Microbiological diagnosis of community-acquired respiratory tract infections by nucleic acid detection.

Background: Microbiological diagnostic procedures have changed significantly over the last decade. Initially the implementation of the polymerase chain reaction (PCR) resulted in improved detection tests for microbes that were difficult or even impossible to detect by conventional methods such as culture and serology, especially in community-acquired respiratory tract infections (CA-RTI). A further improvement was the development of real-time PCR, which allows end point detection and quantification, and many diagnostic laboratories have now implemented this powerful method. Objective: At present, new performant and convenient molecular tests have emerged targeting in parallel many viruses and bacteria responsible for lower and/or upper respiratory tract infections. The range of test formats and microbial agents detected is evolving very quickly and the added value of these new tests needs to be studied in terms of better use of antibiotics, better patient management, duration of hospitalization and overall costs. Conclusions: Molecular tools for a better microbial documentation of CA-RTI are now available. Controlled studies are now required to address the relevance issue of these new methods, such as, for example, the role of some newly detected respiratory viruses or of the microbial DNA load in a particular patient at a particular time. The future challenge for molecular diagnosis will be to become easy to handle, highly efficient and cost-effective, delivering rapid results with a direct impact on clinical management.

Treeline and climate change : analyzing and modeling patterns and shifts in the Swiss Alps

Summary: Global warming has led to an average earth surface temperature increase of about 0.7 °C in the 20th century, according to the 2007 IPCC report. In Switzerland, the temperature increase in the same period was even higher: 1.3 °C in the Northern Alps anal 1.7 °C in the Southern Alps. The impacts of this warming on ecosystems aspecially on climatically sensitive systems like the treeline ecotone -are already visible today. Alpine treeline species show increased growth rates, more establishment of young trees in forest gaps is observed in many locations and treelines are migrating upwards. With the forecasted warming, this globally visible phenomenon is expected to continue. This PhD thesis aimed to develop a set of methods and models to investigate current and future climatic treeline positions and treeline shifts in the Swiss Alps in a spatial context. The focus was therefore on: 1) the quantification of current treeline dynamics and its potential causes, 2) the evaluation and improvement of temperaturebased treeline indicators and 3) the spatial analysis and projection of past, current and future climatic treeline positions and their respective elevational shifts. The methods used involved a combination of field temperature measurements, statistical modeling and spatial modeling in a geographical information system. To determine treeline shifts and assign the respective drivers, neighborhood relationships between forest patches were analyzed using moving window algorithms. Time series regression modeling was used in the development of an air-to-soil temperature transfer model to calculate thermal treeline indicators. The indicators were then applied spatially to delineate the climatic treeline, based on interpolated temperature data. Observation of recent forest dynamics in the Swiss treeline ecotone showed that changes were mainly due to forest in-growth, but also partly to upward attitudinal shifts. The recent reduction in agricultural land-use was found to be the dominant driver of these changes. Climate-driven changes were identified only at the uppermost limits of the treeline ecotone. Seasonal mean temperature indicators were found to be the best for predicting climatic treelines. Applying dynamic seasonal delimitations and the air-to-soil temperature transfer model improved the indicators' applicability for spatial modeling. Reproducing the climatic treelines of the past 45 years revealed regionally different attitudinal shifts, the largest being located near the highest mountain mass. Modeling climatic treelines based on two IPCC climate warming scenarios predicted major shifts in treeline altitude. However, the currently-observed treeline is not expected to reach this limit easily, due to lagged reaction, possible climate feedback effects and other limiting factors. Résumé: Selon le rapport 2007 de l'IPCC, le réchauffement global a induit une augmentation de la température terrestre de 0.7 °C en moyenne au cours du 20e siècle. En Suisse, l'augmentation durant la même période a été plus importante: 1.3 °C dans les Alpes du nord et 1.7 °C dans les Alpes du sud. Les impacts de ce réchauffement sur les écosystèmes - en particuliers les systèmes sensibles comme l'écotone de la limite des arbres - sont déjà visibles aujourd'hui. Les espèces de la limite alpine des forêts ont des taux de croissance plus forts, on observe en de nombreux endroits un accroissement du nombre de jeunes arbres s'établissant dans les trouées et la limite des arbres migre vers le haut. Compte tenu du réchauffement prévu, on s'attend à ce que ce phénomène, visible globalement, persiste. Cette thèse de doctorat visait à développer un jeu de méthodes et de modèles pour étudier dans un contexte spatial la position présente et future de la limite climatique des arbres, ainsi que ses déplacements, au sein des Alpes suisses. L'étude s'est donc focalisée sur: 1) la quantification de la dynamique actuelle de la limite des arbres et ses causes potentielles, 2) l'évaluation et l'amélioration des indicateurs, basés sur la température, pour la limite des arbres et 3) l'analyse spatiale et la projection de la position climatique passée, présente et future de la limite des arbres et des déplacements altitudinaux de cette position. Les méthodes utilisées sont une combinaison de mesures de température sur le terrain, de modélisation statistique et de la modélisation spatiale à l'aide d'un système d'information géographique. Les relations de voisinage entre parcelles de forêt ont été analysées à l'aide d'algorithmes utilisant des fenêtres mobiles, afin de mesurer les déplacements de la limite des arbres et déterminer leurs causes. Un modèle de transfert de température air-sol, basé sur les modèles de régression sur séries temporelles, a été développé pour calculer des indicateurs thermiques de la limite des arbres. Les indicateurs ont ensuite été appliqués spatialement pour délimiter la limite climatique des arbres, sur la base de données de températures interpolées. L'observation de la dynamique forestière récente dans l'écotone de la limite des arbres en Suisse a montré que les changements étaient principalement dus à la fermeture des trouées, mais aussi en partie à des déplacements vers des altitudes plus élevées. Il a été montré que la récente déprise agricole était la cause principale de ces changements. Des changements dus au climat n'ont été identifiés qu'aux limites supérieures de l'écotone de la limite des arbres. Les indicateurs de température moyenne saisonnière se sont avérés le mieux convenir pour prédire la limite climatique des arbres. L'application de limites dynamiques saisonnières et du modèle de transfert de température air-sol a amélioré l'applicabilité des indicateurs pour la modélisation spatiale. La reproduction des limites climatiques des arbres durant ces 45 dernières années a mis en évidence des changements d'altitude différents selon les régions, les plus importants étant situés près du plus haut massif montagneux. La modélisation des limites climatiques des arbres d'après deux scénarios de réchauffement climatique de l'IPCC a prédit des changements majeurs de l'altitude de la limite des arbres. Toutefois, l'on ne s'attend pas à ce que la limite des arbres actuellement observée atteigne cette limite facilement, en raison du délai de réaction, d'effets rétroactifs du climat et d'autres facteurs limitants.

Improvement of chronic congestive heart-failure by oral captopril

Catopril, an inhibitor of angiotensin converting enzyme, was given orally during cardiac catheterisation to 6 normotensive patients with refractory congestive heart-failure. 60--180 minutes after administration of 25 mg captopril, arterial pressure fell by 25%, cardiac index rose by 38%, and left-ventricular pressure and right-atrial pressure fell by 25% and 40% respectively. Plasma-renin activity rose while plasma noradrenaline and aldosterone fell. These data suggest that, in the short term, captopril can reduce both preload and afterload, and improve cardiac function, in refractory congestive heart-failure.

Infections nosocomiales : interventions et qualité des soins [Maladies infectieuses = Nosocomial infections : quality improvement interventions]

Le développement de méthodes de mesure de la qualité des soins et les actions visant à son amélioration font partie intégrante de la pratique quotidienne. Les infections nosocomiales représentent un Indicateur Incontournable et leur prévention apparaît comme un champ d'action privilégié du concept de l'amélioration continue de la qualité des soins. Dans ce domaine, de nombreuses mesures ont fait l'objet d'études répondant à des critères méthodologiques de qualité élevée, dont les résultats sont hautement significatifs au plan statistique. Les interventions complexes concernant par exemple l'ensemble des patients d'un service ou d'une institution ne permettent que difficilement de satisfaire à ces exigences méthodologiques. Sous certaines conditions, elle peuvent cependant avoir un impact réel et parfois durable sur la qualité des soins. A titre d'exemple, nous discutons deux interventions complexes dans le domaine de la prévention des infections nosocomiales, résultant de démarches visant à une amélioration globale et durable de la qualité des soins. L'utilisation d'outils épidémiologiques adaptés permet également d'estimer le bénéfice des Interventions en milieu hospitalier conformément à ce que la communauté scientifique reconnaît comme étant une recherche clinique de qualité.

The E.coli RuvAB proteins branch migrate Holliday junctions through heterologous DNA sequences in a reaction facilitated by SSB.

During genetic recombination a heteroduplex joint is formed between two homologous DNA molecules. The heteroduplex joint plays an important role in recombination since it accommodates sequence heterogeneities (mismatches, insertions or deletions) that lead to genetic variation. Two Escherichia coli proteins, RuvA and RuvB, promote the formation of heteroduplex DNA by catalysing the branch migration of crossovers, or Holliday junctions, which link recombining chromosomes. We show that RuvA and RuvB can promote branch migration through 1800 bp of heterologous DNA, in a reaction facilitated by the presence of E.coli single-stranded DNA binding (SSB) protein. Reaction intermediates, containing unpaired heteroduplex regions bound by SSB, were directly visualized by electron microscopy. In the absence of SSB, or when SSB was replaced by a single-strand binding protein from bacteriophage T4 (gene 32 protein), only limited heterologous branch migration was observed. These results show that the RuvAB proteins, which are induced as part of the SOS response to DNA damage, allow genetic recombination and the recombinational repair of DNA to occur in the presence of extensive lengths of heterology.

Manual therapy followed by specific active exercises versus a placebo followed by specific active exercises on the improvement of functional disability in patients with chronic non specific low back pain: a randomized controlled trial.

BACKGROUND: Recent clinical recommendations still propose active exercises (AE) for CNSLBP. However, acceptance of exercises by patients may be limited by pain-related manifestations. Current evidences suggest that manual therapy (MT) induces an immediate analgesic effect through neurophysiologic mechanisms at peripheral, spinal and cortical levels. The aim of this pilot study was first, to assess whether MT has an immediate analgesic effect, and second, to compare the lasting effect on functional disability of MT plus AE to sham therapy (ST) plus AE. METHODS: Forty-two CNSLBP patients without co-morbidities, randomly distributed into 2 treatment groups, received either spinal manipulation/mobilization (first intervention) plus AE (MT group; n = 22), or detuned ultrasound (first intervention) plus AE (ST group; n = 20). Eight therapeutic sessions were delivered over 4 to 8 weeks. Immediate analgesic effect was obtained by measuring pain intensity (Visual Analogue Scale) before and immediately after the first intervention of each therapeutic session. Pain intensity, disability (Oswestry Disability Index), fear-avoidance beliefs (Fear-Avoidance Beliefs Questionnaire), erector spinae and abdominal muscles endurance (Sorensen and Shirado tests) were assessed before treatment, after the 8th therapeutic session, and at 3- and 6-month follow-ups. RESULTS: Thirty-seven subjects completed the study. MT intervention induced a better immediate analgesic effect that was independent from the therapeutic session (VAS mean difference between interventions: -0.8; 95% CI: -1.2 to -0.3). Independently from time after treatment, MT + AE induced lower disability (ODI mean group difference: -7.1; 95% CI: -12.8 to -1.5) and a trend to lower pain (VAS mean group difference: -1.2; 95% CI: -2.4 to -0.30). Six months after treatment, Shirado test was better for the ST group (Shirado mean group difference: -61.6; 95% CI: -117.5 to -5.7). Insufficient evidence for group differences was found in remaining outcomes. CONCLUSIONS: This study confirmed the immediate analgesic effect of MT over ST. Followed by specific active exercises, it reduces significantly functional disability and tends to induce a larger decrease in pain intensity, compared to a control group. These results confirm the clinical relevance of MT as an appropriate treatment for CNSLBP. Its neurophysiologic mechanisms at cortical level should be investigated more thoroughly. TRIAL REGISTRATION: Trial registration number: NCT01496144.

The effect of thrombolysis on short-term improvement depends on initial stroke severity.

A large number of parameters have been identified as predictors of early outcome in patients with acute ischemic stroke. In the present work we analyzed a wide range of demographic, metabolic, physiological, clinical, laboratory and neuroimaging parameters in a large population of consecutive patients with acute ischemic stroke with the aim of identifying independent predictors of the early clinical course. We used prospectively collected data from the Acute Stroke Registry and Analysis of Lausanne. All consecutive patients with ischemic stroke admitted to our stroke unit and/or intensive care unit between 1 January 2003 and 12 December 2008 within 24 h after last-well time were analyzed. Univariate and multivariate analyses were performed to identify significant associations with the National Institute of Health Stroke Scale (NIHSS) score at admission and 24 h later. We also sought any interactions between the identified predictors. Of the 1,730 consecutive patients with acute ischemic stroke who were included in the analysis, 260 (15.0%) were thrombolyzed (mostly intravenously) within the recommended time window. In multivariate analysis, the NIHSS score at 24 h after admission was associated with the NIHSS score at admission (β = 1, p < 0.001), initial glucose level (β = 0.05, p < 0.002) and thrombolytic intervention (β = -2.91, p < 0.001). There was a significant interaction between thrombolysis and the NIHSS score at admission (p < 0.001), indicating that the short-term effect of thrombolysis decreases with increasing initial stroke severity. Thrombolytic treatment, lower initial glucose level and lower initial stroke severity predict a favorable early clinical course. The short-term effect of thrombolysis appears mainly in minor and moderate strokes, and decreases with increasing initial stroke severity.

Therapeutic Drug Monitoring of the new targeted anticancer agents imatinib, nilotinib, dasatinib, sunitinib, sorafenib and lapatinib by LC tandem mass spectrometry.

The treatment of some cancer patients has shifted from traditional, non-specific cytotoxic chemotherapy to chronic treatment with molecular targeted therapies. Imatinib mesylate, a selective inhibitor of tyrosine kinases (TKIs) is the most prominent example of this new era and has opened the way to the development of several additional TKIs, including sunitinib, nilotinib, dasatinib, sorafenib and lapatinib, in the treatment of various hematological malignancies and solid tumors. All these agents are characterized by an important inter-individual pharmacokinetic variability, are at risk for drug interactions, and are not devoid of toxicity. Additionally, they are administered for prolonged periods, anticipating the careful monitoring of their plasma exposure via Therapeutic Drug Monitoring (TDM) to be an important component of patients' follow-up. We have developed a liquid chromatography-tandem mass spectrometry method (LC-MS/MS) requiring 100 microL of plasma for the simultaneous determination of the six major TKIs currently in use. Plasma is purified by protein precipitation and the supernatant is diluted in ammonium formate 20 mM (pH 4.0) 1:2. Reverse-phase chromatographic separation of TKIs is obtained using a gradient elution of 20 mM ammonium formate pH 2.2 and acetonitrile containing 1% formic acid, followed by rinsing and re-equilibration to the initial solvent composition up to 20 min. Analyte quantification, using matrix-matched calibration samples, is performed by electro-spray ionization-triple quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effects variability (<9.6%), overall process efficiency (87.1-104.2%), as well as TKIs short- and long-term stability in plasma. The method is precise (inter-day CV%: 1.3-9.4%), accurate (-9.2 to +9.9%) and sensitive (lower limits of quantification comprised between 1 and 10 ng/mL). This is the first broad-range LC-MS/MS assay covering the major currently in-use TKIs. It is an improvement over previous methods in terms of convenience (a single extraction procedure for six major TKIs, reducing significantly the analytical time), sensitivity, selectivity and throughput. It may contribute to filling the current knowledge gaps in the pharmacokinetics/pharmacodynamics relationships of the latest TKIs developed after imatinib and better define their therapeutic ranges in different patient populations in order to evaluate whether a systematic TDM-guided dose adjustment of these anticancer drugs could contribute to minimize the risk of major adverse reactions and to increase the probability of efficient, long lasting, therapeutic response.

Improvement of the HIV/AIDS vaccine candidate NYVAC-C by deletion of the viral type I IFN inhibitor and/or acquisition of replication competence

Background: Recombinant viruses based on the attenuated vaccinia virus strain NYVAC are promising HIV vaccine candidates as phase I/II clinical trials have shown good safety and immunogenicity profiles. However, this NYVAC strain is non-replicating in most human cell lines and encodes viral inhibitors of the immune system. Methods: With the aim to increase the immune potency of the current NYVAC-C vector (expressing the codon optimized clade C HIV-1 genes encoding gp120 and Gag-Pol-Nef polyprotein), we have generated and characterized three NYVAC-C-based vectors by, 1) deletion of the viral type I IFN inhibitor gene (NYVAC-CdeltaB19R), 2) restoration of virus replication competence in human cells by re-inserting K1L and C7L host range genes (NYVAC-C-KC) and, 3) combination of both strategies (NYVACC- KC-deltaB19R). Results: Insertion of the KC fragment restored the replication competence of the viruses in human cells (HeLa cells and primary dermal fibroblasts and keratinocytes), increased the expression of HIV antigens by more than 3-fold compared to the non-replicating homologs, inhibited apoptosis induced by the parental NYVAC-C and retained attenuation in a newborn mouse model. In adult mice, replication-competent viruses showed a limited capacity to replicate in tissues surrounding the inoculation site (ovaries and lymph nodes). After infection of keratinocytes, PBMCs and dendritic cells these viruses induced differential modulation in specific host cell signal transduction pathways, triggering genes important in immune modulation. Conclusion: We have developed improved NYVAC-C-based vectors with enhanced HIV-1 antigen expression, with the ability to replicate in cultured human cells and partially in some tissues, with an induced expression of cellular genes relevant to immune system activation, and which trigger IFN-dependent and independent signalling pathways, while maintaining a safety phenotype. These new vectors are promising new HIV vaccine candidates. These studies were performed within the Poxvirus Tcell Vaccine Discovery Consortium (PTVDC) which is part of the CAVD program.

Reverse transcription quantitative real-time polymerase chain reaction reference genes in the spared nerve injury model of neuropathic pain : validation and literature search

La douleur neuropathique est définie comme une douleur causée par une lésion du système nerveux somato-sensoriel. Elle se caractérise par des douleurs exagérées, spontanées, ou déclenchées par des stimuli normalement non douloureux (allodynie) ou douloureux (hyperalgésie). Bien qu'elle concerne 7% de la population, ses mécanismes biologiques ne sont pas encore élucidés. L'étude des variations d'expressions géniques dans les tissus-clés des voies sensorielles (notamment le ganglion spinal et la corne dorsale de la moelle épinière) à différents moments après une lésion nerveuse périphérique permettrait de mettre en évidence de nouvelles cibles thérapeutiques. Elles se détectent de manière sensible par reverse transcription quantitative real-time polymerase chain reaction (RT- qPCR). Pour garantir des résultats fiables, des guidelines ont récemment recommandé la validation des gènes de référence utilisés pour la normalisation des données ("Minimum information for publication of quantitative real-time PCR experiments", Bustin et al 2009). Après recherche dans la littérature des gènes de référence fréquemment utilisés dans notre modèle de douleur neuropathique périphérique SNI (spared nerve injury) et dans le tissu nerveux en général, nous avons établi une liste de potentiels bons candidats: Actin beta (Actb), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal proteins 18S (18S), L13a (RPL13a) et L29 (RPL29), hypoxanthine phosphoribosyltransferase 1 (HPRT1) et hydroxymethyl-bilane synthase (HMBS). Nous avons évalué la stabilité d'expression de ces gènes dans le ganglion spinal et dans la corne dorsale à différents moments après la lésion nerveuse (SNI) en calculant des coefficients de variation et utilisant l'algorithme geNorm qui compare les niveaux d'expression entre les différents candidats et détermine la paire de gènes restante la plus stable. Il a aussi été possible de classer les gènes selon leur stabilité et d'identifier le nombre de gènes nécessaires pour une normalisation la plus précise. Les gènes les plus cités comme référence dans le modèle SNI ont été GAPDH, HMBS, Actb, HPRT1 et 18S. Seuls HPRT1 and 18S ont été précédemment validés dans des arrays de RT-qPCR. Dans notre étude, tous les gènes testés dans le ganglion spinal et dans la corne dorsale satisfont au critère de stabilité exprimé par une M-value inférieure à 1. Par contre avec un coefficient de variation (CV) supérieur à 50% dans le ganglion spinal, 18S ne peut être retenu. La paire de gènes la plus stable dans le ganglion spinal est HPRT1 et Actb et dans la corne dorsale il s'agit de RPL29 et RPL13a. L'utilisation de 2 gènes de référence stables suffit pour une normalisation fiable. Nous avons donc classé et validé Actb, RPL29, RPL13a, HMBS, GAPDH, HPRT1 et 18S comme gènes de référence utilisables dans la corne dorsale pour le modèle SNI chez le rat. Dans le ganglion spinal 18S n'a pas rempli nos critères. Nous avons aussi déterminé que la combinaison de deux gènes de référence stables suffit pour une normalisation précise. Les variations d'expression génique de potentiels gènes d'intérêts dans des conditions expérimentales identiques (SNI, tissu et timepoints post SNI) vont pouvoir se mesurer sur la base d'une normalisation fiable. Non seulement il sera possible d'identifier des régulations potentiellement importantes dans la genèse de la douleur neuropathique mais aussi d'observer les différents phénotypes évoluant au cours du temps après lésion nerveuse.

Reaction to novelty and spatial orientation in ALPHA1B(-/-) KO mice

Knockout mice lacking alphalb noradrenergic receptors were tested in behavioural experiments to test a possible effect of the absence of this receptor in reaction to novelty and spatial orientation. Reaction to novelty was tested in two experiments. In the first one the mice' latency to exit the first part of a two compartment set-up was measured. The knockout mice were faster to emerge then their littermate controls. Then they were tested in an open-field, in which new objects were added at the second trial. In the open-field without objects (first trial), the knockout mice showed a greater locomotor activity (path length). Then the same mice showed enhanced exploration of the newly introduced objects, relative to the control. The spatial orientation experiments were done on a homing board and in the water maze. The homing board did not yield a significant difference between the knock-out and the control mice. Both groups showed impaired results when the proximal (olfactory) and distal (visual) cues were disrupted by the rotation of the table. In the water maze however, the alphalb(-/-) mice were unable to solve the task (acquisition and retention), whereas the control mice showed a good acquisition and retention behaviour. The knockout mice' incapacity to learn to reach the submerged platform was not due to an incapacity to swim, as they were as good as their control littermates to reach the platform when it was visible.

Clinical outcome after trimipramine in patients with delusional depression - a pilot study

The treatment of delusional depression is a major challenge in psychopharmacology. Hypothalamic-pituitary-adrenocortical (HPA) overdrive may contribute, via increased dopaminergic activity, to the pathophysiology of the disorder. Trimipramine appears to be an interesting potential candidate, since it is an atypical antidepressant that is known to inhibit HPA activity. In a four-week open trial we investigated its effects in 15 inpatients with delusional depression. The dosage was increased within 7 days up to 300 - 400 mg/d and was then maintained for three weeks. Psychometric assessments and safety monitoring were conducted weekly. Assessment of the HPA activity was achieved by a combined dexamethasone suppression/corticotropin-releasing hormone stimulation (Dex/CRH) test before and after four weeks of treatment. Therapeutic response was defined as a decrease in the HAMD-score of at least 50 %. Eight out of 13 completers were rated as responders. Therapeutic response was associated with L, D-trimipramine concentrations higher than 160 ng/ml. Intent-to-treat analysis showed significant improvement in psychometric variables. Despite the high dosage, the substance was generally well tolerated, with the exception of one patient who suffered from a hypotensive reaction. Mean +/- SD concentration of L-trimipramine and D-trimipramine were 138 +/- 61 ng/ml and 119 +/- 50 ng/ml at a final dose of 346 +/- 50 mg/d. The ACTH and cortisol area under the curve in the Dex/CRH tests decreased significantly, reflecting a decrease of activity in the HPA system. We suggest that the clinical use of high-dose trimipramine in delusional depression seems to be a promising treatment strategy.