A robust SNP barcode for typing Mycobacterium tuberculosis complex strains

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 ~92k 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 ~7k 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

First insights into the phylogenetic diversity of Mycobacterium tuberculosis in Nepal

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 reg

Two new rapid SNP-typing methods for classifying Mycobacterium tuberculosis complex into the main phylogenetic lineages

There is increasing evidence that strain variation in Mycobacterium tuberculosis complex (MTBC) might influence the outcome of tuberculosis infection and disease. To assess genotype-phenotype associations, phylogenetically robust molecular markers and appropriate genotyping tools are required. Most current genotyping methods for MTBC are based on mobile or repetitive DNA elements. Because these elements are prone to convergent evolution, the corresponding genotyping techniques are suboptimal for phylogenetic studies and strain classification. By contrast, single nucleotide polymorphisms (SNP) are ideal markers for classifying MTBC into phylogenetic lineages, as they exhibit very low degrees of homoplasy. In this study, we developed two complementary SNP-based genotyping methods to classify strains into the six main human-associated lineages of MTBC, the 'Beijing' sublineage, and the clade comprising Mycobacterium bovis and Mycobacterium caprae. Phylogenetically informative SNPs were obtained from 22 MTBC whole-genome sequences. The first assay, referred to as MOL-PCR, is a ligation-dependent PCR with signal detection by fluorescent microspheres and a Luminex flow cytometer, which simultaneously interrogates eight SNPs. The second assay is based on six individual TaqMan real-time PCR assays for singleplex SNP-typing. We compared MOL-PCR and TaqMan results in two panels of clinical MTBC isolates. Both methods agreed fully when assigning 36 well-characterized strains into the main phylogenetic lineages. The sensitivity in allele-calling was 98.6% and 98.8% for MOL-PCR and TaqMan, respectively. Typing of an additional panel of 78 unknown clinical isolates revealed 99.2% and 100% sensitivity in allele-calling, respectively, and 100% agreement in lineage assignment between both methods. While MOL-PCR and TaqMan are both highly sensitive and specific, MOL-PCR is ideal for classification of isolates with no previous information, whereas TaqMan is faster for confirmation. Furthermore, both methods are rapid, flexible and comparably inexpensive

Fact and fiction in housing research: utilizing the creative imagination

As much of our conceptual framework is informed by the experience of the imagination, there is much to be learnt from a study of various creative forms. Narrative fiction can be one such form, allowing us to gain a useful insight into complex features of social life. The purpose of this article is to investigate the treatment of housing issues in contemporary literature in order to gain insights into attitudes, experiences and interpretations from the perspective of a broad cultural milieu. Discussions of professionalism, housing tenure and homelessness have tended to be conducted within a narrow framework and adopted orthodox modes of evaluation. Consequently, the neglect of housing within a wider cultural context has reinforced the isolation of housing issues. The article argues that although discussions of housing and housing policy have been seriously limited within the contemporary novel, there are a number of key insights that can be gained from a discussion of issues within a fictional setting

Associations between human leukocyte antigen class I variants and the Mycobacterium tuberculosis subtypes causing disease

BACKGROUND. The development of active tuberculosis disease has been shown to be multifactorial. Interactions between host and bacterial genotype may influence disease outcome, with some studies indicating the adaptation of M. tuberculosis strains to specific human populations. Here we investigate the role of the human leukocyte antigen (HLA) class I genes in this biological process. METHODS. Three hundred patients with tuberculosis from South Africa were typed for their HLA class I alleles by direct sequencing. Mycobacterium tuberculosis genotype classification was done by IS6110 restriction fragment length polymorphism genotyping and spoligotyping. RESULTS. We showed that Beijing strain occurred more frequently in individuals with multiple disease episodes (P < .001) with the HLA-B27 allele lowering the odds of having an additional episode (odds ratio, 0.21; P = .006). Associations were also identified for specific HLA types and disease caused by the Beijing, LAM, LCC, and Quebec strains. HLA types were also associated with disease caused by strains from the Euro-American or East Asian lineages, and the frequencies of these alleles in their sympatric human populations identified potential coevolutionary events between host and pathogen. CONCLUSIONS. This is the first report of the association of human HLA types and M. tuberculosis strain genotype, highlighting that both host and pathogen genetics need to be taken into consideration when studying tuberculosis disease development.Web of Scienc

Six Studies in Nineteenth-Century English Literature and Thought

122 p. 23 cm. Includes bibliography University of Kansas autho

Intensified Antituberculosis Therapy in Adults with Tuberculous Meningitis

BACKGROUND Tuberculous meningitis is often lethal. Early antituberculosis treatment and adjunctive treatment with glucocorticoids improve survival, but nearly one third of patients with the condition still die. We hypothesized that intensified antituberculosis treatment would enhance the killing of intracerebral Mycobacterium tuberculosis organisms and decrease the rate of death among patients. METHODS We performed a randomized, double-blind, placebo-controlled trial involving human immunodeficiency virus (HIV)-infected adults and HIV-uninfected adults with a clinical diagnosis of tuberculous meningitis who were admitted to one of two Vietnamese hospitals. We compared a standard, 9-month antituberculosis regimen (which included 10 mg of rifampin per kilogram of body weight per day) with an intensified regimen that included higher-dose rifampin (15 mg per kilogram per day) and levofloxacin (20 mg per kilogram per day) for the first 8 weeks of treatment. The primary outcome was death by 9 months after randomization. RESULTS A total of 817 patients (349 of whom were HIV-infected) were enrolled; 409 were randomly assigned to receive the standard regimen, and 408 were assigned to receive intensified treatment. During the 9 months of follow-up, 113 patients in the intensified-treatment group and 114 patients in the standard-treatment group died (hazard ratio, 0.94; 95% confidence interval, 0.73 to 1.22; P=0.66). There was no evidence of a significant differential effect of intensified treatment in the overall population or in any of the subgroups, with the possible exception of patients infected with isoniazid-resistant M. tuberculosis. There were also no significant differences in secondary outcomes between the treatment groups. The overall number of adverse events leading to treatment interruption did not differ significantly between the treatment groups (64 events in the standard-treatment group and 95 events in the intensified-treatment group, P=0.08). CONCLUSIONS Intensified antituberculosis treatment was not associated with a higher rate of survival among patients with tuberculous meningitis than standard treatment. (Funded by the Wellcome Trust and the Li Ka Shing Foundation; Current Controlled Trials number, ISRCTN61649292.)

Investigating heparin affinity chromatography for extracellular vesicle purification and fractionation

The purification of extracellular vesicles (EVs) remains a major hurdle in the progression of fundamental research and the commercial application of EV-based products. In this study, we evaluated the potential of heparin affinity chromatography (HAC) to purify neural stem cell-derived EVs as part of a multistep process. Bind-elute chromatography, such as HAC, is an attractive method of purification because it is highly scalable, robust and can be automated. Our findings support an interaction between EVs and heparin. The recovery of EVs using HAC based on particle counts was a minimum of 68.7%. We found HAC could remove on average 98.8% and 99.0% of residual protein and DNA respectively. In addition to EV purification, HAC was used to separate EVs into three populations based on their affinity to the heparin column. Within these populations, we detected differences in the expression of the exosome-associated protein TSG101 and the tetraspanin immunophenotype. However, the significance of these observations is not clear. Overall HAC shows promise as a potential purification method to capture EVs and this study proposes a novel application of HAC for EV fractionation. Moving forward, a better understanding of the heparin-EV interaction would be required before HAC can be more widely adopted for these applications

An exploration of the use of simple statistics to measure consensus and stability in Delphi studies

<p>Abstract</p> <p>Background</p> <p>The criteria for stopping Delphi studies are often subjective. This study aimed to examine whether consensus and stability in the Delphi process can be ascertained by descriptive evaluation of trends in participants' views.</p> <p>Methods</p> <p>A three round email-based Delphi required participants (n = 12) to verify their level of agreement with 8 statements, write comments on each if they considered it necessary and rank the statements for importance. Each statement was analysed quantitatively by the percentage of agreement ratings, importance rankings and the amount of comments made for each statement, and qualitatively using thematic analysis. Importance rankings between rounds were compared by calculating Kappa values to observe trends in how the process impacts on subject's views.</p> <p>Results</p> <p>Evolution of consensus was shown by increase in agreement percentages, convergence of range with standard deviations of importance ratings, and a decrease in the number of comments made. Stability was demonstrated by a trend of increasing Kappa values.</p> <p>Conclusion</p> <p>Following the original use of Delphi in social sciences, Delphi is suggested to be an effective way to gain and measure group consensus in healthcare. However, the proposed analytical process should be followed to ensure maximum validity of results in Delphi methodology for improved evidence of consensual decision-making.</p

Extensive transmission of isoniazid resistant M. tuberculosis and its association with increased multidrug-resistant TB in two rural counties of eastern China: A molecular epidemiological study

<p>Abstract</p> <p>Background</p> <p>The aim of this study was to investigate the molecular characteristics of isoniazid resistant <it>Mycobacterium tuberculosis </it>(MTB), as well as its contribution to the dissemination of multi-drug resistant TB (MDR-TB) in rural areas of eastern China.</p> <p>Methods</p> <p>A population-based epidemiological study was conducted in two rural counties of eastern China from 2004 to 2005. In total, 131 isoniazid resistant MTB isolates were molecularly characterized by DNA sequencing and genotyped by IS<it>6110 </it>restriction fragment length polymorphism (RFLP) and spoligotyping.</p> <p>Results</p> <p>The <it>katG</it>315Thr mutation was observed in 74 of 131 isoniazid resistant isolates and more likely to be MDR-TB (48.6%) and have mutations in <it>rpoB </it>gene (47.3%). Spoligotyping identified 80.2% of isoniazid resistant MTB isolates as belonging to the Beijing family. Cluster analysis by genotyping based on IS<it>6110 </it>RFLP, showed that 48.1% isoniazid resistant isolates were grouped into 26 clusters and <it>katG</it>315Thr mutants had a significantly higher clustering proportion compared to those with <it>katG </it>wild type (73%.vs.18%; OR, 12.70; 95%CI, 6.357-14.80). Thirty-one of the 53 MDR-TB isolates were observed in 19 clusters. Of these clusters, isoniazid resistance in MDR-TB isolates was all due to the <it>katG</it>315Thr mutation; 18 clusters also contained mono-isoniazid resistant and other isoniazid resistant isolates.</p> <p>Conclusions</p> <p>These results highlighted that isoniazid resistant MTB especially with <it>katG</it>315Thr is likely to be clustered in a community, develop extra resistance to rifampicin and become MDR-TB in Chinese rural settings.</p