos, J. L.; Kahn, J. G.; Strathdee, S. A.; Valencia-Mendoza, A.; Bautista-Arredondo, S.; Laniado-Laborin, R.; Castañeda, R.; Deiss, R.; Garfein, R. S.

2009-01-01

SUMMARY OBJECTIVE To assess the cost-effectiveness of screening for latent tuberculosis infection (LTBI) using a commercially available detection test and treating individuals at high risk for human immunodeficiency virus (HIV) infection in a middle-income country. DESIGN We developed a Markov model to evaluate the cost per LTBI case detected, TB case averted and quality-adjusted life year (QALY) gained for a cohort of 1000 individuals at high risk for HIV infection over 20 years. Baseline model inputs for LTBI prevalence were obtained from published literature and cross-sectional data from tuberculosis (TB) screening using QuantiFERON®-TB Gold In-Tube (QFT-GIT) testing among sex workers and illicit drug users at high risk for HIV recruited through street outreach in Tijuana, Mexico. Costs are reported in 2007 US dollars. Future costs and QALYs were discounted at 3% per year. Sensitivity analyses were performed to evaluate model robustness. RESULTS Over 20 years, we estimate the program would prevent 78 cases of active TB and 55 TB-related deaths. The incremental cost per case of LTBI detected was US$730, cost per active TB averted was US$529 and cost per QALY gained was US$108. CONCLUSIONS In settings of endemic TB and escalating HIV incidence, targeting LTBI screening and treatment among high-risk groups may be highly cost-effective. PMID:19723375

Correlates of tuberculosis risk: predictive biomarkers for progression to active tuberculosis

PubMed Central

Petruccioli, Elisa; Scriba, Thomas J.; Petrone, Linda; Hatherill, Mark; Cirillo, Daniela M.; Joosten, Simone A.; Ottenhoff, Tom H.; Denkinger, Claudia M.; Goletti, Delia

2016-01-01

New approaches to control the spread of tuberculosis (TB) are needed, including tools to predict development of active TB from latent TB infection (LTBI). Recent studies have described potential correlates of risk, in order to inform the development of prognostic tests for TB disease progression. These efforts have included unbiased approaches employing “omics” technologies, as well as more directed, hypothesis-driven approaches assessing a small set or even individual selected markers as candidate correlates of TB risk. Unbiased high-throughput screening of blood RNAseq profiles identified signatures of active TB risk in individuals with LTBI, ≥1 year before diagnosis. A recent infant vaccination study identified enhanced expression of T-cell activation markers as a correlate of risk prior to developing TB; conversely, high levels of Ag85A antibodies and high frequencies of interferon (IFN)-γ specific T-cells were associated with reduced risk of disease. Others have described CD27−IFN-γ+CD4+ T-cells as possibly predictive markers of TB disease. T-cell responses to TB latency antigens, including heparin-binding haemagglutinin and DosR-regulon-encoded antigens have also been correlated with protection. Further studies are needed to determine whether correlates of risk can be used to prevent active TB through targeted prophylactic treatment, or to allow targeted enrolment into efficacy trials of new TB vaccines and therapeutic drugs. PMID:27836953

Detection of Bacterial Meningitis Pathogens by PCR-Mass Spectrometry in Cerebrospinal Fluid.

PubMed

Jing-Zi, Piao; Zheng-Xin, He; Wei-Jun, Chen; Yong-Qiang, Jiang

2018-06-01

Acute bacterial meningitis remains a life-threatening infectious disease with considerable morbidity and mortality. DNA-based detection methods are an urgent requisite for meningitis-causing bacterial pathogens for the prevention of outbreaks and control of infections. We proposed a novel PCR-mass spectrometry (PCR-Mass) assay for the simultaneous detection of four meningitis-causing agents, Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae, and Mycobacterium tuberculosis in the present study. A total of 138 cerebrospinal fluid (CSF) samples (including 56 CSF culture positive, 44 CSF culture negative, and 38 CSF control) were enrolled and analyzed by PCR/Mass. Results were compared to real-time PCR detection. These four targeting pathogens could be discriminated without cross-reaction by the accurate detection of the corresponding extension products with different masses. The limits of detection were 102 copies/reaction for S. pneumoniae, H. influenzae, and N. meningitidis and 103 for M. tuberculosis. The evaluation of the culture-positive CSF specimens from the meningitis patients provided an overall agreement rate of 85.7% with PCR-Mass and real-time PCR. The PCR-Mass was also able to detect the targeting pathogens from culture-negative CSF specimens from meningitis patients receiving early antibiotic treatment. PCR-Mass could be used for the molecular detection of bacterial meningitis and tuberculosis, especially when early antibiotic treatment has been administered to the suspected patients.

Advances in nanotechnology-based carrier systems for targeted delivery of bioactive drug molecules with special emphasis on immunotherapy in drug resistant tuberculosis - a critical review.

PubMed

Singh, Jagdeep; Garg, Tarun; Rath, Goutam; Goyal, Amit K

2015-08-11

From the early sixteenth and seventeenth centuries to the present day of life, tuberculosis (TB) still is a global health threat with some new emergence of resistance. This type of emergence poses a vital challenge to control TB cases across the world. Mortality and morbidity rates are high due to this new face of TB. The newer nanotechnology-based drug-delivery approaches involving micro-metric and nano-metric carriers are much needed at this stage. These delivery systems would provide more advantages over conventional systems of treatment by producing enhanced therapeutic efficacy, uniform distribution of drug molecule to the target site, sustained and controlled release of drug molecules and lesser side effects. The main aim to develop these novel drug-delivery systems is to improve the patient compliance and reduce therapy time. This article reviews and elaborates the new concepts and drug-delivery approaches for the treatment of TB involving solid-lipid particulate drug-delivery systems (solid-lipid micro- and nanoparticles, nanostructured lipid carriers), vesicular drug-delivery systems (liposomes, niosomes and liposphere), emulsion-based drug-delivery systems (micro and nanoemulsion) and some other novel drug-delivery systems for the effective treatment of tuberculosis and role of immunomodulators as an adjuvant therapy for management of MDR-TB and XDR-TB.

Differentiation of Mycobacterium tuberculosis complex from non-tubercular mycobacteria by nested multiplex PCR targeting IS6110, MTP40 and 32kD alpha antigen encoding gene fragments.

PubMed

Sinha, Pallavi; Gupta, Anamika; Prakash, Pradyot; Anupurba, Shampa; Tripathi, Rajneesh; Srivastava, G N

2016-03-12

Control of the global burden of tuberculosis is obstructed due to lack of simple, rapid and cost effective diagnostic techniques that can be used in resource poor-settings. To facilitate the early diagnosis of TB directly from clinical specimens, we have standardized and validated the use of nested multiplex PCR, targeting gene fragments IS6110, MTP40 and 32kD α-antigen encoding genes specific for Mycobacterium tuberculosis complex and non-tubercular mycobacteria (NTM), in comparison to smear microscopy, solid culture and single step multiplex PCR. The results were evaluated in comparison to a composite reference standard (CRS) comprising of microbiological results (smear and culture), clinical, radiological and cytopathological findings, clinical treatment and response to anti-tubercular therapy. The nested multiplex PCR (nMPCR) assay was evaluated to test its utility in 600 (535 pulmonary and 65 extra-pulmonary specimens) clinically suspected TB cases. All specimens were processed for smear, culture, single step multiplex PCR and nested multiplex PCR testing. Out of 535 screened pulmonary and 65 extra-pulmonary specimens, 329 (61.5%) and 19 (29.2%) cases were culture positive for M. tuberculosis. Based on CRS, 450 patients had "clinical TB" (definitive-TB, probable-TB and possible-TB). Remaining 150 were confirmed "non-TB" cases. For culture, the sensitivity was low, 79.3% for pulmonary and 54.3% for extra-pulmonary cases. The sensitivity and specificity results for nMPCR test were evaluated taken composite reference standard as a gold standard. The sensitivity of the nMPCR assay was 97.1% for pulmonary and 91.4% for extra-pulmonary TB cases with specificity of 100% and 93.3% respectively. Nested multiplex PCR using three gene primers is a rapid, reliable and highly sensitive and specific diagnostic technique for the detection and differentiation of M. tuberculosis complex from NTM genome and will be useful in diagnosing paucibacillary samples. Nested multiplex PCR assay was found to be better than single step multiplex PCR for assessing the diagnosis of TB.

Autophagy protects type II alveolar epithelial cells from Mycobacterium tuberculosis infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guo, Xu-Guang; Department of Laboratory Medicine, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou; Ji, Tian-Xing

Highlights: ► We investigated the protective effect of autophagy pathway against MTB infection. ► MTB-infected A549 cells had higher LDH release. ► Inhibition of autophagy signaling significantly enhanced the MTB-induced necrosis. ► Autophagy prevents apoptosis and promotes cell survival in infected cells. -- Abstract: This study was designed to investigate the protective effect of the autophagy signaling pathway against Mycobacterium tuberculosis infection in type II alveolar epithelial cells. An in vitro M. tuberculosis system was established using human A549 cells. Infection-induced changes in the expression of the autophagic marker LC3 were assessed by reverse transcription-PCR and Western blotting. Morphological changesmore » in autophagosomes were detected by transmission electron microscopy (TEM). The function of the autophagy signaling pathway during infection was assessed by measuring the level of cell death and the amount of lactate dehydrogenase (LDH) released in the presence or absence of the inhibitor 3-methyladenine (3-MA). In addition, effects on LDH release were assessed after the siRNA-mediated knockdown of the essential autophagosomal structural membrane protein Atg5. LC3 mRNA expression was significantly reduced in M.tuberculosis-infected A549 cells (16888.76 ± 1576.34 vs. uninfected: 12744.29 ± 1089.37; P < 0.05). TEM revealed M.tuberculosis bacilli-containing compartments that were surrounded by double membranes characteristic of the autophagic process. M.tuberculosis-infected A549 cells released more LDH (1.45 ± 0.12 vs. uninfected: 0.45 ± 0.04; P < 0.05). The inhibition of autophagy signaling significantly enhanced M.tuberculosis-induced necrosis (3-MA: 75 ± 5% vs. untreated: 15 ± 1%; P < 0.05) and LDH release (3-MA: 2.50 ± 0.24 vs. untreated: 0.45 ± 0.04; Atg5 knockdown: 3.19 ± 0.29 vs. untreated: 1.28 ± 0.11; P < 0.05). Our results indicate that autophagy signaling pathway prevents apoptosis in type II alveolar epithelial cells infected with M.tuberculosis and may represent a molecular target for promoting cell survival during infection by respiratory pathogens.« less

Rifampicin-induced disseminated intravascular coagulation in pulmonary tuberculosis treatment: A case report and literature review.

PubMed

Chen, Guo; He, Jian-Qing

2017-02-01

Disseminated intravascular coagulation (DIC) induced by daily rifampicin therapy is rare, especially the patient is absent of malignancy, severe infection, and prior exposure to rifampicin. We report a case of DIC induced by daily rifampicin treatment for pulmonary tuberculosis. A 22-year-old, previously healthy man received an anti-tuberculosis therapy consisting of isoniazid, rifampicin, ethambutol, and pyrazinamide on the daily dose recommended by the World Health Organization tuberculosis guidelines after a diagnosis of pulmonary tuberculosis. Two weeks later, he was transferred to the West China Hospital with nasal hemorrhage for 1 week, hematochezia, hematuria, and petechiae for 5 days. Laboratory data and symptoms on admission indicated DIC. The anti-tuberculosis drugs were discontinued after admission and he was initiated with targeted treatment for DIC, omeprazole and polyene hosphatidylcholine infusion, as well as nutrition supportive treatment. Five days after admission, ethambutol, moxifloxacin, and amikacin were added to the patient without further active hemorrhage. Eight days after admission, the platelet count had risen gradually. Isoniazid was administered on 24 days after admission, while his liver function tests and platelet counts returned to normal. No recurrence of DIC occurred. The diagnosis of rifampicin-induced DIC was confirmed. The patient recovered and left hospital with isoniazid, ethambutol, levofloxacin, and streptomycin after 4 weeks of hospitalization. There was no recurrence of DIC or hemorrhage during the 8 months of follow-up. The literature review revealed that there were 10 other cases of rifampicin-induced DIC. Only 4 cases received rifampicin on a daily basis for pulmonary tuberculosis treatment and the others were on intermittent dosing schedule for pulmonary tuberculosis or leprosy treatment. As a rare adverse effect, DIC induced by rifampicin occurs irregularly and unpredictably, which is reported to be more associated with the intermittent usage of rifampicin, but can occur with rifampicin daily administration. Identification of early symptoms, drug discontinuation, supportive management, and regular monitoring are the key points to correct this adverse effect, which may contribute to severe even fetal results in patients and deserves more attention.

Global Introduction of New Multidrug-Resistant Tuberculosis Drugs—Balancing Regulation with Urgent Patient Needs

PubMed Central

Sullivan, Timothy

2016-01-01

New treatments for multidrug-resistant tuberculosis (MDR TB) are urgently needed. Two new drugs, bedaquiline and delamanid, have recently been released, and several new drugs and treatment regimens are in the pipeline. Misuse of TB drugs is a principal cause of drug resistance. As new drugs and regimens reach the market, the need to make them available to patients must be balanced with regulation of their use so that resistance to the new drugs can be prevented. To foster the rational use of new drugs, we propose 1) expanding/strengthening the capacity for drug susceptibility testing, beginning with countries with a high TB burden; 2) regulating prescribing practices by banning over-the-counter sale of TB drugs and enacting an accreditation system whereby providers must be certified to prescribe new drugs; and 3) decentralizing MDR TB care in rural communities by employing trained community health workers, using promising mobile technologies, and enlisting the aid of civil society organizations. PMID:26889711

[Micro and Macro Social Work Practice in the Context of Tuberculosis].

PubMed

Moya, Eva M; Chavez-Baray, Silvia M; Martínez, Omar

2017-07-01

The study of tuberculosis (TB) in the U.S.-Mexico border involves the consideration of three key components, which are complex and interrelated: the difficulty tracking and providing appropriate TB care due to the epidemiological and pathological characteristics of TB; the border itself is a geographical and epidemiological area that interweaves two nations, two cultures, two health systems and different laws; and the need for prevention and treatment approaches to TB that involve comprehensive clinical care and prevention while also taking into consideration stigma, social context and knowledge. This manuscript describes the work of ten studies conducted in Mexico and in the U.S.-Mexico border region from 2006 to 2013. It also provides an understanding of the social and public health implications of TB, the environment and communities, as well as the identification and application of advocacy and social mobilization practices. The work presented provides an understanding of different interventions related to adherence, stigma reduction, person-centered approaches, and effective micro and macro practices in social work.

Tuberculosis and latent infection in employees of different prison unit types

PubMed Central

Nogueira, Péricles Alves; Abrahão, Regina Maura Cabral de Melo; Galesi, Vera Maria Neder; López, Rossana Verónica Mendoza

2018-01-01

ABSTRACT OBJECTIVE Estimate the prevalence of active tuberculosis and latent tuberculosis infection among the staff that is in contact and the staff that is not in contact with prisoners, and investigate factors associated with latent tuberculosis infection in this population. METHODS Observational cross-sectional study, conducted from 2012 to 2015, in employees of different prison units in the municipality of Franco da Rocha, SP. It consisted of the application of a questionnaire, application and reading of the tuberculin test, sputum smear microscopy, sputum culture, and radiological examination. The association between the qualitative variables was calculated by the Pearson's chi-squared test. The sociodemographic and clinical-epidemiological factors related to the latent tuberculosis infection were evaluated by the logistic regression with the odds ratios (OR) calculation and their respective intervals with 95% of confidence (95%CI). RESULTS A total of 1,059 employees were examined, 657 (62.0%) of prisons, 249 (23.5%) of CASA Foundation units and 153 (14.5%) of custodial and psychiatric treatment hospitals. The tuberculin test was applied and read for 945 (89.2%) professionals. Of these, 797 (84.3%) were contacts of detainees and 148 (15.7%) were not. Among prison staff, the factors associated with latent tuberculosis infection were: contact with detainee (OR = 2.12, 95%CI 1.21–3.71); male gender (OR = 1.97, 95%CI 1.19–3.27); between 30 and 39 years old (OR = 2.98, 95%CI 1.34–6.63), 40 to 49 years old (OR = 4.32, 95%CI 1.94–9.60), and 50 to 59 years old (OR = 3.98, 95%CI 1.68–9.43); nonwhite color or race (OR = 1.89, 95%CI 1.29–2.78); and smoker (OR = 1.64, 95%CI 1.05–2.55). There were no positive test on sputum smear microscopy and culture. Of the 241 (22.8%) professionals who underwent radiological examination, 48 (19.9%) presented alterations of which 11 were suspected of tuberculosis. CONCLUSIONS Prison employees who have direct contact with detainees are 2.12 times more likely to become infected with Mycobacterium tuberculosis in the work environment and consequently to become ill with tuberculosis and should be targeted for disease prevention and control. PMID:29412377

Systems level mapping of metabolic complexity in Mycobacterium tuberculosis to identify high-value drug targets.

PubMed

Vashisht, Rohit; Bhat, Ashwini G; Kushwaha, Shreeram; Bhardwaj, Anshu; Brahmachari, Samir K

2014-10-11

The effectiveness of current therapeutic regimens for Mycobacterium tuberculosis (Mtb) is diminished by the need for prolonged therapy and the rise of drug resistant/tolerant strains. This global health threat, despite decades of basic research and a wealth of legacy knowledge, is due to a lack of systems level understanding that can innovate the process of fast acting and high efficacy drug discovery. The enhanced functional annotations of the Mtb genome, which were previously obtained through a crowd sourcing approach was used to reconstruct the metabolic network of Mtb in a bottom up manner. We represent this information by developing a novel Systems Biology Spindle Map of Metabolism (SBSM) and comprehend its static and dynamic structure using various computational approaches based on simulation and design. The reconstructed metabolism of Mtb encompasses 961 metabolites, involved in 1152 reactions catalyzed by 890 protein coding genes, organized into 50 pathways. By accounting for static and dynamic analysis of SBSM in Mtb we identified various critical proteins required for the growth and survival of bacteria. Further, we assessed the potential of these proteins as putative drug targets that are fast acting and less toxic. Further, we formulate a novel concept of metabolic persister genes (MPGs) and compared our predictions with published in vitro and in vivo experimental evidence. Through such analyses, we report for the first time that de novo biosynthesis of NAD may give rise to bacterial persistence in Mtb under conditions of metabolic stress induced by conventional anti-tuberculosis therapy. We propose such MPG's as potential combination of drug targets for existing antibiotics that can improve their efficacy and efficiency for drug tolerant bacteria. The systems level framework formulated by us to identify potential non-toxic drug targets and strategies to circumvent the issue of bacterial persistence can substantially aid in the process of TB drug discovery and translational research.

Detection of Mycobacterium tuberculosis peptides in the exosomes of patients with active and latent M. tuberculosis infection using MRM-MS.

PubMed

Kruh-Garcia, Nicole A; Wolfe, Lisa M; Chaisson, Lelia H; Worodria, William O; Nahid, Payam; Schorey, Jeff S; Davis, J Lucian; Dobos, Karen M

2014-01-01

The identification of easily measured, accurate diagnostic biomarkers for active tuberculosis (TB) will have a significant impact on global TB control efforts. Because of the host and pathogen complexities involved in TB pathogenesis, identifying a single biomarker that is adequately sensitive and specific continues to be a major hurdle. Our previous studies in models of TB demonstrated that exosomes, such as those released from infected macrophages, contain mycobacterial products, including many Mtb proteins. In this report, we describe the development of targeted proteomics assays employing multiplexed multiple reaction monitoring mass spectrometry (MRM-MS) in order to allow us to follow those proteins previously identified by western blot or shotgun mass spectrometry, and enhance biomarker discovery to include detection of Mtb proteins in human serum exosomes. Targeted MRM-MS assays were applied to exosomes isolated from human serum samples obtained from culture-confirmed active TB patients to detect 76 peptides representing 33 unique Mtb proteins. Our studies revealed the first identification of bacteria-derived biomarker candidates of active TB in exosomes from human serum. Twenty of the 33 proteins targeted for detection were found in the exosomes of TB patients, and included multiple peptides from 8 proteins (Antigen 85B, Antigen 85C, Apa, BfrB, GlcB, HspX, KatG, and Mpt64). Interestingly, all of these proteins are known mycobacterial adhesins and/or proteins that contribute to the intracellular survival of Mtb. These proteins will be included as target analytes in future validation studies as they may serve as markers for persistent active and latent Mtb infection. In summary, this work is the first step in identifying a unique and specific panel of Mtb peptide biomarkers encapsulated in exosomes and reveals complex biomarker patterns across a spectrum of TB disease states.

Detection of Mycobacterium tuberculosis Peptides in the Exosomes of Patients with Active and Latent M. tuberculosis Infection Using MRM-MS

PubMed Central

Kruh-Garcia, Nicole A.; Wolfe, Lisa M.; Chaisson, Lelia H.; Worodria, William O.; Nahid, Payam; Schorey, Jeff S.; Davis, J. Lucian; Dobos, Karen M.

2014-01-01

The identification of easily measured, accurate diagnostic biomarkers for active tuberculosis (TB) will have a significant impact on global TB control efforts. Because of the host and pathogen complexities involved in TB pathogenesis, identifying a single biomarker that is adequately sensitive and specific continues to be a major hurdle. Our previous studies in models of TB demonstrated that exosomes, such as those released from infected macrophages, contain mycobacterial products, including many Mtb proteins. In this report, we describe the development of targeted proteomics assays employing multiplexed multiple reaction monitoring mass spectrometry (MRM-MS) in order to allow us to follow those proteins previously identified by western blot or shotgun mass spectrometry, and enhance biomarker discovery to include detection of Mtb proteins in human serum exosomes. Targeted MRM-MS assays were applied to exosomes isolated from human serum samples obtained from culture-confirmed active TB patients to detect 76 peptides representing 33 unique Mtb proteins. Our studies revealed the first identification of bacteria-derived biomarker candidates of active TB in exosomes from human serum. Twenty of the 33 proteins targeted for detection were found in the exosomes of TB patients, and included multiple peptides from 8 proteins (Antigen 85B, Antigen 85C, Apa, BfrB, GlcB, HspX, KatG, and Mpt64). Interestingly, all of these proteins are known mycobacterial adhesins and/or proteins that contribute to the intracellular survival of Mtb. These proteins will be included as target analytes in future validation studies as they may serve as markers for persistent active and latent Mtb infection. In summary, this work is the first step in identifying a unique and specific panel of Mtb peptide biomarkers encapsulated in exosomes and reveals complex biomarker patterns across a spectrum of TB disease states. PMID:25080351

Identification of Mycobacterium tuberculosis BioA inhibitors by using structure-based virtual screening

PubMed Central

Singh, Swati; Khare, Garima; Bahal, Ritika Kar; Ghosh, Prahlad C; Tyagi, Anil K

2018-01-01

Background 7,8-Diaminopelargonic acid synthase (BioA), an enzyme of biotin biosynthesis pathway, is a well-known promising target for anti-tubercular drug development. Methods In this study, structure-based virtual screening was employed against the active site of BioA to identify new chemical entities for BioA inhibition and top ranking compounds were evaluated for their ability to inhibit BioA enzymatic activity. Results Seven compounds inhibited BioA enzymatic activity by greater than 60% at 100 μg/mL with most potent compounds being A36, A35 and A65, displaying IC50 values of 10.48 μg/mL (28.94 μM), 33.36 μg/mL (88.16 μM) and 39.17 μg/mL (114.42 μM), respectively. Compounds A65 and A35 inhibited Mycobacterium tuberculosis (M. tuberculosis) growth with MIC90 of 20 μg/mL and 80 μg/mL, respectively, whereas compound A36 exhibited relatively weak inhibition of M. tuberculosis growth (83% inhibition at 200 μg/mL). Compound A65 emerged as the most potent compound identified in our study that inhibited BioA enzymatic activity and growth of the pathogen and possessed drug-like properties. Conclusion Our study has identified a few hit molecules against M. tuberculosis BioA that can act as potential candidates for further development of potent anti-tubercular therapeutic agents. PMID:29750019

Development of sandwich-form biosensor to detect Mycobacterium tuberculosis complex in clinical sputum specimens.

PubMed

Shojaei, Taha Roodbar; Mohd Salleh, Mohamad Amran; Tabatabaei, Meisam; Ekrami, Alireza; Motallebi, Roya; Rahmani-Cherati, Tavoos; Hajalilou, Abdollah; Jorfi, Raheleh

2014-01-01

Mycobacterium tuberculosis, the causing agent of tuberculosis, comes second only after HIV on the list of infectious agents slaughtering many worldwide. Due to the limitations behind the conventional detection methods, it is therefore critical to develop new sensitive sensing systems capable of quick detection of the infectious agent. In the present study, the surface modified cadmium-telluride quantum dots and gold nanoparticles conjunct with two specific oligonucleotides against early secretory antigenic target 6 were used to develop a sandwich-form fluorescence resonance energy transfer-based biosensor to detect M. tuberculosis complex and differentiate M. tuberculosis and M. bovis Bacille Calmette-Guerin simultaneously. The sensitivity and specificity of the newly developed biosensor were 94.2% and 86.6%, respectively, while the sensitivity and specificity of polymerase chain reaction and nested polymerase chain reaction were considerably lower, 74.2%, 73.3% and 82.8%, 80%, respectively. The detection limits of the sandwich-form fluorescence resonance energy transfer-based biosensor were far lower (10 fg) than those of the polymerase chain reaction and nested polymerase chain reaction (100 fg). Although the cost of the developed nanobiosensor was slightly higher than those of the polymerase chain reaction-based techniques, its unique advantages in terms of turnaround time, higher sensitivity and specificity, as well as a 10-fold lower detection limit would clearly recommend this test as a more appropriate and cost-effective tool for large scale operations. Copyright © 2014 Elsevier Editora Ltda. All rights reserved.

Motivations and concerns about adolescent tuberculosis vaccine trial participation in rural Uganda: a qualitative study.

PubMed

Buregyeya, Esther; Kulane, Asli; Kiguli, Juliet; Musoke, Phillipa; Mayanja, Harriet; Mitchell, Ellen Maeve Hanlon

2015-01-01

Research is being carried out to develop and test new potentially more effective tuberculosis vaccines. Among the vaccines being developed are those that target adolescents. This study explored the stakeholders' perceptions about adolescent participation in a hypothetical tuberculosis vaccine trial in Ugandan adolescents. Focus group discussions with adolescents, parents of infants and adolescents, and key informant interviews with community leaders and traditional healers were conducted. The majority of the respondents expressed potential willingness to allow their children participate in a tuberculosis vaccine trial. Main motivations for potential participation would be being able to learn about health-related issues. Hesitations included the notion that trial participation would distract the youths from their studies, fear of possible side effects of an investigational product, and potential for being sexually exploited by researchers. In addition, bad experiences from participation in previous research and doubts about the importance of research were mentioned. Suggested ways to motivate participation included: improved clarity on study purpose, risks, benefits and better scheduling of study procedures to minimize disruption to participants' academic schedules. Findings from this study suggest that the community is open to potential participation of adolescents in a tuberculosis vaccine trial. However, there is a need to communicate more effectively with the community about the purpose of the trial and its effects, including safety data, in a low-literacy, readily understood format. This raises a challenge to researchers, who cannot know all the potential effects of a trial product before it is tested.

CFP-10 from Mycobacterium tuberculosis Selectively Activates Human Neutrophils through a Pertussis Toxin-Sensitive Chemotactic Receptor

PubMed Central

Björnsdottir, Halla; Winther, Malene; Christenson, Karin; Oprea, Tudor; Karlsson, Anna; Forsman, Huamei; Dahlgren, Claes; Bylund, Johan

2014-01-01

Upon infection with Mycobacterium tuberculosis, neutrophils are massively recruited to the lungs, but the role of these cells in combating the infection is poorly understood. Through a type VII secretion system, M. tuberculosis releases a heterodimeric protein complex, containing a 6-kDa early secreted antigenic target (ESAT-6) and a 10-kDa culture filtrate protein (CFP-10), that is essential for virulence. Whereas the ESAT-6 component possesses multiple virulence-related activities, no direct biological activity of CFP-10 has been shown, and CFP-10 has been described as a chaperone protein for ESAT-6. We here show that the ESAT-6:CFP-10 complex induces a transient release of Ca2+ from intracellular stores in human neutrophils. Surprisingly, CFP-10 rather than ESAT-6 was responsible for triggering the Ca2+ response, in a pertussis toxin-sensitive manner, suggesting the involvement of a G-protein-coupled receptor. In line with this, the response was accompanied by neutrophil chemotaxis and activation of the superoxide-producing NADPH-oxidase. Neutrophils were unique among leukocytes in responding to CFP-10, as monocytes and lymphocytes failed to produce a Ca2+ signal upon stimulation with the M. tuberculosis protein. Hence, CFP-10 may contribute specifically to neutrophil recruitment and activation during M. tuberculosis infection, representing a novel biological role for CFP-10 in the ESAT-6:CFP-10 complex, beyond the previously described chaperone function. PMID:25332123

Cost-effectiveness of a comprehensive programme for drug-resistant tuberculosis in China.

PubMed

Fitzpatrick, Christopher; Hui, Zhang; Lixia, Wang; Renzhong, Li; Yunzhou, Ruan; Mingting, Chen; Yanlin, Zhao; Jin, Zhao; Wei, Su; Caihong, Xu; Cheng, Chen; Alston, Timothy; Yan, Qu; Chengfei, Lv; Yunting, Fu; Shitong, Huan; Qiang, Sun; Scano, Fabio; Chin, Daniel P; Floyd, Katherine

2015-11-01

To investigate the cost-effectiveness of a comprehensive programme for drug-resistant tuberculosis launched in four sites in China in 2011. In 2011-2012, we reviewed the records of 172 patients with drug-resistant tuberculosis who enrolled in the comprehensive programme and we collected relevant administrative data from hospitals and China's public health agency. For comparison, we examined a cohort of 81 patients who were treated for drug-resistant tuberculosis in 2006-2009. We performed a cost-effectiveness analysis, from a societal perspective, that included probabilistic uncertainty. We measured early treatment outcomes based on three-month culture results and modelled longer-term outcomes to facilitate estimation of the comprehensive programme's cost per disability-adjusted life-year (DALY) averted. The comprehensive programme cost 8837 United States dollars (US$) per patient treated. Low enrolment rates meant that some fixed costs were higher, per patient, than expected. Although the comprehensive programme appeared 30 times more costly than the previous one, it resulted in greater health benefits. The comprehensive programme, which cost US$ 639 (95% credible interval: 112 to 1322) per DALY averted, satisfied the World Health Organization's criterion for a very cost-effective intervention. The comprehensive programme, which included rapid screening, standardized care and financial protection, improved individual outcomes for MDR tuberculosis in a cost-effective manner. To support post-2015 global heath targets, the comprehensive programme should be expanded to non-residents and other areas of China.

Mastering Mobile Security

ERIC Educational Resources Information Center

Panettieri, Joseph C.

2007-01-01

Without proper security, mobile devices are easy targets for worms, viruses, and so-called robot ("bot") networks. Hackers increasingly use bot networks to launch massive attacks against eCommerce websites--potentially targeting one's online tuition payment or fundraising/financial development systems. How can one defend his mobile systems against…

Mycobacterium tuberculosis Maltosyltransferase GlgE, a Genetically Validated Antituberculosis Target, Is Negatively Regulated by Ser/Thr Phosphorylation*

PubMed Central

Leiba, Jade; Syson, Karl; Baronian, Grégory; Zanella-Cléon, Isabelle; Kalscheuer, Rainer; Kremer, Laurent; Bornemann, Stephen; Molle, Virginie

2013-01-01

GlgE is a maltosyltransferase involved in the biosynthesis of α-glucans that has been genetically validated as a potential therapeutic target against Mycobacterium tuberculosis. Despite also making α-glucan, the GlgC/GlgA glycogen pathway is distinct and allosterically regulated. We have used a combination of genetics and biochemistry to establish how the GlgE pathway is regulated. M. tuberculosis GlgE was phosphorylated specifically by the Ser/Thr protein kinase PknB in vitro on one serine and six threonine residues. Furthermore, GlgE was phosphorylated in vivo when expressed in Mycobacterium bovis bacillus Calmette–Guérin (BCG) but not when all seven phosphorylation sites were replaced by Ala residues. The GlgE orthologues from Mycobacterium smegmatis and Streptomyces coelicolor were phosphorylated by the corresponding PknB orthologues in vitro, implying that the phosphorylation of GlgE is widespread among actinomycetes. PknB-dependent phosphorylation of GlgE led to a 2 orders of magnitude reduction in catalytic efficiency in vitro. The activities of phosphoablative and phosphomimetic GlgE derivatives, where each phosphorylation site was substituted with either Ala or Asp residues, respectively, correlated with negative phosphoregulation. Complementation studies of a M. smegmatis glgE mutant strain with these GlgE derivatives, together with both classical and chemical forward genetics, were consistent with flux through the GlgE pathway being correlated with GlgE activity. We conclude that the GlgE pathway appears to be negatively regulated in actinomycetes through the phosphorylation of GlgE by PknB, a mechanism distinct from that known in the classical glycogen pathway. Thus, these findings open new opportunities to target the GlgE pathway therapeutically. PMID:23609448

Rapid Identification of Mycobacteria and Drug-Resistant Mycobacterium tuberculosis by Use of a Single Multiplex PCR and DNA Sequencing

PubMed Central

Pérez-Osorio, Ailyn C.; Boyle, David S.; Ingham, Zachary K.; Ostash, Alla; Gautom, Romesh K.; Colombel, Craig; Houze, Yolanda

2012-01-01

Tuberculosis (TB) remains a significant global health problem for which rapid diagnosis is critical to both treatment and control. This report describes a multiplex PCR method, the Mycobacterial IDentification and Drug Resistance Screen (MID-DRS) assay, which allows identification of members of the Mycobacterium tuberculosis complex (MTBC) and the simultaneous amplification of targets for sequencing-based drug resistance screening of rifampin-resistant (rifampinr), isoniazidr, and pyrazinamider TB. Additionally, the same multiplex reaction amplifies a specific 16S rRNA gene target for rapid identification of M. avium complex (MAC) and a region of the heat shock protein 65 gene (hsp65) for further DNA sequencing-based confirmation or identification of other mycobacterial species. Comparison of preliminary results generated with MID-DRS versus culture-based methods for a total of 188 bacterial isolates demonstrated MID-DRS sensitivity and specificity as 100% and 96.8% for MTBC identification; 100% and 98.3% for MAC identification; 97.4% and 98.7% for rifampinr TB identification; 60.6% and 100% for isoniazidr TB identification; and 75.0% and 98.1% for pyrazinamider TB identification. The performance of the MID-DRS was also tested on acid-fast-bacterium (AFB)-positive clinical specimens, resulting in sensitivity and specificity of 100% and 78.6% for detection of MTBC and 100% and 97.8% for detection of MAC. In conclusion, use of the MID-DRS reduces the time necessary for initial identification and drug resistance screening of TB specimens to as little as 2 days. Since all targets needed for completing the assay are included in a single PCR amplification step, assay costs, preparation time, and risks due to user errors are also reduced. PMID:22162548

Evaluating treatment outcomes and durations among cases of smear-positive pulmonary tuberculosis in Yemen: a prospective follow-up study.

PubMed

Jaber, Ammar Ali Saleh; Khan, Amer Hayat; Sulaiman, Syed Azhar Syed

2017-01-01

Evaluating outcomes after tuberculosis (TB) treatment can help identify the primary reasons for treatment success or failure. However, Yemen has a treatment success rate that remains below the World Health Organization's target. This study aimed to identify factors that were associated with unsuccessful treatment and prolonged treatment (>1 year). Newly diagnosed cases of smear-positive pulmonary TB were prospectively followed at two centers (Taiz and Alhodidah, Yemen) between April 2014 and March 2015. Standardized forms were used to obtain information from the patients regarding their socio-demographic and clinical characteristics, treatment duration, and TB-related information. Multivariate logistic regression analyses were performed to identify factors that were associated with unsuccessful treatment and prolonged treatment (>1 year). The study included data from 273 cases of newly diagnosed TB, with treatment being successful in 227 cases (83.1%) and unsuccessful in 46 cases (16.9%). Among the 46 patients with unsuccessful treatment, 29 patients (10.6%) stopped treatment, 6 patients (2.2%) transferred to another facility, 6 patients (2.2%) experienced treatment failure, and 5 patients (1.8%) died. The multivariate logistic regression analyses revealed that unsuccessful treatment was associated with female sex, illiterate status, and the presence of comorbidities. Prolonged treatment durations were associated with living in a rural area, smoking, chewing khat, a cough that lasted for >3 weeks at the beginning of treatment, and bilateral cavities during radiography. These results confirm that the treatment success rate in Yemen is lower than the World Health Organization's target for smear-positive pulmonary tuberculosis. Targeting the risk factors that we identified may help improve treatment outcomes. Furthermore, it may not be prudent to re-treat patients using first-line TB drugs after an initial treatment failure.

MIR144* inhibits antimicrobial responses against Mycobacterium tuberculosis in human monocytes and macrophages by targeting the autophagy protein DRAM2.

PubMed

Kim, Jin Kyung; Lee, Hye-Mi; Park, Ki-Sun; Shin, Dong-Min; Kim, Tae Sung; Kim, Yi Sak; Suh, Hyun-Woo; Kim, Soo Yeon; Kim, In Soo; Kim, Jin-Man; Son, Ji-Woong; Sohn, Kyung Mok; Jung, Sung Soo; Chung, Chaeuk; Han, Sang-Bae; Yang, Chul-Su; Jo, Eun-Kyeong

2017-02-01

Autophagy is an important antimicrobial effector process that defends against Mycobacterium tuberculosis (Mtb), the human pathogen causing tuberculosis (TB). MicroRNAs (miRNAs), endogenous noncoding RNAs, are involved in various biological functions and act as post-transcriptional regulators to target mRNAs. The process by which miRNAs affect antibacterial autophagy and host defense mechanisms against Mtb infections in human monocytes and macrophages is largely uncharacterized. In this study, we show that Mtb significantly induces the expression of MIR144*/hsa-miR-144-5p, which targets the 3'-untranslated region of DRAM2 (DNA damage regulated autophagy modulator 2) in human monocytes and macrophages. Mtb infection downregulated, whereas the autophagy activators upregulated, DRAM2 expression in human monocytes and macrophages by activating AMP-activated protein kinase. In addition, overexpression of MIR144* decreased DRAM2 expression and formation of autophagosomes in human monocytes, whereas inhibition of MIR144* had the opposite effect. Moreover, the levels of MIR144* were elevated, whereas DRAM2 levels were reduced, in human peripheral blood cells and tissues in TB patients, indicating the clinical significance of MIR144* and DRAM2 in human TB. Notably, DRAM2 interacted with BECN1 and UVRAG, essential components of the autophagic machinery, leading to displacement of RUBCN from the BECN1 complex and enhancement of Ptdlns3K activity. Furthermore, MIR144* and DRAM2 were critically involved in phagosomal maturation and enhanced antimicrobial effects against Mtb. Our findings identify a previously unrecognized role of human MIR144* in the inhibition of antibacterial autophagy and the innate host immune response to Mtb. Additionally, these data reveal that DRAM2 is a key coordinator of autophagy activation that enhances antimicrobial activity against Mtb.

Mycobacterium tuberculosis maltosyltransferase GlgE, a genetically validated antituberculosis target, is negatively regulated by Ser/Thr phosphorylation.

PubMed

Leiba, Jade; Syson, Karl; Baronian, Grégory; Zanella-Cléon, Isabelle; Kalscheuer, Rainer; Kremer, Laurent; Bornemann, Stephen; Molle, Virginie

2013-06-07

GlgE is a maltosyltransferase involved in the biosynthesis of α-glucans that has been genetically validated as a potential therapeutic target against Mycobacterium tuberculosis. Despite also making α-glucan, the GlgC/GlgA glycogen pathway is distinct and allosterically regulated. We have used a combination of genetics and biochemistry to establish how the GlgE pathway is regulated. M. tuberculosis GlgE was phosphorylated specifically by the Ser/Thr protein kinase PknB in vitro on one serine and six threonine residues. Furthermore, GlgE was phosphorylated in vivo when expressed in Mycobacterium bovis bacillus Calmette-Guérin (BCG) but not when all seven phosphorylation sites were replaced by Ala residues. The GlgE orthologues from Mycobacterium smegmatis and Streptomyces coelicolor were phosphorylated by the corresponding PknB orthologues in vitro, implying that the phosphorylation of GlgE is widespread among actinomycetes. PknB-dependent phosphorylation of GlgE led to a 2 orders of magnitude reduction in catalytic efficiency in vitro. The activities of phosphoablative and phosphomimetic GlgE derivatives, where each phosphorylation site was substituted with either Ala or Asp residues, respectively, correlated with negative phosphoregulation. Complementation studies of a M. smegmatis glgE mutant strain with these GlgE derivatives, together with both classical and chemical forward genetics, were consistent with flux through the GlgE pathway being correlated with GlgE activity. We conclude that the GlgE pathway appears to be negatively regulated in actinomycetes through the phosphorylation of GlgE by PknB, a mechanism distinct from that known in the classical glycogen pathway. Thus, these findings open new opportunities to target the GlgE pathway therapeutically.

In silico discovery and in vitro activity of inhibitors against Mycobacterium tuberculosis 7,8-diaminopelargonic acid synthase (Mtb BioA).

PubMed

Billones, Junie B; Carrillo, Maria Constancia O; Organo, Voltaire G; Sy, Jamie Bernadette A; Clavio, Nina Abigail B; Macalino, Stephani Joy Y; Emnacen, Inno A; Lee, Alexandra P; Ko, Paul Kenny L; Concepcion, Gisela P

2017-01-01

Computer-aided drug discovery and development approaches such as virtual screening, molecular docking, and in silico drug property calculations have been utilized in this effort to discover new lead compounds against tuberculosis. The enzyme 7,8-diaminopelargonic acid aminotransferase (BioA) in Mycobacterium tuberculosis ( Mtb ), primarily involved in the lipid biosynthesis pathway, was chosen as the drug target due to the fact that humans are not capable of synthesizing biotin endogenously. The computational screening of 4.5 million compounds from the Enamine REAL database has ultimately yielded 45 high-scoring, high-affinity compounds with desirable in silico absorption, distribution, metabolism, excretion, and toxicity properties. Seventeen of the 45 compounds were subjected to bioactivity validation using the resazurin microtiter assay. Among the 4 actives, compound 7 (( Z )- N -(2-isopropoxyphenyl)-2-oxo-2-((3-(trifluoromethyl)cyclohexyl)amino)acetimidic acid) displayed inhibitory activity up to 83% at 10 μg/mL concentration against the growth of the Mtb H37Ra strain.

In silico discovery and in vitro activity of inhibitors against Mycobacterium tuberculosis 7,8-diaminopelargonic acid synthase (Mtb BioA)

PubMed Central

Billones, Junie B; Carrillo, Maria Constancia O; Organo, Voltaire G; Sy, Jamie Bernadette A; Clavio, Nina Abigail B; Macalino, Stephani Joy Y; Emnacen, Inno A; Lee, Alexandra P; Ko, Paul Kenny L; Concepcion, Gisela P

2017-01-01

Computer-aided drug discovery and development approaches such as virtual screening, molecular docking, and in silico drug property calculations have been utilized in this effort to discover new lead compounds against tuberculosis. The enzyme 7,8-diaminopelargonic acid aminotransferase (BioA) in Mycobacterium tuberculosis (Mtb), primarily involved in the lipid biosynthesis pathway, was chosen as the drug target due to the fact that humans are not capable of synthesizing biotin endogenously. The computational screening of 4.5 million compounds from the Enamine REAL database has ultimately yielded 45 high-scoring, high-affinity compounds with desirable in silico absorption, distribution, metabolism, excretion, and toxicity properties. Seventeen of the 45 compounds were subjected to bioactivity validation using the resazurin microtiter assay. Among the 4 actives, compound 7 ((Z)-N-(2-isopropoxyphenyl)-2-oxo-2-((3-(trifluoromethyl)cyclohexyl)amino)acetimidic acid) displayed inhibitory activity up to 83% at 10 μg/mL concentration against the growth of the Mtb H37Ra strain. PMID:28280303

Metformin: Candidate host-directed therapy for tuberculosis in diabetes and non-diabetes patients.

PubMed

Restrepo, Blanca I

2016-12-01

Despite major advances in tuberculosis (TB) control, TB continues to be a leading cause of death worldwide. The discovery of new anti-TB treatment drugs and regimens that target drug-sensitive and drug-resistant TB are being complemented with a search for adjunct host-directed therapies that synergize for Mycobacterium tuberculosis (Mtb) elimination. The goal of host-directed therapies is to boost immune mechanisms that diminish excess inflammation to reduce lung tissue damage and limit Mtb growth. Metformin is the most commonly-used medication for type 2 diabetes, and a candidate for host-directed therapy for TB. Preliminary data suggests metformin may be beneficial for TB control by reducing the deleterious inflammation associated with immune pathology and enhancing the anti-mycobacterial activity of immune cells. In this review I summarize current findings, knowledge gaps and the potential benefits as well as points of caution for using metformin as adjunct therapy for TB in patients with and without type 2 diabetes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Tuberculosis beliefs among recent Vietnamese refugees in New York State.

PubMed Central

Carey, J W; Oxtoby, M J; Nguyen, L P; Huynh, V; Morgan, M; Jeffery, M

1997-01-01

OBJECTIVE: To identify newly arrived Vietnamese refugees' beliefs about tuberculosis (TB) and TB education needs. METHODS: In 1994, the New York State Health Department and the Centers for Disease Control and Prevention conducted a survey of 51 newly arrived adult Vietnamese refugees in two New York counties. After being trained in interview methods, two bilingual researchers asked 32 open-ended questions on the causes of TB, TB treatment, and the disease's impact on work and social relationships. RESULTS: Respondents correctly viewed TB as an infectious lung disease with symptoms such as cough, weakness, and weight loss. Hard manual labor, smoking, alcohol consumption, and poor nutrition were believed to be risk factors. Many respondents incorrectly believed that asymptomatic latent infection is not possible and that infection inevitably leads to disease. Nearly all respondents anticipated that having tuberculosis would adversely impact their work, family, and community activities and relationships. CONCLUSIONS: Targeted patient education is needed to address misconceptions about TB among Vietnamese refugees and to help ensure adherence to prescribed treatment regimens. PMID:9018292

Determinants of Poor Adherence to Anti-Tuberculosis Treatment in Mumbai, India

PubMed Central

Bagchi, Suparna; Ambe, Guirish; Sathiakumar, Nalini

2010-01-01

Objectives: In this study, we investigated the determinants of poor adherence with anti-tuberculosis therapy among pulmonary tuberculosis (TB) patients in Mumbai, India, receiving Directly Observed Treatment Short Course (DOTS) therapy. Methods: A cross-sectional study on 538 patients receiving DOTS I and II regimen was conducted. Patients were interviewed and clinical and laboratory data were collected. Eighty seven patients were considered non-adherent. Multivariable logistic regression was used to determine risk factors associated with non-adherence. Results: Factors associated with non-adherence were found to be different among the newly-diagnosed patients and all the other residual groups. Smoking during treatment and travel-related cost factors were significantly associated with non-adherence in the newly-diagnosed patients, while alcohol consumption and short-age of drugs were significant in the residual groups. Conclusions: An approach, targeting easier access to drugs, an ensured drug supply, effective solutions for travel-related concerns and modification of smoking and alcohol related behaviors are essential for treatment adherence. PMID:21566777

DNA replication fidelity in Mycobacterium tuberculosis is mediated by an ancestral prokaryotic proofreader.

PubMed

Rock, Jeremy M; Lang, Ulla F; Chase, Michael R; Ford, Christopher B; Gerrick, Elias R; Gawande, Richa; Coscolla, Mireia; Gagneux, Sebastien; Fortune, Sarah M; Lamers, Meindert H

2015-06-01

The DNA replication machinery is an important target for antibiotic development in increasingly drug-resistant bacteria, including Mycobacterium tuberculosis. Although blocking DNA replication leads to cell death, disrupting the processes used to ensure replication fidelity can accelerate mutation and the evolution of drug resistance. In Escherichia coli, the proofreading subunit of the replisome, the ɛ exonuclease, is essential for high-fidelity DNA replication; however, we find that the corresponding subunit is completely dispensable in M. tuberculosis. Rather, the mycobacterial replicative polymerase DnaE1 itself encodes an editing function that proofreads DNA replication, mediated by an intrinsic 3'-5' exonuclease activity within its PHP domain. Inactivation of the DnaE1 PHP domain increases the mutation rate by more than 3,000-fold. Moreover, phylogenetic analysis of DNA replication proofreading in the bacterial kingdom suggests that E. coli is a phylogenetic outlier and that PHP domain-mediated proofreading is widely conserved and indeed may be the ancestral prokaryotic proofreader.

DNA replication fidelity in Mycobacterium tuberculosis is mediated by an ancestral prokaryotic proofreader

PubMed Central

Rock, Jeremy M.; Lang, Ulla F.; Chase, Michael R.; Ford, Christopher B.; Gerrick, Elias R.; Gawande, Richa; Coscolla, Mireia; Gagneux, Sebastien; Fortune, Sarah M.; Lamers, Meindert H.

2015-01-01

The DNA replication machinery is an important target for antibiotic development for increasingly drug resistant bacteria including Mycobacterium tuberculosis1. While blocking DNA replication leads to cell death, disrupting the processes used to ensure replication fidelity can accelerate mutation and the evolution of drug resistance. In E. coli, the proofreading subunit of the replisome, the ε-exonuclease, is essential for high fidelity DNA replication2; however, we find that it is completely dispensable in M. tuberculosis. Rather, the mycobacterial replicative polymerase, DnaE1, encodes a novel editing function that proofreads DNA replication, mediated by an intrinsic 3′-5′ exonuclease activity within its PHP domain. Inactivation of the DnaE1 PHP domain increases the mutation rate by greater than 3,000 fold. Moreover, phylogenetic analysis of DNA replication proofreading in the bacterial kingdom suggests that E. coli is a phylogenetic outlier and that PHP-domain mediated proofreading is widely conserved and indeed may be the ancestral prokaryotic proofreader. PMID:25894501

Biofabrication of polyphenols stabilized reduced graphene oxide and its anti-tuberculosis activity.

PubMed

Han, Wei; Niu, Wen-Yi; Sun, Bing; Shi, Guang-Can; Cui, Xiu-Qin

2016-12-01

A facile one step eco-friendly method for the reduction graphene oxide by Cinnamomumverum (C. verum) bark extract is reported in this work. This approach avoids the utilization of hazardous chemical reagents. The characterization results of various spectroscopic and microscopic techniques for the prepared graphene oxide (GO) and reduced graphene oxide (RGO) afford a strong indication of the removal of oxygen functionalities of GO after reduction and following stabilization by the oxidised polyphenols. Fourier transform infrared spectral results showed the capping of oxidised polyphenols onto the surface of reduced graphene oxide which further prevent their aggregation. Additionally, the prepared graphene nanosheets were tested for their antituberculosis activity against standard strain such as M. tuberculosis H37Ra. The obtained results suggested that the synthesized graphene acts as an effective growth inhibitors against M. tuberculosis H37Ra making it applicable for targeted drug delivery by combining with other chemical drugs as a therapeutic index. Copyright © 2016 Elsevier B.V. All rights reserved.