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Find video protocols related to scientific articles indexed in Pubmed.
Unraveling Mycobacterium tuberculosis genomic diversity and evolution in Lisbon, Portugal, a highly drug resistant setting.
BMC Genomics
PUBLISHED: 11-18-2014
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Multidrug- (MDR) and extensively drug resistant (XDR) tuberculosis (TB) presents a challenge to disease control and elimination goals. In Lisbon, Portugal, specific and successful XDR-TB strains have been found in circulation for almost two decades.
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A robust SNP barcode for typing Mycobacterium tuberculosis complex strains.
Nat Commun
PUBLISHED: 09-01-2014
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Strain-specific genomic diversity in the Mycobacterium tuberculosis complex (MTBC) is an important factor in pathogenesis that may affect virulence, transmissibility, host response and emergence of drug resistance. Several systems have been proposed to classify MTBC strains into distinct lineages and families. Here, we investigate single-nucleotide polymorphisms (SNPs) as robust (stable) markers of genetic variation for phylogenetic analysis. We identify ~92 k SNP across a global collection of 1,601 genomes. The SNP-based phylogeny is consistent with the gold-standard regions of difference (RD) classification system. Of the ~7 k strain-specific SNPs identified, 62 markers are proposed to discriminate known circulating strains. This SNP-based barcode is the first to cover all main lineages, and classifies a greater number of sublineages than current alternatives. It may be used to classify clinical isolates to evaluate tools to control the disease, including therapeutics and vaccines whose effectiveness may vary by strain type.
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Mycobacterial interspersed repetitive unit typing and mutational profile for multidrug-resistant and extensively drug-resistant tuberculosis surveillance in Portugal: a 3-year period overview.
Int. J. Antimicrob. Agents
PUBLISHED: 04-10-2014
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Multidrug tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) cases constitute a serious health problem in Portugal, of which the majority of isolates belong to the Lisboa family and the Q1 cluster, highly related to the Lisboa family. Here we sought to investigate the molecular basis of resistant TB as well as to determine the prevalence of specific drug resistance mutations and their association with MDR-TB and/or XDR-TB. In total, 74 Mycobacterium tuberculosis clinical isolates collected in Lisbon Health Region were genotyped by 24-loci mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR), and the mutational profile associated with first- and second-line drug resistance was studied. Seven new mutations were found, whilst the remaining 28 mutations had been previously associated with drug resistance. None of the mutations was specifically associated with MDR-TB. The mutational patterns observed among isolates belonging to Lisboa3 and Q1 clusters were also observed in isolates with unique MIRU-VNTR patterns but closely related to these strains. Such data suggest that the genotyping technique employed discriminates isolates with the same mutational profile. To establish the most adequate genotyping technique, the discriminatory power of three different MIRU-VNTR sets was analysed. The 15-loci MIRU-VNTR set showed adequate discriminatory power, comparable with the 24-loci set, allowing clustering of 60% and 86% of the MDR-TB and XDR-TB isolates, respectively, the majority of which belonged to the Lisboa3 and Q1 clusters. From an epidemiological standpoint, this study suggests combined mutational and genotyping analysis as a valuable tool for drug resistance surveillance.
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PolyTB: a genomic variation map for Mycobacterium tuberculosis.
Tuberculosis (Edinb)
PUBLISHED: 02-08-2014
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Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is the second major cause of death from an infectious disease worldwide. Recent advances in DNA sequencing are leading to the ability to generate whole genome information in clinical isolates of M. tuberculosis complex (MTBC). The identification of informative genetic variants such as phylogenetic markers and those associated with drug resistance or virulence will help barcode Mtb in the context of epidemiological, diagnostic and clinical studies. Mtb genomic datasets are increasingly available as raw sequences, which are potentially difficult and computer intensive to process, and compare across studies. Here we have processed the raw sequence data (>1500 isolates, eight studies) to compile a catalogue of SNPs (n = 74,039, 63% non-synonymous, 51.1% in more than one isolate, i.e. non-private), small indels (n = 4810) and larger structural variants (n = 800). We have developed the PolyTB web-based tool (http://pathogenseq.lshtm.ac.uk/polytb) to visualise the resulting variation and important meta-data (e.g. in silico inferred strain-types, location) within geographical map and phylogenetic views. This resource will allow researchers to identify polymorphisms within candidate genes of interest, as well as examine the genomic diversity and distribution of strains. PolyTB source code is freely available to researchers wishing to develop similar tools for their pathogen of interest.
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Pre-Multidrug-Resistant Mycobacterium tuberculosis Beijing Strain Associated with Disseminated Tuberculosis in a Pet Dog.
J. Clin. Microbiol.
PUBLISHED: 10-23-2013
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Resistance to isoniazid, ethambutol, and streptomycin was detected in a Mycobacterium tuberculosis strain, belonging to the Beijing family lineage, isolated from two nodule exudates of a Yorkshire terrier with generalized tuberculosis. This report alerts medical practitioners to the risk of dissemination of pre-multidrug-resistant tuberculosis (preMDR-TB) through exposure to M. tuberculosis-shedding pets.
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Exploring the contribution of mycobacteria characteristics in their interaction with human macrophages.
Microsc. Microanal.
PUBLISHED: 06-24-2013
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Tuberculosis (TB) is a major health problem. The emergence of multidrug resistant (MDR) Mycobacterium tuberculosis (Mtb) isolates confounds treatment strategies. In Portugal, cases of MDR-TB are reported annually with an increased incidence noted in Lisbon. The majority of these MDR-TB cases are due to closely related mycobacteria known collectively as the Lisboa family and Q1 cluster. Genetic determinants linked to drug resistance have been exhaustively studied resulting in the identification of family and cluster specific mutations. Nevertheless, little is known about other factors involved in development of mycobacteria drug resistance. Here, we complement genetic analysis with the study of morphological and structural features of the Lisboa family and Q1 cluster isolates by using scanning and transmission electron microscopy. This analysis allowed the identification of structural differences, such as cell envelope thickness, between Mtb clinical isolates that are correlated with antibiotic resistance. The infection of human monocyte derived macrophages allowed us to document the relative selective advantage of the Lisboa family isolates over other circulating Mtb isolates.
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High-level resistance to isoniazid and ethionamide in multidrug-resistant Mycobacterium tuberculosis of the Lisboa family is associated with inhA double mutations.
J. Antimicrob. Chemother.
PUBLISHED: 03-28-2013
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The purpose of this study was to determine the levels of isoniazid and ethionamide resistance and to identify associated mutations in endemic multidrug-resistant (MDR) strains of Mycobacterium tuberculosis from the Lisbon metropolitan area, Portugal.
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Au-nanoprobes for detection of SNPs associated with antibiotic resistance in Mycobacterium tuberculosis.
Nanotechnology
PUBLISHED: 09-16-2010
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Tuberculosis (TB) is one of the leading causes of infection in humans, causing high morbility and mortality all over the world. The rate of new cases of multidrug resistant tuberculosis (MDRTB) continues to increase, and since these infections are very difficult to manage, they constitute a serious health problem. In most cases, drug resistance in Mycobacterium tuberculosis has been related to mutations in several loci within the pathogens genome. The development of fast, cheap and simple screening methodologies would be of paramount relevance for the early detection of these mutations, essential for the timely and effective diagnosis and management of MDRTB patients. The use of gold nanoparticles derivatized with thiol-modified oligonucleotides (Au-nanoprobes) has led to new approaches in molecular diagnostics. Based on the differential non-cross-linking aggregation of Au-nanoprobes, we were able to develop a colorimetric method for the detection of specific sequences and to apply this approach to pathogen identification and single base mutations/single nucleotide polymorphisms (SNP) discrimination. Here we report on the development of Au-nanoprobes for the specific identification of SNPs within the beta subunit of the RNA polymerase (rpoB locus), responsible for resistance to rifampicin in over 95% of rifampicin resistant M. tuberculosis strains.
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Molecular tools for rapid identification and novel effective therapy against MDRTB/XDRTB infections.
Expert Rev Anti Infect Ther
PUBLISHED: 04-10-2010
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Tuberculosis (TB) is mainly an intracellular infection of the lung alveolar macrophages, and any anti-TB agent must therefore be active at the macrophage. Among the available therapies, isoniazid and rifampicin are the most effective drugs against susceptible Mycobacterium tuberculosis, but they are ineffective against multidrug-resistant TB (MDRTB) strains. Rates of MDRTB in Portugal are the highest in Western Europe, demanding effective measures for their control. Our application of molecular techniques for the early identification of MDRTB assisted in the reduction of these rates. Further examination revealed that a large number of MDRTB cases were extensively-drug resistant (XDRTB), providing evidence for the urgent need of new and effective anti-MDRTB/XDRTB therapeutic strategies. This review describes in detail: the characteristics of the main M. tuberculosis strains circulating in Portugal; the creation of a Task Force for TB control, based on molecular tools that allow 1-day identification of an MDRTB patient; the usefulness of evaluating the ex vivo activity of anti-tubercular agents against the M. tuberculosis isolated from the patients sputum; and the mode of action by which phenothiazines have been shown to promote the killing of intracellular MDRTB/XDRTB by nonkilling macrophages.
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Genetic analysis of extensively drug-resistant Mycobacterium tuberculosis strains in Lisbon, Portugal.
J. Antimicrob. Chemother.
PUBLISHED: 12-22-2009
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Extensively drug-resistant (XDR) tuberculosis (TB) threatens the global control of TB worldwide. Lisbon has a high XDR-TB rate [50% of the multidrug-resistant tuberculosis (MDR-TB)], which is mainly associated with Lisboa family strains. Few studies have addressed the identification of mutations associated with resistance to second-line injectable drugs, and the relative frequency of such mutations varies geographically. The aim of this study was to characterize the genetic changes associated with the high number of XDR-TB cases in Lisbon.
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Genotypic analysis of Mycobacterium tuberculosis isolates from a Lisbon hospital in Portugal.
Rev Port Pneumol
PUBLISHED: 08-04-2009
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Portugal has one of the highest tuberculosis notification rates of the European Union with Lisbon Health Region having an incidence rate well above the national average. The present study analyses the transmission, drug susceptibility and characteristics of a study population from a Central Lisbons Hospital. One hundred and thirty -two Mycobacterium tuberculosis clinical isolates were previously tested for drug susceptibility to first -line drugs. The multidrug (MDR) resistance rate was found to be 3.0%, while 13.6% of the isolates were resistant to one or more first -line drugs. HIV serology was available for 98 patients, 26 (26.5%) were positive. Genotyping was performed by MIRU -VNTR and 53 (40.2%) out of the 132 isolates were found to be distributed through 17 MIRU -VNTR clusters of two or more isolates. Lisboa strains accounted for 25.8% of all strains. We conclude that transmission of resistant and susceptible Mycobacterium tuberculosis strains is occurring, with special concern for Lisboa strains.
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Genetic characterisation of the ethambutol resistance-determining region in Mycobacterium tuberculosis: prevalence and significance of embB306 mutations.
Int. J. Antimicrob. Agents
PUBLISHED: 05-02-2009
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Ethambutol (EMB) is a first-line antitubercular drug that inhibits arabinogalactan and lipoarabinomannan biosynthesis. Resistance to EMB has traditionally been associated with embB mutations, especially in the Met306 codon. In this study, the region encompassing the embB306 codon in 109 Mycobacterium tuberculosis clinical isolates (49 EMB-susceptible and 60 EMB-resistant) was characterised. The occurrence of embB306 mutations was verified not only in EMB-resistant isolates (55.0%) but also in EMB-susceptible isolates (16.3%), which questions the role of embB306 mutations as determinants of EMB resistance. Subsequently, four different embB alleles were created by in vitro mutagenesis and were introduced into Mycobacterium smegmatis on a multicopy plasmid. To assess the contribution of embB306 mutations to EMB resistance, the EMB minimum inhibitory concentration (MIC) of these strains was determined. Strains carrying mutant embB306 alleles were able to grow at slightly higher MICs compared with the strain carrying the wild-type embB gene. The small MIC increase obtained here does not appear to be sufficient to cause high-level EMB resistance. The results obtained in the present study suggest that acquisition of EMB resistance might be a multistep process in which embB306 mutations may represent a first-step in EMB resistance acquisition.
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A rapid and simple method for identifying Mycobacterium tuberculosis W-Beijing strains based on detection of a unique mutation in Rv0927c by PCR-SSCP.
Microbes Infect.
PUBLISHED: 01-11-2009
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Recently we have found that W-Beijing Mycobacterium tuberculosis strains have a unique in-frame trinucleotide (AGC) deletion at position 421 of Rv0927c and a -127G-->A mutation in Rv0927c-pstS3 intergenic region. Based on detecting the 421 trinucleotide deletion of these two mutations which can alter the ssDNA conformation more extensively than the other, we developed a PCR-SSCP method for rapid identification of W-Beijing strains among non-Beijing strains. Altogether, 104 clinical isolates were analyzed, including 68 W-Beijing strains and 36 non-Beijing strains. We found that PCR-SSCP successfully differentiated all the W-Beijing strains from the non-Beijing strains. In addition, we unexpectedly discovered that SDS-PAGE protein gels had better resolving power than conventional TBE polyacrylamide gel in detecting the AGC deletion mutation in the SSCP analysis.
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From multidrug-resistant to extensively drug-resistant tuberculosis in Lisbon, Portugal: the stepwise mode of resistance acquisition.
J. Antimicrob. Chemother.
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The development and transmission of extensively drug-resistant (XDR) tuberculosis (TB) constitutes a serious threat to the effective control of TB in several countries. Here, in an attempt to further elucidate the dynamics of the acquisition of resistance to second-line drugs and investigate an eventual role for eis promoter mutations in aminoglycoside resistance, we have studied a set of multidrug-resistant (MDR)/XDR-TB isolates circulating in Lisbon, Portugal.
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Contribution of efflux to the emergence of isoniazid and multidrug resistance in Mycobacterium tuberculosis.
PLoS ONE
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Multidrug resistant (MDR) tuberculosis is caused by Mycobacterium tuberculosis resistant to isoniazid and rifampicin, the two most effective drugs used in tuberculosis therapy. Here, we investigated the mechanism by which resistance towards isoniazid develops and how overexpression of efflux pumps favors accumulation of mutations in isoniazid targets, thus establishing a MDR phenotype. The study was based on the in vitro induction of an isoniazid resistant phenotype by prolonged serial exposure of M. tuberculosis strains to the critical concentration of isoniazid employed for determination of drug susceptibility testing in clinical isolates. Results show that susceptible and rifampicin monoresistant strains exposed to this concentration become resistant to isoniazid after three weeks; and that resistance observed for the majority of these strains could be reduced by means of efflux pumps inhibitors. RT-qPCR assessment of efflux pump genes expression showed overexpression of all tested genes. Enhanced real-time efflux of ethidium bromide, a common efflux pump substrate, was also observed, showing a clear relation between overexpression of the genes and increased efflux pump function. Further exposure to isoniazid resulted in the selection and stabilization of spontaneous mutations and deletions in the katG gene along with sustained increased efflux activity. Together, results demonstrate the relevance of efflux pumps as one of the factors of isoniazid resistance in M. tuberculosis. These results support the hypothesis that activity of efflux pumps allows the maintenance of an isoniazid resistant population in a sub-optimally treated patient from which isoniazid genetically resistant mutants emerge. Therefore, the use of inhibitors of efflux should be considered in the development of new therapeutic strategies for preventing the emergence of MDR-TB during treatment.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.