Combined species identification, genotyping, and drug resistance detection of mycobacterium tuberculosis cultures by mlpa on a bead-based array

The population structure of Mycobacterium tuberculosis is typically clonal therefore genotypic lineages can be unequivocally identified by characteristic markers such as mutations or genomic deletions. In addition, drug resistance is mainly mediated by mutations. These issues make multiplexed detection of selected mutations potentially a very powerful tool to characterise Mycobacterium tuberculosis. We used Multiplex Ligation-dependent Probe Amplification (MLPA) to screen for dispersed mutations, which can be successfully applied to Mycobacterium tuberculosis as was previously shown. Here we selected 47 discriminative and informative markers and designed MLPA probes accordingly to allow analysis with a liquid bead array and robust reader (Luminex MAGPIX technology). To validate the bead-based MLPA, we screened a panel of 88 selected strains, previously characterised by other methods with the developed multiplex assay using automated positive and negative calling. In total 3059 characteristics were screened and 3034 (99.2%) were consistent with previous molecular characterizations, of which 2056 (67.2%) were directly supported by other molecular methods, and 978 (32.0%) were consistent with but not directly supported by previous molecular characterizations. Results directly conflicting or inconsistent with previous methods, were obtained for 25 (0.8%) of the characteristics tested. Here we report the validation of the bead-based MLPA and demonstrate its potential to simultaneously identify a range of drug resistance markers, discriminate the species within the Mycobacterium tuberculosis complex, determine the genetic lineage and detect and identify the clinically most relevant non-tuberculous mycobacterial species. The detection of multiple genetic markers in clinically derived Mycobacterium tuberculosis strains with a multiplex assay could reduce the number of TB-dedicated screening methods needed for full characterization. Additionally, as a proportion of the markers screened are specific to certain Mycobacterium tuberculosis lineages each profile can be checked for internal consistency. Strain characterization can allow selection of appropriate treatment and thereby improve treatment outcome and patient management

Mycobacterium tuberculosis ecology in Venezuela: epidemiologic correlates of common spoligotypes and a large clonal cluster defined by MIRU-VNTR-24

<p>Abstract</p> <p>Background</p> <p>Tuberculosis remains an endemic public health problem, but the ecology of the TB strains prevalent, and their transmission, can vary by country and by region. We sought to investigate the prevalence of <it>Mycobacterium tuberculosis </it>strains in different regions of Venezuela. A previous study identified the most prevalent strains in Venezuela but did not show geographical distribution nor identify clonal genotypes. To better understand local strain ecology, we used spoligotyping to analyze 1298 <it>M. tuberculosis </it>strains isolated in Venezuela from 1997 to 2006, predominantly from two large urban centers and two geographically distinct indigenous areas, and then studied a subgroup with MIRU-VNTR 24 loci.</p> <p>Results</p> <p>The distribution of spoligotype families is similar to that previously reported for Venezuela and other South American countries: LAM 53%, T 10%, Haarlem 5%, S 1.9%, X 1.2%, Beijing 0.4%, and EAI 0.2%. The six most common shared types (SIT's 17, 93, 605, 42, 53, 20) accounted for 49% of the isolates and were the most common in almost all regions, but only a minority were clustered by MIRU-VNTR 24. One exception was the third most frequent overall, SIT 605, which is the most common spoligotype in the state of Carabobo but infrequent in other regions. MIRU-VNTR homogeneity suggests it is a clonal group of strains and was named the "Carabobo" genotype. Epidemiologic comparisons showed that patients with SIT 17 were younger and more likely to have had specimens positive for Acid Fast Bacilli on microscopy, and patients with SIT 53 were older and more commonly smear negative. Female TB patients tended to be younger than male patients. Patients from the high incidence, indigenous population in Delta Amacuro state were younger and had a nearly equal male:female distribution.</p> <p>Conclusion</p> <p>Six SIT's cause nearly half of the cases of tuberculosis in Venezuela and dominate in nearly all regions. Strains with SIT 17, the most common pattern overall may be more actively transmitted and SIT 53 strains may be less virulent and associated with reactivation of past infections in older patients. In contrast to other common spoligotypes, strains with SIT 605 form a clonal group centered in the state of Carabobo.</p

Diseño de un horno eléctrico de convección mecánica tipo laboratorio de temperatura controlada

Tesis (Ingeniero mecánico)--Universidad Autónoma de Occidente, 1993PregradoIngeniero(a) Mecánico(a

Diseño de un horno eléctrico de convección mecánica tipo laboratorio de temperatura controlada

Tesis (Ingeniero mecánico)--Universidad Autónoma de Occidente, 199

Gobernanza del agua. Una mirada desde la ecología política y la justicia hídrica

La gobernanza del agua, en la actualidad, presenta notables retos en términos de equidad, justicia y sustentabilidad. Para poder abordar estos temas consideramos necesario entender los discursos, políticas y relaciones de poder que dan forma a los procesos de toma de decisiones e intervención, al manejo de la información y conocimientos y, por ende, al control del agua y sus territorios. En Latinoamérica, la gobernanza ambiental se ha transformado con base en la descentralización política y la restructuración liberal. Bajo este modelo se priman mecanismos de participación de los diversos actores en condiciones desiguales de poder, lo que en muchas ocasiones vulnera la justicia hídrica. Los estudios de la gobernanza del agua, desde el enfoque de la ecología política que este libro presenta, abren posiciones críticas para el debate interdisciplinario. Entendemos así, las diversas aportaciones de este libro como una construcción de puentes de diálogo entre la comunidad académica y las poblaciones locales. De igual manera, pretendemos crear nuevas ideas y perspectivas analíticas que sirvan de fuente de inspiración para todos aquellos que se empeñan en investigar y comprender la problemática del agua y sus injusticias

Los caminos del agua

El agua fluye y a su paso siembra vida. Desde siempre, los territorios dependen de este importante recurso y sus dinámicas sociales se organizan en torno a la gestión del agua. Por eso, el acceso al agua, si bien es un derecho, implica también una disputa entre los diferentes actores de un territorio. En el escenario actual, que tiene como marco un modelo de desarrollo capitalista y globalizado, se priorizan los intereses de la agroindustria de exportación y la mneria, funcionales al capital, en detrimento de los sectores campesinos y de pequeños productores

Algorithm applied to all strains analysed for species identification of <i>M. tuberculosis</i> complex and non-tuberculous mycobacteria.

<p>MLPA markers are framed and final NTM species, MTBC lineages or sublineages are shown in bold. The species identification of a sample always starts with the MTBC 16SrRNA marker. As an example the call for the Beijing lineage K1 is highlighted with bold arrows. The following markers are present or absent in a strain belonging to the Beijing K1 lineage: MTBC 16S rRNA (present), TbD1 (present), RD750 (absent), pks15/1–7 (absent), RD105 (present), fbpB-238 (present), muT2-58 (present), acs-1551 (absent), RD131 (present). * as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043240#pone.0043240-Comas2" target="_blank">[25]</a>.</p

Summary of the MLPA probes designed and used in this study^a.

a<p>only probes that were functional in this study are shown. Probes are named after the gene and specific codon, nucleotide position (bold), or region they target. Probes are either targeting the mutation (mut) or the wild type (wt) sequence or the presence or absence of an RD. Bacterial DNA sequences are targeted with the left oligo (capital letters), spanning oligo (bold), right oligo (italics), iii =  inosine. xTAG sequences are not shown. RD  =  region of difference.</p

Reproducibility of the MLPA assay.

<p>DNA from <i>M. tuberculosis</i> strains 16, 27, 42 and 75 was analysed by MLPA in duplicate and in three experimental replicates. Mean Median Fluorescence Intensity (MFI) values with standard error of the mean (SEM) are shown. Each bar represents one experiment. The dashed line at MFI 150 indicates the set threshold.</p