NEW METHODS FOR DETECTION, SUSCEPTIBILITY TESTING AND BIOFILM ERADICATION OF DIFFICULT ORGANISMS

Aujourd'hui, les problèmes des maladies infectieuses concernent l'émergence d'infections difficiles à traiter, telles que les infections associées aux implants et les infections fongiques invasives chez les patients immunodéprimés. L'objectif de cette thèse était de développer des stratégies pour l'éradication des biofilms bactériens (partie 1), ainsi que d'étudier des méthodes innovantes pour la détection microbienne, pour l'établissement de nouveaux tests de sensibilité (partie 2). Le traitement des infections associées aux implants est difficile car les biofilms bactériens peuvent résister à des niveaux élevés d'antibiotiques. A ce jour, il n'y a pas de traitement optimal défini contre des infections causées par des bactéries de prévalence moindre telles que Enterococcus faecalis ou Propionibacterium acnés. Dans un premier temps, nous avons démontré une excellente activité in vitro de la gentamicine sur une souche de E. faecalis en phase stationnaire de croissance Nous avons ensuite confirmé l'activité de la gentamicine sur un biofilm précoce en modèle expérimental animal à corps étranger avec un taux de guérison de 50%. De plus, les courbes de bactéricidie ainsi que les résultats de calorimétrie ont prouvé que l'ajout de gentamicine améliorait l'activité in vitro de la daptomycine, ainsi que celle de la vancomycine. In vivo, le schéma thérapeutique le plus efficace était l'association daptomycine/gentamicine avec un taux de guérison de 55%. En établissant une nouvelle méthode pour l'évaluation de l'activité des antimicrobiens vis-à-vis de micro-organismes en biofilm, nous avons démontré que le meilleur antibiotique actif sur les biofilms à P. acnés était la rifampicine, suivi par la penicilline G, la daptomycine et la ceftriaxone. Les études conduites en modèle expérimental animal ont confirmé l'activité de la rifampicine seule avec un taux de guérison 36%. Le meilleur schéma thérapeutique était au final l'association rifampicine/daptomycine avec un taux de guérison 63%. Les associations de rifampicine avec la vancomycine ou la levofloxacine présentaient des taux de guérisons respectivement de 46% et 25%. Nous avons ensuite étudié l'émergence in vitro de la résistance à la rifampicine chez P. acnés. Nous avons observé un taux de mutations de 10"9. La caractérisation moléculaire de la résistance chez les mutant-résistants a mis en évidence l'implication de 5 mutations ponctuelles dans les domaines I et II du gène rpoB. Ce type de mutations a déjà été décrit au préalable chez d'autres espèces bactériennes, corroborant ainsi la validité de nos résultats. La deuxième partie de cette thèse décrit une nouvelle méthode d'évaluation de l'efficacité des antifongiques basée sur des mesures de microcalorimétrie isotherme. En utilisant un microcalorimètre, la chaleur produite par la croissance microbienne peut être-mesurée en temps réel, très précisément. Nous avons évalué l'activité de l'amphotéricine B, des triazolés et des échinocandines sur différentes souches de Aspergillus spp. par microcalorimétrie. La présence d'amphotéricine Β ou de triazole retardait la production de chaleur de manière concentration-dépendante. En revanche, pour les échinochandines, seule une diminution le pic de « flux de chaleur » a été observé. La concordance entre la concentration minimale inhibitrice de chaleur (CMIC) et la CMI ou CEM (définie par CLSI M38A), avec une marge de 2 dilutions, était de 90% pour l'amphotéricine B, 100% pour le voriconazole, 90% pour le pozoconazole et 70% pour la caspofongine. La méthode a été utilisée pour définir la sensibilité aux antifongiques pour d'autres types de champignons filamenteux. Par détermination microcalorimétrique, l'amphotéricine B s'est avéré être l'agent le plus actif contre les Mucorales et les Fusarium spp.. et le voriconazole le plus actif contre les Scedosporium spp. Finalement, nous avons évalué l'activité d'associations d'antifongiques vis-à-vis de Aspergillus spp. Une meilleure activité antifongique était retrouvée avec l'amphotéricine B ou le voriconazole lorsque ces derniers étaient associés aux échinocandines vis-à-vis de A. fumigatus. L'association échinocandine/amphotéricine B a démontré une activité antifongique synergique vis-à-vis de A. terreus, contrairement à l'association échinocandine/voriconazole qui ne démontrait aucune amélioration significative de l'activité antifongique. - The diagnosis and treatment of infectious diseases are today increasingly challenged by the emergence of difficult-to-manage situations, such as infections associated with medical devices and invasive fungal infections, especially in immunocompromised patients. The aim of this thesis was to address these challenges by developing new strategies for eradication of biofilms of difficult-to-treat microorganisms (treatment, part 1) and investigating innovative methods for microbial detection and antimicrobial susceptibility testing (diagnosis, part 2). The first part of the thesis investigates antimicrobial treatment strategies for infections caused by two less investigated microorganisms, Enterococcus faecalis and Propionibacterium acnes, which are important pathogens causing implant-associated infections. The treatment of implant-associated infections is difficult in general due to reduced susceptibility of bacteria when present in biofilms. We demonstrated an excellent in vitro activity of gentamicin against E. faecalis in stationary growth- phase and were able to confirm the activity against "young" biofilms (3 hours) in an experimental foreign-body infection model (cure rate 50%). The addition of gentamicin improved the activity of daptomycin and vancomycin in vitro, as determined by time-kill curves and microcalorimetry. In vivo, the most efficient combination regimen was daptomycin plus gentamicin (cure rate 55%). Despite a short duration of infection, the cure rates were low, highlighting that enterococcal biofilms remain difficult to treat despite administration of newer antibiotics, such as daptomycin. By establishing a novel in vitro assay for evaluation of anti-biofilm activity (microcalorimetry), we demonstrated that rifampin was the most active antimicrobial against P. acnes biofilms, followed by penicillin G, daptomycin and ceftriaxone. In animal studies we confirmed the anti-biofilm activity of rifampin (cure rate 36% when administered alone), as well as in combination with daptomycin (cure rate 63%), whereas in combination with vancomycin or levofloxacin it showed lower cure rates (46% and 25%, respectively). We further investigated the emergence of rifampin resistance in P. acnes in vitro. Rifampin resistance progressively emerged during exposure to rifampin, if the bacterial concentration was high (108 cfu/ml) with a mutation rate of 10"9. In resistant isolates, five point mutations of the rpoB gene were found in cluster I and II, as previously described for staphylococci and other bacterial species. The second part of the thesis describes a novel real-time method for evaluation of antifungals against molds, based on measurements of the growth-related heat production by isothermal microcalorimetry. Current methods for evaluation of antifungal agents against molds, have several limitations, especially when combinations of antifungals are investigated. We evaluated the activity of amphotericin B, triazoles (voriconazole, posaconazole) and echinocandins (caspofungin and anidulafungin) against Aspergillus spp. by microcalorimetry. The presence of amphotericin Β or a triazole delayed the heat production in a concentration-dependent manner and the minimal heat inhibition concentration (MHIC) was determined as the lowest concentration inhibiting 50% of the heat produced at 48 h. Due to the different mechanism of action echinocandins, the MHIC for this antifungal class was determined as the lowest concentration lowering the heat-flow peak with 50%. Agreement within two 2-fold dilutions between MHIC and MIC or MEC (determined by CLSI M38A) was 90% for amphotericin B, 100% for voriconazole, 90% for posaconazole and 70% for caspofungin. We further evaluated our assay for antifungal susceptibility testing of non-Aspergillus molds. As determined by microcalorimetry, amphotericin Β was the most active agent against Mucorales and Fusarium spp., whereas voriconazole was the most active agent against Scedosporium spp. Finally, we evaluated the activity of antifungal combinations against Aspergillus spp. Against A. jumigatus, an improved activity of amphotericin Β and voriconazole was observed when combined with an echinocandin. Against A. terreus, an echinocandin showed a synergistic activity with amphotericin B, whereas in combination with voriconazole, no considerable improved activity was observed.

Changes in Running Mechanics and Spring-Mass Behaviour during a 5-km Time Trial

Research into the biomechanical manifestation of fatigue during exhaustive runs is increasingly popular but additional understanding of the adaptation of the spring-mass behaviour during the course of strenuous, self-paced exercises continues to be a challenge in order to develop optimized training and injury prevention programs. This study investigated continuous changes in running mechanics and spring-mass behaviour during a 5-km run. 12 competitive triathletes performed a 5-km running time trial (mean performance: 17 min 30 s) on a 200 m indoor track. Vertical and anterior-posterior ground reaction forces were measured every 200 m by a 5-m long force platform system, and used to determine spring-mass model characteristics. After a fast start, running velocity progressively decreased (- 11.6%; P<0.001) in the middle part of the race before an end spurt in the final 400-600 m. Stride length (- 7.4%; P<0.001) and frequency (- 4.1%; P=0.001) decreased over the 25 laps, while contact time (+ 8.9%; P<0.001) and total stride duration (+ 4.1%; P<0.001) progressively lengthened. Peak vertical forces (- 2.0%; P<0.01) and leg compression (- 4.3%; P<0.05), but not centre of mass vertical displacement (+ 3.2%; P>0.05), decreased with time. As a result, vertical stiffness decreased (- 6.0%; P<0.001) during the run, whereas leg stiffness changes were not significant (+ 1.3%; P>0.05). Spring-mass behaviour progressively changes during a 5-km time trial towards deteriorated vertical stiffness, which alters impact and force production characteristics.

Adaptive Continuous time Markov Chain Approximation Model to General Jump-Diusions

We propose a non-equidistant Q rate matrix formula and an adaptive numerical algorithm for a continuous time Markov chain to approximate jump-diffusions with affine or non-affine functional specifications. Our approach also accommodates state-dependent jump intensity and jump distribution, a flexibility that is very hard to achieve with other numerical methods. The Kolmogorov-Smirnov test shows that the proposed Markov chain transition density converges to the one given by the likelihood expansion formula as in Ait-Sahalia (2008). We provide numerical examples for European stock option pricing in Black and Scholes (1973), Merton (1976) and Kou (2002).

Large Time-Varying Parameter VARs

In this paper we develop methods for estimation and forecasting in large timevarying parameter vector autoregressive models (TVP-VARs). To overcome computational constraints with likelihood-based estimation of large systems, we rely on Kalman filter estimation with forgetting factors. We also draw on ideas from the dynamic model averaging literature and extend the TVP-VAR so that its dimension can change over time. A final extension lies in the development of a new method for estimating, in a time-varying manner, the parameter(s) of the shrinkage priors commonly-used with large VARs. These extensions are operationalized through the use of forgetting factor methods and are, thus, computationally simple. An empirical application involving forecasting inflation, real output, and interest rates demonstrates the feasibility and usefulness of our approach.

Time Variation in the Dynamics of Worker Flows: Evidence from the US and Canada

VAR methods have been used to model the inter-relationships between inflows and outfl ows into unemployment and vacancies using tools such as impulse response analysis. In order to investigate whether such impulse responses change over the course of the business cycle or or over time, this paper uses TVP-VARs for US and Canadian data. For the US, we find interesting differences between the most recent recession and earlier recessions and expansions. In particular, we find the immediate effect of a negative shock on both in ow and out flow hazards to be larger in 2008 than in earlier times. Furthermore, the effect of this shock takes longer to decay. For Canada, we fi nd less evidence of time-variation in impulse responses.

Model Switching and Model Averaging in Time- Varying Parameter Regression Models

This paper investigates the usefulness of switching Gaussian state space models as a tool for implementing dynamic model selecting (DMS) or averaging (DMA) in time-varying parameter regression models. DMS methods allow for model switching, where a different model can be chosen at each point in time. Thus, they allow for the explanatory variables in the time-varying parameter regression model to change over time. DMA will carry out model averaging in a time-varying manner. We compare our exact approach to DMA/DMS to a popular existing procedure which relies on the use of forgetting factor approximations. In an application, we use DMS to select different predictors in an in ation forecasting application. We also compare different ways of implementing DMA/DMS and investigate whether they lead to similar results.

A Case of Framing Effects: The Elicitation of Time Preferences.

We compare three methods for the elicitation of time preferences in an experimental setting: the Becker-DeGroot-Marschak procedure (BDM); the second price auction; and the multiple price list format. The first two methods have been used rarely to elicit time preferences. All methods used are perfectly equivalent from a decision theoretic point of view, and they should induce the same ‘truthful’ revelation i dominant strategies. In spite of this, we find that framing does matter: the money discount rates elicited with the multiple price list tend to be higher than those elicited with the other two methods. In addition, our results shed some light on attitudes towards time, and they permit a broad classification of subjects depending on how the size of the elicited values varies with the time horizon.

Time Varying Dimension Models

Time varying parameter (TVP) models have enjoyed an increasing popularity in empirical macroeconomics. However, TVP models are parameter-rich and risk over-fitting unless the dimension of the model is small. Motivated by this worry, this paper proposes several Time Varying dimension (TVD) models where the dimension of the model can change over time, allowing for the model to automatically choose a more parsimonious TVP representation, or to switch between different parsimonious representations. Our TVD models all fall in the category of dynamic mixture models. We discuss the properties of these models and present methods for Bayesian inference. An application involving US inflation forecasting illustrates and compares the different TVD models. We find our TVD approaches exhibit better forecasting performance than several standard benchmarks and shrink towards parsimonious specifications.

Hierarchical Shrinkage in Time-Varying Parameter Models

In this paper, we forecast EU-area inflation with many predictors using time-varying parameter models. The facts that time-varying parameter models are parameter-rich and the time span of our data is relatively short motivate a desire for shrinkage. In constant coefficient regression models, the Bayesian Lasso is gaining increasing popularity as an effective tool for achieving such shrinkage. In this paper, we develop econometric methods for using the Bayesian Lasso with time-varying parameter models. Our approach allows for the coefficient on each predictor to be: i) time varying, ii) constant over time or iii) shrunk to zero. The econometric methodology decides automatically which category each coefficient belongs in. Our empirical results indicate the benefits of such an approach.

A first attempt of geographically-distributed Multi-scale integrated analysis of societal and ecosystem metabolism (MuSIASEM): mapping human time and energy throughput in metropolitan Barcelona

This study presents a first attempt to extend the “Multi-scale integrated analysis of societal and ecosystem metabolism (MuSIASEM)” approach to a spatial dimension using GIS techniques in the Metropolitan area of Barcelona. We use a combination of census and commercial databases along with a detailed land cover map to create a layer of Common Geographic Units that we populate with the local values of human time spent in different activities according to MuSIASEM hierarchical typology. In this way, we mapped the hours of available human time, in regards to the working hours spent in different locations, putting in evidence the gradients in spatial density between the residential location of workers (generating the work supply) and the places where the working hours are actually taking place. We found a strong three-modal pattern of clumps of areas with different combinations of values of time spent on household activities and on paid work. We also measured and mapped spatial segregation between these two activities and put forward the conjecture that this segregation increases with higher energy throughput, as the size of the functional units must be able to cope with the flow of exosomatic energy. Finally, we discuss the effectiveness of the approach by comparing our geographic representation of exosomatic throughput to the one issued from conventional methods.

A Modulated Closed Form solution for Quantitative Susceptibility Mapping - A thorough evaluation and comparison to iterative methods based on edge prior knowledge.

The aim of this study is to perform a thorough comparison of quantitative susceptibility mapping (QSM) techniques and their dependence on the assumptions made. The compared methodologies were: two iterative single orientation methodologies minimizing the l2, l1TV norm of the prior knowledge of the edges of the object, one over-determined multiple orientation method (COSMOS) and anewly proposed modulated closed-form solution (MCF). The performance of these methods was compared using a numerical phantom and in-vivo high resolution (0.65mm isotropic) brain data acquired at 7T using a new coil combination method. For all QSM methods, the relevant regularization and prior-knowledge parameters were systematically changed in order to evaluate the optimal reconstruction in the presence and absence of a ground truth. Additionally, the QSM contrast was compared to conventional gradient recalled echo (GRE) magnitude and R2* maps obtained from the same dataset. The QSM reconstruction results of the single orientation methods show comparable performance. The MCF method has the highest correlation (corrMCF=0.95, r(2)MCF =0.97) with the state of the art method (COSMOS) with additional advantage of extreme fast computation time. The l-curve method gave the visually most satisfactory balance between reduction of streaking artifacts and over-regularization with the latter being overemphasized when the using the COSMOS susceptibility maps as ground-truth. R2* and susceptibility maps, when calculated from the same datasets, although based on distinct features of the data, have a comparable ability to distinguish deep gray matter structures.

Real-time PCR for type-specific identification of herpes simplex in clinical samples: evaluation of type-specific results in the context of CNS diseases.

BACKGROUND: HSV-1 and HSV-2 cause CNS infections of dissimilar clinico-pathological characteristics with prognostic and therapeutic implications. OBJECTIVES: To validate a type-specific real-time PCR that uses MGB/LNA Taqman probes and to review the virologico-clinical data of 25 eligible patients with non-neonatal CNS infections. RESULTS: This real-time PCR was evaluated against conventional PCR (26 CSF and 20 quality controls), and LightCycler assay (51 mucocutaneous, 8 CSF and 32 quality controls) and culture/immunofluorescence (75 mucocutaneous) to assess typing with independent methods. Taqman real-time PCR detected 240 HSV genomes per ml CSF, a level appropriate for the management of patients, and provided unambiguous typing for the 104 positive (62 HSV-1 and 42 HSV-2) out the 160 independent clinical samples tested. HSV type diagnosed by Taqman real-time PCR predicted final diagnosis (meningitis versus encephalitis/meningoencephalitis, p<0.001) in 24/25 patients at time of presentation, in contrast to clinical evaluation. CONCLUSIONS: Our real-time PCR, as a sensitive and specific means for type-specific HSV diagnosis, provided rapid prognostic information for patient management.

Quantitative comparison of reconstruction methods for intra-voxel fiber recovery from diffusion MRI.

Validation is arguably the bottleneck in the diffusion magnetic resonance imaging (MRI) community. This paper evaluates and compares 20 algorithms for recovering the local intra-voxel fiber structure from diffusion MRI data and is based on the results of the "HARDI reconstruction challenge" organized in the context of the "ISBI 2012" conference. Evaluated methods encompass a mixture of classical techniques well known in the literature such as diffusion tensor, Q-Ball and diffusion spectrum imaging, algorithms inspired by the recent theory of compressed sensing and also brand new approaches proposed for the first time at this contest. To quantitatively compare the methods under controlled conditions, two datasets with known ground-truth were synthetically generated and two main criteria were used to evaluate the quality of the reconstructions in every voxel: correct assessment of the number of fiber populations and angular accuracy in their orientation. This comparative study investigates the behavior of every algorithm with varying experimental conditions and highlights strengths and weaknesses of each approach. This information can be useful not only for enhancing current algorithms and develop the next generation of reconstruction methods, but also to assist physicians in the choice of the most adequate technique for their studies.

Decreasing population selection rates of resistance mutation K65R over time in HIV-1 patients receiving combination therapy including tenofovir.

OBJECTIVES: The use of tenofovir is highly associated with the emergence of mutation K65R, which confers broad resistance to nucleoside/nucleotide analogue reverse transcriptase inhibitors (NRTIs), especially when tenofovir is combined with other NRTIs also selecting for K65R. Although recent HIV-1 treatment guidelines discouraging these combinations resulted in reduced K65R selection with tenofovir, updated information on the impact of currently recommended regimens on the population selection rate of K65R is presently lacking. METHODS: In this study, we evaluated changes over time in the selection rate of resistance mutation K65R in a large population of 2736 HIV-1-infected patients failing combination antiretroviral treatment between 2002 and 2010. RESULTS: The K65R resistance mutation was detected in 144 patients, a prevalence of 5.3%. A large majority of observed K65R cases were explained by the use of tenofovir, reflecting its wide use in clinical practice. However, changing patterns over time in NRTIs accompanying tenofovir resulted in a persistent decreasing probability of K65R selection by tenofovir-based therapy. The currently recommended NRTI combination tenofovir/emtricitabine was associated with a low probability of K65R emergence. For any given dual NRTI combination including tenofovir, higher selection rates of K65R were consistently observed with a non-nucleoside reverse transcriptase inhibitor than with a protease inhibitor as the third agent. DISCUSSION: Our finding of a stable time trend of K65R despite elevated use of tenofovir illustrates increased potency of current HIV-1 therapy including tenofovir.

A cohort-based analysis of the evolution of lifetime prevalence of recourse to prostitution by men in the general population of Switzerland, 1987-2000

Background: Despite their relevance to the prevention of sexually transmitted infections, there are few data on the frequency of recourse to prostitution in the male population in Switzerland. Using data gathered for the evaluation of the Swiss AIDS prevention strategy, we analysed net aggregate change and cohort-based change in lifetime prevalence of recourse to prostitution. Methods: Seven repeated cross-sectional telephone surveys of the general population aged 17-45 years (17-30 years only for the 1987 and 1988 surveys) were undertaken from 1987 to 2000 providing information on sexual behaviour including men's recourse to prostitution (total n¼9318). Age categories were: 17-20, 21-25, 26-30, 31-35, 36-40 and 41-45 years. Prevalence at 17-30 years was available in all surveys and prevalence at 41-45 was available for 1989-2000, though not for the same cohorts. Intra-cohort increase in prevalence over 10 years was analysed using truncated information for cohorts aged 21-25 and 26-30 years in 1987 and 1990. Population estimates were computed with 95% confidence intervals (CI). Results: No net change occurred in the 17-45 years male population prevalence between 1989 (17.6%, CI ¼ 15.4; 20.0) and 2000 (17.7%, CI ¼ 15.6; 20.0). The median starting prevalence of recourse to prostitution at age 17-20 was 4.8% (in 1989, CI ¼ 2.0; 9.7) and the range was from 1.8 (in 1994) to 10.4% (in 1990). The median ending prevalence at age 41-45 was 21.9% (in 1994, CI 16.7; 27.9) and the range was from 17.9 (in 2000) to 26.1% (in 1992). No clear trend was observed in either starting or ending prevalence. Intra-cohort evolution of the 1997 and 1990 cohorts was very similar. Conclusions: Based on available data, there was no net (aggregate) change in the prevalence of recourse to prostitution by males in Switzerland between 1989 and 2000. Within the time frame available, intra-cohort evolution was also very similar.