Detection of micrometastases in sentinel lymph nodes from melanoma patients: direct comparison of multimarker molecular and immunopathological methods.

The technique of sentinel lymph node (SLN) dissection is a reliable predictor of metastatic disease in the lymphatic basin draining the primary melanoma. Reverse transcription-polymerase chain reaction (RT-PCR) is emerging as a highly sensitive technique to detect micrometastases in SLNs, but its specificity has been questioned. A prospective SLN study in melanoma patients was undertaken to compare in detail immunopathological versus molecular detection methods. Sentinel lymphadenectomy was performed on 57 patients, with a total of 71 SLNs analysed. SLNs were cut in slices, which were alternatively subjected to parallel multimarker analysis by microscopy (haematoxylin and eosin and immunohistochemistry for HMB-45, S100, tyrosinase and Melan-A/MART-1) and RT-PCR (for tyrosinase and Melan-A/MART-1). Metastases were detected by both methods in 23% of the SLNs (28% of the patients). The combined use of Melan-A/MART-1 and tyrosinase amplification increased the sensitivity of PCR detection of microscopically proven micrometastases. Of the 55 immunopathologically negative SLNs, 25 were found to be positive on RT-PCR. Notably, eight of these SLNs contained naevi, all of which were positive for tyrosinase and/or Melan-A/MART-1, as detected at both mRNA and protein level. The remaining 41% of the SLNs were negative on both immunohistochemistry and RT-PCR. Analysis of a series of adjacent non-SLNs by RT-PCR confirmed the concept of orderly progression of metastasis. Clinical follow-up showed disease recurrence in 12% of the RT-PCR-positive immunopathology-negative SLNs, indicating that even an extensive immunohistochemical analysis may underestimate the presence of micrometastases. However, molecular analyses, albeit more sensitive, need to be further improved in order to attain acceptable specificity before they can be applied diagnostically.

Association of serum homocysteine with major depressive disorder: results from a large population-based study.

BACKGROUND: Studies on the association between homocysteine levels and depression have shown conflicting results. To examine the association between serum total homocysteine (tHcy) levels and major depressive disorder (MDD) in a large community sample with an extended age range. METHODS: A total of 3392 men and women aged 35-66 years participating in the CoLaus study and its psychiatric arm (PsyCoLaus) were included in the analyses. High tHcy measured from fasting blood samples was defined as a concentration ≥15μmol/L. MDD was assessed using the semi-structured Diagnostic Interview for Genetics Studies. RESULTS: In multivariate analyses, elevated tHcy levels were associated with greater odds of meeting the diagnostic criteria for lifetime MDD among men (OR=1.71; 95% CI, 1.18-2.50). This was particularly the case for remitted MDD. Among women, there was no significant association between tHcy levels and MDD and the association tended to be in the opposite direction (OR=0.61; 95% CI, 0.34-1.08). CONCLUSIONS: In this large population-based study, elevated tHcy concentrations are associated with lifetime MDD and particularly with remitted MDD among men.

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.

Evaluating sampling strategies and logistic regression methods for modelling complex land cover changes

The role of land cover change as a significant component of global change has become increasingly recognized in recent decades. Large databases measuring land cover change, and the data which can potentially be used to explain the observed changes, are also becoming more commonly available. When developing statistical models to investigate observed changes, it is important to be aware that the chosen sampling strategy and modelling techniques can influence results. We present a comparison of three sampling strategies and two forms of grouped logistic regression models (multinomial and ordinal) in the investigation of patterns of successional change after agricultural land abandonment in Switzerland. Results indicated that both ordinal and nominal transitional change occurs in the landscape and that the use of different sampling regimes and modelling techniques as investigative tools yield different results. Synthesis and applications. Our multimodel inference identified successfully a set of consistently selected indicators of land cover change, which can be used to predict further change, including annual average temperature, the number of already overgrown neighbouring areas of land and distance to historically destructive avalanche sites. This allows for more reliable decision making and planning with respect to landscape management. Although both model approaches gave similar results, ordinal regression yielded more parsimonious models that identified the important predictors of land cover change more efficiently. Thus, this approach is favourable where land cover change pattern can be interpreted as an ordinal process. Otherwise, multinomial logistic regression is a viable alternative.

Chronic beryllium disease as a re-emerging occupational disorder possibly misdiagnosed as sarcoidosis : a Swiss study project

Background: Beryllium (Be) is increasingly used worldwide for numerous industrial applications. Occupational exposure to Be may lead to Be sensitization (BeS), a CD4-mediated immune response. BeS may progress to chronic beryllium disease (CBD), a granulomatous lung disorder closely resembling sarcoidosis. The recognition of CBD requires detection of Be exposure at occupational history, and detection of BeS on blood or BAL lymphocytes. Since methods for CBD detection are not routinely available in Switzerland, we hypothesized that CBD cases are not recognized but misdiagnosis as sarcoidosis. Objective: To present an ongoing Swiss study screening patients with sarcoidosis in search of Be exposure, BeS, and CBD. Methods: Both a prospective and a retrospective cohort are being studied. In the prospective cohort, the main steps include: 1) recruitment of 100 consecutive patients with newly diagnosed pulmonary sarcoidosis at 2 centers (Lausanne, Bern). 2) screening for possible occupational Be exposure by self-administered patient questionnaire. 3) standardized detailed occupational interview and clinical visit by occupational health specialist. If step 3 is positive, then 4) blood and BAL sampling for detection of BeS by specifically developed Elispot assay and CFSE flow cytometry, with subsequent comparison to the classical Be lymphocyte proliferation test. If step 4 is positive, then 5) review of medical records and diagnostic revision from sarcoidosis to CBD. 6) appropriate measures for exposure cessation and case reporting to SUVA as occupational disease. The retrospective cohort will include 400 patients with previously diagnosed pulmonary sarcoidosis, either treated or untreated, recruited through the SIOLD Registries. Steps 2 to 5 will be peformed as above, except for a) end of study after step 2 if screening questionnaire does not reveal Be exposure, and b) step 4 done on blood sample only (BAL not needed). Current status: Self-administered screening questionnaire and tools for standardized occupational interview have been developed. BeS testing has been implemented and undergoes validation. Inclusions in the prospective phase have started at both study sites. The retrospective phase is in preparation. Conclusion: The current study status allows to conclude to technical feasibility of the project. The prospective phase if this study is funded by the SUVA. The SIOLD Registries are supported by the Swiss Pulmonary League.

Surfactant protein-D and exposure to bioaerosols in wastewater and garbage workers.

PURPOSE: Bioaerosols and their constituents, such as endotoxins, are capable of causing an inflammatory reaction at the level of the lung-blood barrier, which becomes more permeable. Thus, it was hypothesized that occupational exposure to bioaerosols can increase leakage of surfactant protein-D (SP-D), a lung-specific protein, into the bloodstream. METHODS: SP-D was determined by ELISA in 316 wastewater workers, 67 garbage collectors, and 395 control subjects. Exposure was assessed with four interview-based indicators and by preliminary endotoxin measurements using the Limulus amoebocyte lysate assay. Influence of exposure on serum SP-D was assessed by multiple linear regression considering smoking, glomerular function, lung diseases, obesity, and other confounders. RESULTS: Overall, mean exposure levels to endotoxins were below 100 EU/m(3). However, special tasks of wastewater workers caused higher endotoxin exposure. SP-D concentration was slightly increased in this occupational group and associated with the occurrence of splashes and contact to raw sewage. No effect was found in garbage collectors. Smoking increased serum SP-D. No clinically relevant correlation between spirometry results and SP-D concentrations appeared. CONCLUSIONS: These results support the hypothesis that inhalation of bioaerosols, even at low concentrations, has a subclinical effect on the lung-blood barrier, the permeability of which increases without associated spirometric changes.

Quelle assistance psychologique pour les couples requérant une aide médicale à la procréation [Which psychological support for couples seeking medical assistance for procreation?]

OBJECTIVES: The objective of our qualitative study was to define modalities of psychological support to be offered to couples seeking medically assisted procreation. MATERIAL AND METHODS: Forty couples participated in a semi-structured videotaped interview, which touched on themes such as personal and family histories, the couple's relationship, etc. We focused on the "narrative mobility", that is the way in which the couples transmit their personal and family history during the interview and the interviewer's impression that he may or may not share this with the couples. RESULTS: Observed differences in narrative mobility led us to distinguish three groups of couples and to propose various types of psychological support. DISCUSSION AND CONCLUSION: This exploratory study, with its interest for the narrative mobility, concerns the couples' capacity to stand back from their own story as responsible interlocutors. We made the hypothesis that this capacity is linked to their capacity to handle their emotional stress, to act as partners to the medical team and to prepare themselves for their future parenthood.

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.

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.