Safety of human immunisation with a live-attenuated Mycobacterium tuberculosis vaccine: a randomised, double-blind, controlled phase I trial.

BACKGROUND: Tuberculosis remains one of the world's deadliest transmissible diseases despite widespread use of the BCG vaccine. MTBVAC is a new live tuberculosis vaccine based on genetically attenuated Mycobacterium tuberculosis that expresses most antigens present in human isolates of M tuberculosis. We aimed to compare the safety of MTBVAC with BCG in healthy adult volunteers. METHODS: We did this single-centre, randomised, double-blind, controlled phase 1 study at the Centre Hospitalier Universitaire Vaudois (CHUV; Lausanne, Switzerland). Volunteers were eligible for inclusion if they were aged 18-45 years, clinically healthy, HIV-negative and tuberculosis-negative, and had no history of active tuberculosis, chemoprophylaxis for tuberculosis, or BCG vaccination. Volunteers fulfilling the inclusion criteria were randomly assigned to three cohorts in a dose-escalation manner. Randomisation was done centrally by the CHUV Pharmacy and treatments were masked from the study team and volunteers. As participants were recruited within each cohort, they were randomly assigned 3:1 to receive MTBVAC or BCG. Of the participants allocated MTBVAC, those in the first cohort received 5 × 10(3) colony forming units (CFU) MTBVAC, those in the second cohort received 5 × 10(4) CFU MTBVAC, and those in the third cohort received 5 × 10(5) CFU MTBVAC. In all cohorts, participants assigned to receive BCG were given 5 × 10(5) CFU BCG. Each participant received a single intradermal injection of their assigned vaccine in 0·1 mL sterile water in their non-dominant arm. The primary outcome was safety in all vaccinated participants. Secondary outcomes included whole blood cell-mediated immune response to live MTBVAC and BCG, and interferon γ release assays (IGRA) of peripheral blood mononuclear cells. This trial is registered with ClinicalTrials.gov, number NCT02013245. FINDINGS: Between Jan 23, 2013, and Nov 6, 2013, we enrolled 36 volunteers into three cohorts, each of which consisted of nine participants who received MTBVAC and three who received BCG. 34 volunteers completed the trial. The safety of vaccination with MTBVAC at all doses was similar to that of BCG, and vaccination did not induce any serious adverse events. All individuals were IGRA negative at the end of follow-up (day 210). After whole blood stimulation with live MTBVAC or BCG, MTBVAC was at least as immunogenic as BCG. At the same dose as BCG (5×10(5) CFU), although no statistical significance could be achieved, there were more responders in the MTBVAC group than in the BCG group, with a greater frequency of polyfunctional CD4+ central memory T cells. INTERPRETATION: To our knowledge, MTBVAC is the first live-attenuated M tuberculosis vaccine to reach clinical assessment, showing similar safety to BCG. MTBVAC seemed to be at least as immunogenic as BCG, but the study was not powered to investigate this outcome. Further plans to use more immunogenicity endpoints in a larger number of volunteers (adults and adolescents) are underway, with the aim to thoroughly characterise and potentially distinguish immunogenicity between MTBVAC and BCG in tuberculosis-endemic countries. Combined with an excellent safety profile, these data support advanced clinical development in high-burden tuberculosis endemic countries. FUNDING: Biofabri and Bill & Melinda Gates Foundation through the TuBerculosis Vaccine Initiative (TBVI).

Systematic evaluation of matrix effects in hydrophilic interaction chromatography versus reversed phase liquid chromatography coupled to mass spectrometry.

Reversed phase liquid chromatography (RPLC) coupled to mass spectrometry (MS) is the gold standard technique in bioanalysis. However, hydrophilic interaction chromatography (HILIC) could represent a viable alternative to RPLC for the analysis of polar and/or ionizable compounds, as it often provides higher MS sensitivity and alternative selectivity. Nevertheless, this technique can be also prone to matrix effects (ME). ME are one of the major issues in quantitative LC-MS bioanalysis. To ensure acceptable method performance (i.e., trueness and precision), a careful evaluation and minimization of ME is required. In the present study, the incidence of ME in HILIC-MS/MS and RPLC-MS/MS was compared for plasma and urine samples using two representative sets of 38 pharmaceutical compounds and 40 doping agents, respectively. The optimal generic chromatographic conditions in terms of selectivity with respect to interfering compounds were established in both chromatographic modes by testing three different stationary phases in each mode with different mobile phase pH. A second step involved the assessment of ME in RPLC and HILIC under the best generic conditions, using the post-extraction addition method. Biological samples were prepared using two different sample pre-treatments, i.e., a non-selective sample clean-up procedure (protein precipitation and simple dilution for plasma and urine samples, respectively) and a selective sample preparation, i.e., solid phase extraction for both matrices. The non-selective pretreatments led to significantly less ME in RPLC vs. HILIC conditions regardless of the matrix. On the contrary, HILIC appeared as a valuable alternative to RPLC for plasma and urine samples treated by a selective sample preparation. Indeed, in the case of selective sample preparation, the compounds influenced by ME were different in HILIC and RPLC, and lower and similar ME occurrence was generally observed in RPLC vs. HILIC for urine and plasma samples, respectively. The complementary of both chromatographic modes was also demonstrated, as ME was observed only scarcely for urine and plasma samples when selecting the most appropriate chromatographic mode.

Fast and sensitive supercritical fluid chromatography - tandem mass spectrometry multi-class screening method for the determination of doping agents in urine.

This study shows the possibility offered by modern ultra-high performance supercritical fluid chromatography combined with tandem mass spectrometry in doping control analysis. A high throughput screening method was developed for 100 substances belonging to the challenging classes of anabolic agents, hormones and metabolic modulators, synthetic cannabinoids and glucocorticoids, which should be detected at low concentrations in urine. To selectively extract these doping agents from urine, a supported liquid extraction procedure was implemented in a 48-well plate format. At the tested concentration levels ranging from 0.5 to 5 ng/mL, the recoveries were better than 70% for 48-68% of the compounds and higher than 50% for 83-87% of the tested substances. Due to the numerous interferences related to isomers of steroids and ions produced by the loss of water in the electrospray source, the choice of SFC separation conditions was very challenging. After careful optimization, a Diol stationary phase was employed. The total analysis time for the screening assay was only 8 min, and interferences as well as susceptibility to matrix effect (ME) were minimized. With the developed method, about 70% of the compounds had relative ME within the range ±20%, at a concentration of 1 and 5 ng/mL. Finally, limits of detection achieved with the above-described strategy including 5-fold preconcentration were below 0.1 ng/mL for the majority of the tested compounds. Therefore, LODs were systematically better than the minimum required performance levels established by the World anti-doping agency, except for very few metabolites.

Inférence de source de traces d'essence retrouvées dans des débris d'incendie: Evaluation de la contribution de la chromatographie en phase gazeuse couplée à la spectrométrie de masse à rapport isotopique

Lorsque de l'essence est employée pour allumer et/ou propager un incendie, l'inférence de la source de l'essence peut permettre d'établir un lien entre le sinistre et une source potentielle. Cette inférence de la source constitue une alternative intéressante pour fournir des éléments de preuve dans ce type d'événements où les preuves matérielles laissées par l'auteur sont rares. Le but principal de cette recherche était le développement d'une méthode d'analyse de spécimens d'essence par GC-IRMS, méthode pas routinière et peu étudiée en science forensique, puis l'évaluation de son potentiel à inférer la source de traces d'essence en comparaison aux performances de la GC-MS. Un appareillage permettant d'analyser simultanément les échantillons par MS et par IRMS a été utilisé dans cette recherche. Une méthode d'analyse a été développée, optimisée et validée pour cet appareillage. Par la suite, des prélèvements d'essence provenant d'un échantillonnage conséquent et représentatif du marché de la région lausannoise ont été analysés. Finalement, les données obtenues ont été traitées et interprétées à l'aide de méthodes chimiométriques. Les analyses effectuées ont permis de montrer que la méthodologie mise en place, aussi bien pour la composante MS que pour l'IRMS, permet de différencier des échantillons d'essence non altérée provenant de différentes stations-service. Il a également pu être démontré qu'à chaque nouveau remplissage des cuves d'une station-service, la composition de l'essence distribuée par cette station est quasi unique. La GC-MS permet une meilleure différenciation d'échantillons prélevés dans différentes stations, alors que la GC-IRMS est plus performante lorsqu'il s'agit de comparer des échantillons collectés après chacun des remplissages d'une cuve. Ainsi, ces résultats indiquent que les deux composantes de la méthode peuvent être complémentaires pour l'analyse d'échantillons d'essence non altérée. Les résultats obtenus ont également permis de montrer que l'évaporation des échantillons d'essence ne compromet pas la possibilité de grouper des échantillons de même source par GC-MS. Il est toutefois nécessaire d'effectuer une sélection des variables afin d'éliminer celles qui sont influencées par le phénomène d'évaporation. Par contre, les analyses effectuées ont montré que l'évaporation des échantillons d'essence a une forte influence sur la composition isotopique des échantillons. Cette influence est telle qu'il n'est pas possible, même en effectuant une sélection des variables, de grouper correctement des échantillons évaporés par GC-IRMS. Par conséquent, seule la composante MS de la méthodologie mise en place permet d'inférer la source d'échantillons d'essence évaporée. _________________________________________________________________________________________________ When gasoline is used to start and / or propagate an arson, source inference of gasoline can allow to establish a link between the fire and a potential source. This source inference is an interesting alternative to provide evidence in this type of events where physical evidence left by the author are rare. The main purpose of this research was to develop a GC-IRMS method for the analysis of gasoline samples, a non-routine method and little investigated in forensic science, and to evaluate its potential to infer the source of gasoline traces compared to the GC-MS performances. An instrument allowing to analyze simultaneously samples by MS and IRMS was used in this research. An analytical method was developed, optimized and validated for this instrument. Thereafter, gasoline samples from a large sampling and representative of the Lausanne area market were analyzed. Finally, the obtained data were processed and interpreted using chemometric methods. The analyses have shown that the methodology, both for MS and for IRMS, allow to differentiate unweathered gasoline samples from different service stations. It has also been demonstrated that each new filling of the tanks of a station generates an almost unique composition of gasoline. GC-MS achieves a better differentiation of samples coming from different stations, while GC-IRMS is more efficient to distinguish samples collected after each filling of a tank. Thus, these results indicate that the two components of the method can be complementary to the analysis of unweathered gasoline samples. The results have also shown that the evaporation of gasoline samples does not compromise the possibility to group samples coming from the same source by GC-MS. It is however necessary to make a selection of variables in order to eliminate those which are influenced by the evaporation. On the other hand, the carried out analyses have shown that the evaporation of gasoline samples has such a strong influence on the isotopic composition of the samples that it is not possible, even by performing a selection of variables, to properly group evaporated samples by GC-IRMS. Therefore, only the MS allows to infer the source of evaporated gasoline samples.