Development and validation of a clinical decision rule for the diagnosis of influenza.

INTRODUCTION: A clinical decision rule to improve the accuracy of a diagnosis of influenza could help clinicians avoid unnecessary use of diagnostic tests and treatments. Our objective was to develop and validate a simple clinical decision rule for diagnosis of influenza. METHODS: We combined data from 2 studies of influenza diagnosis in adult outpatients with suspected influenza: one set in California and one in Switzerland. Patients in both studies underwent a structured history and physical examination and had a reference standard test for influenza (polymerase chain reaction or culture). We randomly divided the dataset into derivation and validation groups and then evaluated simple heuristics and decision rules from previous studies and 3 rules based on our own multivariate analysis. Cutpoints for stratification of risk groups in each model were determined using the derivation group before evaluating them in the validation group. For each decision rule, the positive predictive value and likelihood ratio for influenza in low-, moderate-, and high-risk groups, and the percentage of patients allocated to each risk group, were reported. RESULTS: The simple heuristics (fever and cough; fever, cough, and acute onset) were helpful when positive but not when negative. The most useful and accurate clinical rule assigned 2 points for fever plus cough, 2 points for myalgias, and 1 point each for duration <48 hours and chills or sweats. The risk of influenza was 8% for 0 to 2 points, 30% for 3 points, and 59% for 4 to 6 points; the rule performed similarly in derivation and validation groups. Approximately two-thirds of patients fell into the low- or high-risk group and would not require further diagnostic testing. CONCLUSION: A simple, valid clinical rule can be used to guide point-of-care testing and empiric therapy for patients with suspected influenza.

Identification and Characterization of Natural Anti-Breast Cancer Compound: Costunolide

Breast cancer is the most common cancer among women, 23% (1.3 million) of the total of new cases and the second leading cause of cancer death in women exceeded only by lung cancer. Natural medicines have been proven to be a central source of narrative agents with a pharmaceutical potential. Costunolide is sesquiterpene lactones consisting of diverse plant chemicals that exhibit anti cancer action through cytotoxic effects on various cancer cells. The objectives of present study were to explore the effects of natural compounds on the proliferation of MCF-7 cells and to determine the role of ROS in natural compounds-induced apoptosis in breast cancer cells with a therapeutic potential. Results showed that costunolide screened, possess potent anticancer properties against breast cancer MCF-7 cells, Costunolide was observed as strong anti-proliferative agent with IC50 = 50µM. The anti-proliferative effect of costunolide on MCF cells was confirmed by live/dead assay using fluorescent probes calcein AV/PI. The results demonstrated that treatment of cells with costunolide decreased the viability of MCF-7 cells in a dose-dependent manner. To determine the costunolide-induced apoptosis, flow cytometric analysis was carried out. The results showed that costunolide induced apoptosis in a dose-dependent manner in breast cancer MCF-7cells. ROS are well known mediators of intracellular signaling of cascades. The excessive generation of ROS can induce oxidative stress, loss of cell functioning, and apoptosis. In the present study, we assumed that costunolide might arouse ROS level, which could be involved in induction of apoptosis. Therefore, the intracellular ROS level was measured using the ROS-detecting fluorescence dye 2, 7-dichlorofluorescein diacetate (DCF-DA). Interestingly these effects were significantly abrogated when the cells were pretreated with N-acetyl- cysteine (NAC), a specific ROS inhibitor. Costunolide induces apoptosis through extrinsic pathway in MCF-7 breast cancer cells, In order to examine whether costunolide suppresses cell growth inducing apoptotic cell death, we analyzed DNA contents and apoptosis-related proteins expression level by flow cytometry and western blot, respectively in MCF-7 breast cancer cells we investigated whether costunolide activates extrinsic apoptotic pathway. We examined the expression levels of death receptor signaling-related proteins, caspase-3, and PARP. The results showed that procaspase-3 was cleaved to yield 17 and 20kDa fragments and activation of PARP in treated cells with 25 and 50μM of costunolide. Costunolide induce apoptosis through intrinsic mitochondria pathway in MCF-7 breast cancer Cells. We examined the expression levels of mitochondrial apoptotic pathway related proteins such as anti-apoptotic protein, B-cell lymphoma protein-2 (Bcl2), and pro-apoptotic protein Bax. Costunolide involved in the down regulation of Bcl-2 and up regulation of Bax. These results suggest that costunolide may have beneficial effects for the reduction of breast cancer growth, and new therapeutic strategy for the treatment of human cancers.

Quality assessment of cardiovascular magnetic resonance in the setting of the European CMR registry: description and validation of standardized criteria.

BACKGROUND: Cardiovascular magnetic resonance (CMR) has become an important diagnostic imaging modality in cardiovascular medicine. However, insufficient image quality may compromise its diagnostic accuracy. We aimed to describe and validate standardized criteria to evaluate a) cine steady-state free precession (SSFP), b) late gadolinium enhancement (LGE), and c) stress first-pass perfusion images. These criteria will serve for quality assessment in the setting of the Euro-CMR registry. METHODS: Thirty-five qualitative criteria were defined (scores 0-3) with lower scores indicating better image quality. In addition, quantitative parameters were measured yielding 2 additional quality criteria, i.e. signal-to-noise ratio (SNR) of non-infarcted myocardium (as a measure of correct signal nulling of healthy myocardium) for LGE and % signal increase during contrast medium first-pass for perfusion images. These qualitative and quantitative criteria were assessed in a total of 90 patients (60 patients scanned at our own institution at 1.5T (n=30) and 3T (n=30) and in 30 patients randomly chosen from the Euro-CMR registry examined at 1.5T). Analyses were performed by 2 SCMR level-3 experts, 1 trained study nurse, and 1 trained medical student. RESULTS: The global quality score was 6.7±4.6 (n=90, mean of 4 observers, maximum possible score 64), range 6.4-6.9 (p=0.76 between observers). It ranged from 4.0-4.3 for 1.5T (p=0.96 between observers), from 5.9-6.9 for 3T (p=0.33 between observers), and from 8.6-10.3 for the Euro-CMR cases (p=0.40 between observers). The inter- (n=4) and intra-observer (n=2) agreement for the global quality score, i.e. the percentage of assignments to the same quality tertile ranged from 80% to 88% and from 90% to 98%, respectively. The agreement for the quantitative assessment for LGE images (scores 0-2 for SNR <2, 2-5, >5, respectively) ranged from 78-84% for the entire population, and 70-93% at 1.5T, 64-88% at 3T, and 72-90% for the Euro-CMR cases. The agreement for perfusion images (scores 0-2 for %SI increase >200%, 100%-200%,<100%, respectively) ranged from 81-91% for the entire population, and 76-100% at 1.5T, 67-96% at 3T, and 62-90% for the Euro-CMR registry cases. The intra-class correlation coefficient for the global quality score was 0.83. CONCLUSIONS: The described criteria for the assessment of CMR image quality are robust with a good inter- and intra-observer agreement. Further research is needed to define the impact of image quality on the diagnostic and prognostic yield of CMR studies.

Extraction of soil water by plants: development and validation of a model

A quantitative model of water movement within the immediate vicinity of an individual root is developed and results of an experiment to validate the model are presented. The model is based on the assumption that the amount of water transpired by a plant in a certain period is replaced by an equal volume entering its root system during the same time. The model is based on the Darcy-Buckingham equation to calculate the soil water matric potential at any distance from a plant root as a function of parameters related to crop, soil and atmospheric conditions. The model output is compared against measurements of soil water depletion by rice roots monitored using γ-beam attenuation in a greenhouse of the Escola Superior de Agricultura "Luiz de Queiroz"/Universidade de São Paulo(ESALQ/USP) in Piracicaba, State of São Paulo, Brazil, in 1993. The experimental results are in agreement with the output from the model. Model simulations show that a single plant root is able to withdraw water from more than 0.1 m away within a few days. We therefore can assume that root distribution is a less important factor for soil water extraction efficiency.

Paper 4: EUROCAT statistical monitoring: identification and investigation of ten year trends of congenital anomalies in Europe.

BACKGROUND: As part of EUROCAT's surveillance of congenital anomalies in Europe, a statistical monitoring system has been developed to detect recent clusters or long-term (10 year) time trends. The purpose of this article is to describe the system for the identification and investigation of 10-year time trends, conceived as a "screening" tool ultimately leading to the identification of trends which may be due to changing teratogenic factors.METHODS: The EUROCAT database consists of all cases of congenital anomalies including livebirths, fetal deaths from 20 weeks gestational age, and terminations of pregnancy for fetal anomaly. Monitoring of 10-year trends is performed for each registry for each of 96 non-independent EUROCAT congenital anomaly subgroups, while Pan-Europe analysis combines data from all registries. The monitoring results are reviewed, prioritized according to a prioritization strategy, and communicated to registries for investigation. Twenty-one registries covering over 4 million births, from 1999 to 2008, were included in monitoring in 2010.CONCLUSIONS: Significant increasing trends were detected for abdominal wall anomalies, gastroschisis, hypospadias, Trisomy 18 and renal dysplasia in the Pan-Europe analysis while 68 increasing trends were identified in individual registries. A decreasing trend was detected in over one-third of anomaly subgroups in the Pan-Europe analysis, and 16.9% of individual registry tests. Registry preliminary investigations indicated that many trends are due to changes in data quality, ascertainment, screening, or diagnostic methods. Some trends are inevitably chance phenomena related to multiple testing, while others seem to represent real and continuing change needing further investigation and response by regional/national public health authorities.

Development and Validation of Single Nucleotide Polymorphisms (SNPs) Markers from Two Transcriptome 454-Runs of Turbot (Scophthalmus maximus) Using High-Throughput Genotyping

The turbot (Scophthalmus maximus) is a commercially valuable flatfish and one of the most promising aquaculture species in Europe. Two transcriptome 454-pyrosequencing runs were used in order to detect Single Nucleotide Polymorphisms (SNPs) in genesrelated to immune response and gonad differentiation. A total of 866 true SNPs were detected in 140 different contigs representing 262,093 bp as a whole. Only one true SNP was analyzed in each contig. One hundred and thirteen SNPs out of the 140 analyzed were feasible (genotyped), while Ш were polymorphic in a wild population. Transition/transversion ratio (1.354) was similar to that observed in other fish studies. Unbiased gene diversity (He) estimates ranged from 0.060 to 0.510 (mean = 0.351), minimum allele frequency (MAF) from 0.030 to 0.500 (mean = 0.259) and all loci were in Hardy-Weinberg equilibrium after Bonferroni correction. A large number of SNPs (49) were located in the coding region, 33 representing synonymous and 16 non-synonymous changes. Most SNP-containing genes were related to immune response and gonad differentiation processes, and could be candidates for functional changes leading to phenotypic changes. These markers will be useful for population screening to look for adaptive variation in wild and domestic turbot

Identification and removal of low-complexity sites in allele-specific analysis of ChIP-seq data.

MOTIVATION: High-throughput sequencing technologies enable the genome-wide analysis of the impact of genetic variation on molecular phenotypes at unprecedented resolution. However, although powerful, these technologies can also introduce unexpected artifacts. Results: We investigated the impact of library amplification bias on the identification of allele-specific (AS) molecular events from high-throughput sequencing data derived from chromatin immunoprecipitation assays (ChIP-seq). Putative AS DNA binding activity for RNA polymerase II was determined using ChIP-seq data derived from lymphoblastoid cell lines of two parent-daughter trios. We found that, at high-sequencing depth, many significant AS binding sites suffered from an amplification bias, as evidenced by a larger number of clonal reads representing one of the two alleles. To alleviate this bias, we devised an amplification bias detection strategy, which filters out sites with low read complexity and sites featuring a significant excess of clonal reads. This method will be useful for AS analyses involving ChIP-seq and other functional sequencing assays.

Quality of assessement of CMR images in the setting of the European cardiovascular magnetic resonance registry : description and validation of standardized criteria

l'imagerie par résonance magnétique (IRMC) est une technologie utilisée depuis les aimées quatre¬-vingts dans le monde de la cardiologie. Cette technique d'imagerie non-invasive permet d'acquérir Ses images du coeur en trois dimensions, dans n'importe quel, plan, sans application de radiation, et en haute résolution. Actuellement, cette technique est devenue un référence dans l'évaluation et 'l'investigation de différentes pathologies cardiaques. La morphologie cardiaque, la fonction des ventricules ainsi que leur contraction, la perfusion tissulaire ainsi que la viabilité tissulaire peuvent être caractérisés en utilisant différentes séquences d'imagerie. Cependant, cette technologie repose sur des principes physiques complexes et la mise en pratique de cette technique se heurte à la difficulté d'évaluer un organe en mouvement permanent. L'IRM cardiaque est donc sujette à différents artefacts qui perturbent l'interprétation des examens et peuvent diminuer la précision diagnostique de cette technique. A notre connaissance, la plupart des images d'IRMC sont analysées et interprétées sans évaluation rigoureuse de la qualité intrinsèque de l'examen. Jusqu'à présent, et à notre connaissance, aucun critère d'évaluation de la qualité des examens d'IRMC n'a été clairement déterminé. L'équipe d'IRMC du CHUV, dirigée par le Prof J. Schwitter, a recensé une liste de 35 critères qualitatifs et 12 critères quantitatifs évaluant la qualité d'un examen d'IRMC et les a introduit dans une grille d'évaluation. L'objet de cette étude est de décrire et de valider la reproductibilité des critères figurant dans cette grille d'évaluation, par l'interprétation simultanée d'examens IRMC par différents observateurs (cardiologues spécialisés en IRM, étudiant en médecine, infirmière spécialisée). Notre étude a permis de démontrer que les critères définis pour l'évaluation des examens d'IRMC sont robustes, et permettent une bonne reproductibilité intra- et inter-observateurs. Cette étude valide ainsi l'utilisation de ces critères de qualité dans le cadre de l'imagerie par résonance magnétique cardiaque. D'autres études sont encore nécessaires afin de déterminer l'impact de la qualité de l'image sur la précision diagnostique de cette technique. Les critères standardisés que nous avons validés seront utilisés pour évaluer la qualité des images dans le cadre d'une étude à échelle européenne relative à l'IRMC : "l'EuroCMR registry". Parmi les autres utilités visées par ces critères de qualité, citons notamment la possibilité d'avoir une référence d'évaluation de la qualité d'examen pour toutes les futures études cliniques utilisant la technologie d'IRMC, de permettre aux centres d'IRMC de quantifier leur niveau de qualité, voire de créer un certificat de standard de qualité pour ces centres, d'évaluer la reproductibilité de l'évaluation des images par différents observateurs d'un même centre, ou encore d'évaluer précisément la qualité des séquences développées à l'avenir dans le monde de l'IRMC.

Development and validation of a clinical prediction rule for chest wall syndrome in primary care

Le "Chest wall syndrome" (CWS) est défini comme étant une source bénigne de douleurs thoraciques, localisées sur la paroi thoracique antérieure et provoquées par une affection musculosquelettique. Le CWS représente la cause la plus fréquente de douleurs thoraciques en médecine de premier recours. Le but de cette étude est de développer et valider un score de prédiction clinique pour le CWS. Une revue de la littérature a d'abord été effectuée, d'une part pour savoir si un tel score existait déjà, et d'autre part pour retrouver les variables décrites comme étant prédictives d'un CWS. Le travail d'analyse statistique a été effectué avec les données issues d'une cohorte clinique multicentrique de patients qui avaient consulté en médecine de premier recours en Suisse romande avec une douleur thoracique (59 cabinets, 672 patients). Un diagnostic définitif avait été posé à 12 mois de suivi. Les variables pertinentes ont été sélectionnées par analyses bivariées, et le score de prédiction clinique a été développé par régression logistique multivariée. Une validation externe de ce score a été faite en utilisant les données d'une cohorte allemande (n= 1212). Les analyses bivariées ont permis d'identifier 6 variables caractérisant le CWS : douleur thoracique (ni rétrosternale ni oppressive), douleur en lancées, douleur bien localisée, absence d'antécédent de maladie coronarienne, absence d'inquiétude du médecin et douleur reproductible à la palpation. Cette dernière variable compte pour 2 points dans le score, les autres comptent pour 1 point chacune; le score total s'étend donc de 0 à 7 points. Dans la cohorte de dérivation, l'aire sous la courbe sensibilité/spécificité (courbe ROC) est de 0.80 (95% de l'intervalle de confiance : 0.76-0.83). Avec un seuil diagnostic de > 6 points, le score présente 89% de spécificité et 45% de sensibilité. Parmi tous les patients qui présentaient un CWS (n = 284), 71% (n = 201) avaient une douleur reproductible à la palpation et 45% (n= 127) sont correctement diagnostiqués par le score. Pour une partie (n = 43) de ces patients souffrant de CWS et correctement classifiés, 65 investigations complémentaires (30 électrocardiogrammes, 16 radiographies du thorax, 10 analyses de laboratoire, 8 consultations spécialisées, et une tomodensitométrie thoracique) avaient été réalisées pour parvenir au diagnostic. Parmi les faux positifs (n = 41), on compte trois angors stables (1.8% de tous les positifs). Les résultats de la validation externe sont les suivants : une aire sous la courbe ROC de 0.76 (95% de l'intervalle de confiance : 0.73-0.79) avec une sensibilité de 22% et une spécificité de 93%. Ce score de prédiction clinique pour le CWS constitue un complément utile à son diagnostic, habituellement obtenu par exclusion. En effet, pour les 127 patients présentant un CWS et correctement classifiés par notre score, 65 investigations complémentaires auraient pu être évitées. Par ailleurs, la présence d'une douleur thoracique reproductible à la palpation, bien qu'étant sa plus importante caractéristique, n'est pas pathognomonique du CWS.

Identification and isolation of two ascomycete fungi from spores of the arbuscular mycorrhizal fungus Scutellospora castanea.

Two filamentous fungi with different phenotypes were isolated from crushed healthy spores or perforated dead spores of the arbuscular mycorrhizal fungus (AMF) Scutellospora castanea. Based on comparative sequence analysis of 5.8S ribosomal DNA and internal transcribed spacer fragments, one isolate, obtained from perforated dead spores only, was assigned to the genus Nectria, and the second, obtained from both healthy and dead spores, was assigned to Leptosphaeria, a genus that also contains pathogens of plants in the Brassicaceae. PCR and randomly amplified polymorphic DNA-PCR analyses, however, did not indicate similarities between pathogens and the isolate. The presence of the two isolates in both healthy spores and perforated dead spores of S. castanea was finally confirmed by transmission electron microscopy by using distinctive characteristics of the isolates and S. castanea. The role of this fungus in S. castanea spores remains unclear, but the results serve as a strong warning that sequences obtained from apparently healthy AMF spores cannot be presumed to be of glomalean origin and that this could present problems for studies on AMF genes.

Development, design and validation of solid reference samples.

We developed a method of sample preparation using epoxy compound, which was validated in two steps. First, we studied the homogeneity within samples by scanning tubes filled with radioactive epoxy. We found within-sample homogeneity better than 2%. Then, we studied the homogeneity between samples during a 4.5 h dispensing time. The homogeneity between samples was found to be better than 2%. This study demonstrates that we have a validated method, which assures the traceability of epoxy samples.

Generation and validation of a normative, age-specific reference curve for lumbar spine trabecular bone score (TBS) in French women.

Age-related changes in lumbar vertebral microarchitecture are evaluated, as assessed by trabecular bone score (TBS), in a cohort of 5,942 French women. The magnitude of TBS decline between 45 and 85 years of age is piecewise linear in the spine and averaged 14.5 %. TBS decline rate increases after 65 years by 50 %. INTRODUCTION: This study aimed to evaluate age-related changes in lumbar vertebral microarchitecture, as assessed by TBS, in a cohort of French women aged 45-85 years. METHODS: An all-comers cohort of French Caucasian women was selected from two clinical centers. Data obtained from these centers were cross-calibrated for TBS and bone mineral density (BMD). BMD and TBS were evaluated at L1-L4 and for all lumbar vertebrae combined using GE-Lunar Prodigy densitometer images. Weight, height, and body mass index (BMI) also were determined. To validate our all-comers cohort, the BMD normative data of our cohort and French Prodigy data were compared. RESULTS: A cohort of 5,942 French women aged 45 to 85 years was created. Dual-energy X-ray absorptiometry normative data obtained for BMD from this cohort were not significantly different from French prodigy normative data (p = 0.15). TBS values at L1-L4 were poorly correlated with BMI (r = -0.17) and weight (r = -0.14) and not correlated with height. TBS values obtained for all lumbar vertebra combined (L1, L2, L3, L4) decreased with age. The magnitude of TBS decline at L1-L4 between 45 and 85 years of age was piecewise linear in the spine and averaged 14.5 %, but this rate increased after 65 years by 50 %. Similar results were obtained for other region of interest in the lumbar spine. As opposed to BMD, TBS was not affected by spinal osteoarthrosis. CONCLUSION: The age-specific reference curve for TBS generated here could therefore be used to help clinicians to improve osteoporosis patient management and to monitor microarchitectural changes related to treatment or other diseases in routine clinical practice.

Development and validation of a symptom-based activity index for adults with eosinophilic esophagitis.

BACKGROUND & AIMS: Standardized instruments are needed to assess the activity of eosinophilic esophagitis (EoE) and to provide end points for clinical trials and observational studies. We aimed to develop and validate a patient-reported outcome (PRO) instrument and score, based on items that could account for variations in patient assessments of disease severity. We also evaluated relationships between patient assessment of disease severity and EoE-associated endoscopic, histologic, and laboratory findings. METHODS: We collected information from 186 patients with EoE in Switzerland and the United States (69.4% male; median age, 43 y) via surveys (n = 135), focus groups (n = 27), and semistructured interviews (n = 24). Items were generated for the instruments to assess biologic activity based on physician input. Linear regression was used to quantify the extent to which variations in patient-reported disease characteristics could account for variations in patient assessment of EoE severity. The PRO instrument was used prospectively in 153 adult patients with EoE (72.5% male; median age, 38 y), and validated in an independent group of 120 patients with EoE (60.8% male; median age, 40.5 y). RESULTS: Seven PRO factors that are used to assess characteristics of dysphagia, behavioral adaptations to living with dysphagia, and pain while swallowing accounted for 67% of the variation in patient assessment of disease severity. Based on statistical consideration and patient input, a 7-day recall period was selected. Highly active EoE, based on endoscopic and histologic findings, was associated with an increase in patient-assessed disease severity. In the validation study, the mean difference between patient assessment of EoE severity (range, 0-10) and PRO score (range, 0-8.52) was 0.15. CONCLUSIONS: We developed and validated an EoE scoring system based on 7 PRO items that assess symptoms over a 7-day recall period. Clinicaltrials.gov number: NCT00939263.

Bioinformatics-driven identification and examination of candidate genes for non-alcoholic fatty liver disease.

ObjectiveCandidate genes for non-alcoholic fatty liver disease (NAFLD) identified by a bioinformatics approach were examined for variant associations to quantitative traits of NAFLD-related phenotypes.Research Design and MethodsBy integrating public database text mining, trans-organism protein-protein interaction transferal, and information on liver protein expression a protein-protein interaction network was constructed and from this a smaller isolated interactome was identified. Five genes from this interactome were selected for genetic analysis. Twenty-one tag single-nucleotide polymorphisms (SNPs) which captured all common variation in these genes were genotyped in 10,196 Danes, and analyzed for association with NAFLD-related quantitative traits, type 2 diabetes (T2D), central obesity, and WHO-defined metabolic syndrome (MetS).Results273 genes were included in the protein-protein interaction analysis and EHHADH, ECHS1, HADHA, HADHB, and ACADL were selected for further examination. A total of 10 nominal statistical significant associations (P<0.05) to quantitative metabolic traits were identified. Also, the case-control study showed associations between variation in the five genes and T2D, central obesity, and MetS, respectively. Bonferroni adjustments for multiple testing negated all associations.ConclusionsUsing a bioinformatics approach we identified five candidate genes for NAFLD. However, we failed to provide evidence of associations with major effects between SNPs in these five genes and NAFLD-related quantitative traits, T2D, central obesity, and MetS.