Use of the GenoType® MTBDRplus assay to assess drug resistance of Mycobacterium tuberculosis isolates from patients in rural Uganda

<p>Abstract</p> <p>Background</p> <p>Drug resistance levels and patterns among <it>Mycobacterium tuberculosis </it>isolates from newly diagnosed and previously treated tuberculosis patients in Mbarara Uganda were investigated.</p> <p>Methods</p> <p>We enrolled, consecutively, all newly diagnosed and previously treated smear-positive TB patients aged ≥ 18 years. Isolates were tested for drug resistance against rifampicin (RIF) and isoniazid (INH) using the Genotype^®MDRTBplus assay and results were compared with those obtained by the indirect proportion method on Lowenstein-Jensen media. HIV testing was performed using two rapid HIV tests.</p> <p>Results</p> <p>A total of 125 isolates from 167 TB suspects with a mean age 33.7 years and HIV prevalence of 67.9% (55/81) were analysed. A majority (92.8%) of the participants were newly presenting while only 7.2% were retreatment cases. Resistance mutations to either RIF or INH were detected in 6.4% of the total isolates. Multidrug resistance, INH and RIF resistance was 1.6%, 3.2% and 4.8%, respectively. The <it>rpoβ </it>gene mutations seen in the sample were D516V, S531L, H526Y H526 D and D516V, while one strain had a Δ1 mutation in the wild type probes. There were three strains with <it>katG </it>(codon 315) gene mutations while only one strain showed the <it>inhA </it>promoter region gene mutation.</p> <p>Conclusion</p> <p>The TB resistance rate in Mbarara is relatively low. The GenoType^®MTBDRplus assay can be used for rapid screening of MDR-TB in this setting.</p

Dealing with heterogeneity of treatment effects: is the literature up to the challenge?

<p>Abstract</p> <p>Background</p> <p>Some patients will experience more or less benefit from treatment than the averages reported from clinical trials; such variation in therapeutic outcome is termed heterogeneity of treatment effects (HTE). Identifying HTE is necessary to individualize treatment. The degree to which heterogeneity is sought and analyzed correctly in the general medical literature is unknown. We undertook this literature sample to track the use of HTE analyses over time, examine the appropriateness of the statistical methods used, and explore the predictors of such analyses.</p> <p>Methods</p> <p>Articles were selected through a probability sample of randomized controlled trials (RCTs) published in <it>Annals of Internal Medicine</it>, <it>BMJ</it>, <it>JAMA</it>, <it>The Lancet</it>, and <it>NEJM </it>during odd numbered months of 1994, 1999, and 2004. RCTs were independently reviewed and coded by two abstractors, with adjudication by a third. Studies were classified as reporting: (1) HTE analysis, utilizing a formal test for heterogeneity or treatment-by-covariate interaction, (2) subgroup analysis only, involving no formal test for heterogeneity or interaction; or (3) neither. Chi-square tests and multiple logistic regression were used to identify variables associated with HTE reporting.</p> <p>Results</p> <p>319 studies were included. Ninety-two (29%) reported HTE analysis; another 88 (28%) reported subgroup analysis only, without examining HTE formally. Major covariates examined included individual risk factors associated with prognosis, responsiveness to treatment, or vulnerability to adverse effects of treatment (56%); gender (30%); age (29%); study site or center (29%); and race/ethnicity (7%). Journal of publication and sample size were significant independent predictors of HTE analysis (p < 0.05 and p < 0.001, respectively).</p> <p>Conclusion</p> <p>HTE is frequently ignored or incorrectly analyzed. An iterative process of exploratory analysis followed by confirmatory HTE analysis will generate the data needed to facilitate an individualized approach to evidence-based medicine.</p

Evaluation of qPCR-Based Assays for Leprosy Diagnosis Directly in Clinical Specimens

The increased reliability and efficiency of the quantitative polymerase chain reaction (qPCR) makes it a promising tool for performing large-scale screening for infectious disease among high-risk individuals. To date, no study has evaluated the specificity and sensitivity of different qPCR assays for leprosy diagnosis using a range of clinical samples that could bias molecular results such as difficult-to-diagnose cases. In this study, qPCR assays amplifying different M. leprae gene targets, sodA, 16S rRNA, RLEP and Ag 85B were compared for leprosy differential diagnosis. qPCR assays were performed on frozen skin biopsy samples from a total of 62 patients: 21 untreated multibacillary (MB), 26 untreated paucibacillary (PB) leprosy patients, as well as 10 patients suffering from other dermatological diseases and 5 healthy donors. To develop standardized protocols and to overcome the bias resulted from using chromosome count cutoffs arbitrarily defined for different assays, decision tree classifiers were used to estimate optimum cutoffs and to evaluate the assays. As a result, we found a decreasing sensitivity for Ag 85B (66.1%), 16S rRNA (62.9%), and sodA (59.7%) optimized assay classifiers, but with similar maximum specificity for leprosy diagnosis. Conversely, the RLEP assay showed to be the most sensitive (87.1%). Moreover, RLEP assay was positive for 3 samples of patients originally not diagnosed as having leprosy, but these patients developed leprosy 5–10 years after the collection of the biopsy. In addition, 4 other samples of patients clinically classified as non-leprosy presented detectable chromosome counts in their samples by the RLEP assay suggesting that those patients either had leprosy that was misdiagnosed or a subclinical state of leprosy. Overall, these results are encouraging and suggest that RLEP assay could be useful as a sensitive diagnostic test to detect M. leprae infection before major clinical manifestations

Molecular epidemiology, drug susceptibility and economic aspects of tuberculosis in mubende district, Uganda

<div><p>Background</p><p>Tuberculosis (TB) remains a global public health problem whose effects have major impact in developing countries like Uganda. This study aimed at investigating genotypic characteristics and drug resistance profiles of <i>Mycobacterium tuberculosis</i> isolated from suspected TB patients. Furthermore, risk factors and economic burdens that could affect the current control strategies were studied.</p><p>Methods</p><p>TB suspected patients were examined in a cross-sectional study at the Mubende regional referral hospital between February and July 2011. A questionnaire was administered to each patient to obtain information associated with TB prevalence. Isolates of <i>M. tuberculosis</i> recovered during sampling were examined for drug resistance to first line anti-TB drugs using the BACTEC-MGIT960^TMsystem. All isolates were further characterized using deletion analysis, spoligotyping and MIRU-VNTR analysis. Data were analyzed using different software; MIRU-VNTR <i>plus</i>, SITVITWEB, BioNumerics and multivariable regression models.</p><p>Results</p><p><i>M. tuberculosis</i> was isolated from 74 out of 344 patients, 48 of these were co-infected with HIV. Results from the questionnaire showed that previously treated TB, co-infection with HIV, cigarette smoking, and overcrowding were risk factors associated with TB, while high medical related transport bills were identified as an economic burden. Out of the 67 isolates that gave interpretable results, 23 different spoligopatterns were detected, nine of which were novel patterns. T2 with the sub types Uganda-I and Uganda-II was the most predominant lineage detected. Antibiotic resistance was detected in 19% and multidrug resistance was detected in 3% of the isolates.</p><p>Conclusion</p><p>The study detected <i>M. tuberculosis</i> from 21% of examined TB patients, 62% of whom were also HIV positive. There is a heterogeneous pool of genotypes that circulate in this area, with the T2 lineage being the most predominant. High medical related transport bills and drug resistance could undermine the usefulness of the current TB strategic interventions.</p></div

Leukocytes Are Recruited through the Bronchial Circulation to the Lung in a Spontaneously Hypertensive Rat Model of COPD

Chronic obstructive pulmonary disease (COPD) kills approximately 2.8 million people each year, and more than 80% of COPD cases can be attributed to smoking. Leukocytes recruited to the lung contribute to COPD pathology by releasing reactive oxygen metabolites and proteolytic enzymes. In this work, we investigated where leukocytes enter the lung in the early stages of COPD in order to better understand their effect as a contributor to the development of COPD. We simultaneously evaluated the parenchyma and airways for neutrophil accumulation, as well as increases in the adhesion molecules and chemokines that cause leukocyte recruitment in the early stages of tobacco smoke induced lung disease. We found neutrophil accumulation and increased expression of adhesion molecules and chemokines in the bronchial blood vessels that correlated with the accumulation of leukocytes recovered from the lung. The expression of adhesion molecules and chemokines in other vascular beds did not correlate with leukocytes recovered in bronchoalveolar lavage fluid (BALF). These data strongly suggest leukocytes are recruited in large measure through the bronchial circulation in response to tobacco smoke. Our findings have important implications for understanding the etiology of COPD and suggest that pharmaceuticals designed to reduce leukocyte recruitment through the bronchial circulation may be a potential therapy to treat COPD

Explorative visual analytics on interval-based genomic data and their metadata

Background: With the wide-spreading of public repositories of NGS processed data, the availability of user-friendly and effective tools for data exploration, analysis and visualization is becoming very relevant. These tools enable interactive analytics, an exploratory approach for the seamless "sense-making" of data through on-the-fly integration of analysis and visualization phases, suggested not only for evaluating processing results, but also for designing and adapting NGS data analysis pipelines. Results: This paper presents abstractions for supporting the early analysis of NGS processed data and their implementation in an associated tool, named GenoMetric Space Explorer (GeMSE). This tool serves the needs of the GenoMetric Query Language, an innovative cloud-based system for computing complex queries over heterogeneous processed data. It can also be used starting from any text files in standard BED, BroadPeak, NarrowPeak, GTF, or general tab-delimited format, containing numerical features of genomic regions; metadata can be provided as text files in tab-delimited attribute-value format. GeMSE allows interactive analytics, consisting of on-the-fly cycling among steps of data exploration, analysis and visualization that help biologists and bioinformaticians in making sense of heterogeneous genomic datasets. By means of an explorative interaction support, users can trace past activities and quickly recover their results, seamlessly going backward and forward in the analysis steps and comparative visualizations of heatmaps. Conclusions: GeMSE effective application and practical usefulness is demonstrated through significant use cases of biological interest. GeMSE is available at http://www.bioinformatics.deib.polimi.it/GeMSE/ , and its source code is available at https://github.com/Genometric/GeMSEunder GPLv3 open-source license

Myoblast sensitivity and fibroblast insensitivity to osteogenic conversion by BMP-2 correlates with the expression of Bmpr-1a

<p>Abstract</p> <p>Background</p> <p>Osteoblasts are considered to primarily arise from osseous progenitors within the periosteum or bone marrow. We have speculated that cells from local soft tissues may also take on an osteogenic phenotype. Myoblasts are known to adopt a bone gene program upon treatment with the osteogenic bone morphogenetic proteins (BMP-2,-4,-6,-7,-9), but their osteogenic capacity relative to other progenitor types is unclear. We further hypothesized that the sensitivity of cells to BMP-2 would correlate with BMP receptor expression.</p> <p>Methods</p> <p>We directly compared the BMP-2 sensitivity of myoblastic murine cell lines and primary cells with osteoprogenitors from osseous tissues and fibroblasts. Fibroblasts forced to undergo myogenic conversion by transduction with a MyoD-expressing lentiviral vector (LV-MyoD) were also examined. Outcome measures included alkaline phosphatase expression, matrix mineralization, and expression of osteogenic genes <it>(alkaline phosphatase, osteocalcin </it>and <it>bone morphogenetic protein receptor-1A) </it>as measured by quantitative PCR.</p> <p>Results</p> <p>BMP-2 induced a rapid and robust osteogenic response in myoblasts and osteoprogenitors, but not in fibroblasts. Myoblasts and osteoprogenitors grown in osteogenic media rapidly upregulated <it>Bmpr-1a </it>expression. Chronic BMP-2 treatment resulted in peak <it>Bmpr-1a </it>expression at day 6 before declining, suggestive of a negative feedback mechanism. In contrast, fibroblasts expressed low levels of <it>Bmpr-1a </it>that was only weakly up-regulated by BMP-2 treatment. Bioinformatics analysis confirmed the presence of myogenic responsive elements in the proximal promoter region of human and murine <it>BMPR-1A/Bmpr-1a</it>. Forced myogenic gene expression in fibroblasts was associated with a significant increase in <it>Bmpr-1a </it>expression and a synergistic increase in the osteogenic response to BMP-2.</p> <p>Conclusion</p> <p>These data demonstrate the osteogenic sensitivity of muscle progenitors and provide a mechanistic insight into the variable response of different cell lineages to BMP-2.</p

Cross species comparison of C/EBPα and PPARγ profiles in mouse and human adipocytes reveals interdependent retention of binding sites

<p>Abstract</p> <p>Background</p> <p>The transcription factors peroxisome proliferator activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) are key transcriptional regulators of adipocyte differentiation and function. We and others have previously shown that binding sites of these two transcription factors show a high degree of overlap and are associated with the majority of genes upregulated during differentiation of murine 3T3-L1 adipocytes.</p> <p>Results</p> <p>Here we have mapped all binding sites of C/EBPα and PPARγ in human SGBS adipocytes and compared these with the genome-wide profiles from mouse adipocytes to systematically investigate what biological features correlate with retention of sites in orthologous regions between mouse and human. Despite a limited interspecies retention of binding sites, several biological features make sites more likely to be retained. First, co-binding of PPARγ and C/EBPα in mouse is the most powerful predictor of retention of the corresponding binding sites in human. Second, vicinity to genes highly upregulated during adipogenesis significantly increases retention. Third, the presence of C/EBPα consensus sites correlate with retention of both factors, indicating that C/EBPα facilitates recruitment of PPARγ. Fourth, retention correlates with overall sequence conservation within the binding regions independent of C/EBPα and PPARγ sequence patterns, indicating that other transcription factors work cooperatively with these two key transcription factors.</p> <p>Conclusions</p> <p>This study provides a comprehensive and systematic analysis of what biological features impact on retention of binding sites between human and mouse. Specifically, we show that the binding of C/EBPα and PPARγ in adipocytes have evolved in a highly interdependent manner, indicating a significant cooperativity between these two transcription factors.</p

ECRG4 is a candidate tumor suppressor gene frequently hypermethylated in colorectal carcinoma and glioma

<p>Abstract</p> <p>Background</p> <p>Cancer cells display widespread changes in DNA methylation that may lead to genetic instability by global hypomethylation and aberrant silencing of tumor suppressor genes by focal hypermethylation. In turn, altered DNA methylation patterns have been used to identify putative tumor suppressor genes.</p> <p>Methods</p> <p>In a methylation screening approach, we identified <it>ECRG4 </it>as a differentially methylated gene. We analyzed different cancer cells for <it>ECRG4 </it>promoter methylation by COBRA and bisulfite sequencing. Gene expression analysis was carried out by semi-quantitative RT-PCR. The <it>ECRG4 </it>coding region was cloned and transfected into colorectal carcinoma cells. Cell growth was assessed by MTT and BrdU assays. ECRG4 localization was analyzed by fluorescence microscopy and Western blotting after transfection of an <it>ECRG4-eGFP </it>fusion gene.</p> <p>Results</p> <p>We found a high frequency of <it>ECRG4 </it>promoter methylation in various cancer cell lines. Remarkably, aberrant methylation of <it>ECRG4 </it>was also found in primary human tumor tissues, including samples from colorectal carcinoma and from malignant gliomas. <it>ECRG4 </it>hypermethylation associated strongly with transcriptional silencing and its expression could be re-activated <it>in vitro </it>by demethylating treatment with 5-aza-2'-deoxycytidine. Overexpression of <it>ECRG4 </it>in colorectal carcinoma cells led to a significant decrease in cell growth. In transfected cells, ECRG4 protein was detectable within the Golgi secretion machinery as well as in the culture medium.</p> <p>Conclusions</p> <p><it>ECRG4 </it>is silenced via promoter hypermethylation in different types of human cancer cells. Its gene product may act as inhibitor of cell proliferation in colorectal carcinoma cells and may play a role as extracellular signaling molecule.</p