Discriminatory ability of hypervariable variable number tandem repeat loci in population-based analysis of Mycobacterium tuberculosis strains, London, UK

To address conflicting results about the stability of variable number tandem repeat (VNTR) loci and their value in prospective molecular epidemiology of Mycobacterium tuberculosis, we conducted a large prospective population-based analysis of all M. tuberculosis strains in a metropolitan setting. Optimal and reproducible conditions for reliable PCR and fragment analysis, comprising enzymes, denaturing conditions, and capillary temperature, were identified for a panel of hypervariable loci, including 3232, 2163a, 1982, and 4052. A total of 2,261 individual M. tuberculosis isolates and 265 sets of serial isolates were analyzed by using a standardized 15-loci VNTR panel, then an optimized hypervariable loci panel. The discriminative ability of loci varied substantially; locus VNTR 3232 varied the most, with 19 allelic variants and Hunter-Gaston index value of 0.909 . Hypervariable loci should be included in standardized panels because they can provide consistent comparable results at multiple settings, provided the proposed conditions are adhered to

Reactivity of Particles from Gasoline Direct Injection Engine: Correlation of Engine Parameters and Particle Characteristics

With the introduction of the European emissions legislation for particle number and particle mass (Euro 6c stage), particulate emissions are currently a core issue in the development of Gasoline Direct Injection (GDI) engines. Due to the different mixing process of fuel and air GDI engines emit more particulates than engines with port fuel injection.The present development aims at the reduction of particulate emissions through exhaust gas aftertreatment, e.g. via gasoline particle filters (GPF). To minimize the technical complexity in the aftertreatment system the control of the burn-out of particulates within GPF has an enormous significance. The regeneration behavior of GPF is essentially dependent on the reactivity of the stored soot and its properties that in turn depend on the engine operation conditions. The main objective is the enhancement and control of the reactivity of the emitted soot particles by engine parameters. The basic studies were carried out on a turbocharged 4-cylinder engine with direct injection by varying the engine operating parameters, e.g. engine speed, air fuel ratio, injection timing. The particles are investigated in terms of their reactivity, number and size distribution and microstructure by using thermogravimetric analysis (TGA-FTIR), engine exhaust particle sizer (EEPS) and high-resolution transmission electron microscopy (HR-TEM). The investigations show a direct and indirect impact of engine operation parameters on the soot reactivity and its properties. The results of the parameter variations showed that the reactivity of particles can be controlled and enhanced by the operation conditions of the engine. To maximize the soot reactivity further investigations must be performed

Survival of Civilian and Prisoner Drug-Sensitive, Multi- and Extensive Drug- Resistant Tuberculosis Cohorts Prospectively Followed in Russia

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The Forest behind the Tree: Phylogenetic Exploration of a Dominant Mycobacterium tuberculosis Strain Lineage from a High Tuberculosis Burden Country

BACKGROUND: Genotyping of Mycobacterium tuberculosis isolates is a powerful tool for epidemiological control of tuberculosis (TB) and phylogenetic exploration of the pathogen. Standardized PCR-based typing, based on 15 to 24 mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) loci combined with spoligotyping, has been shown to have adequate resolution power for tracing TB transmission and to be useful for predicting diverse strain lineages in European settings. Its informative value needs to be tested in high TB-burden countries, where the use of genotyping is often complicated by dominance of geographically specific, genetically homogeneous strain lineages. METHODOLOGY/PRINCIPAL FINDINGS: We tested this genotyping system for molecular epidemiological analysis of 369 M. tuberculosis isolates from 3 regions of Brazil, a high TB-burden country. Deligotyping, targeting 43 large sequence polymorphisms (LSPs), and the MIRU-VNTRplus identification database were used to assess phylogenetic predictions. High congruence between the different typing results consistently revealed the countrywide supremacy of the Latin-American-Mediterranean (LAM) lineage, comprised of three main branches. In addition to an already known RDRio branch, at least one other branch characterized by a phylogenetically informative LAM3 spoligo-signature seems to be globally distributed beyond Brazil. Nevertheless, by distinguishing 321 genotypes in this strain population, combined MIRU-VNTR typing and spoligotyping demonstrated the presence of multiple distinct clones. The use of 15 to 24 loci discriminated 21 to 25% more strains within the LAM lineage, compared to a restricted lineage-specific locus set suggested to be used after SNP analysis. Noteworthy, 23 of the 28 molecular clusters identified were exclusively composed of patient isolates from a same region, consistent with expected patterns of mostly local TB transmission. CONCLUSIONS/SIGNIFICANCE: Standard MIRU-VNTR typing combined with spoligotyping can reveal epidemiologically meaningful clonal diversity behind a dominant M. tuberculosis strain lineage in a high TB-burden country and is useful to explore international phylogenetical ramifications

Script-theory virtual case: a novel tool for education and research

Context/Setting: The script theory of diagnostic reasoning proposes that clinicians evaluate cases in the context of an “illness script,” iteratively testing internal hypotheses against new information eventually reaching a diagnosis. We present a novel tool for teaching diagnostic reasoning to undergraduate medical students based on an adaptation of script theory. Intervention: We developed a virtual patient case that used clinically authentic audio and video, interactive three-dimensional (3D) body images, and a simulated electronic medical record. Next, we used interactive slide bars to record respondents’ likelihood estimates of diagnostic possibilities at various stages of the case. Responses were dynamically compared to data from expert clinicians and peers. Comparative frequency distributions were presented to the learner and final diagnostic likelihood estimates were analyzed. Detailed student feedback was collected. Observations: Over two academic years, 322 students participated. Student diagnostic likelihood estimates were similar year to year, but were consistently different from expert clinician estimates. Student feedback was overwhelmingly positive: students found the case was novel, innovative, clinically authentic, and a valuable learning experience. Discussion: We demonstrate the successful implementation of a novel approach to teaching diagnostic reasoning. Future study may delineate reasoning processes associated with differences between novice and expert responses