Performance of matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification of bacterial strains routinely isolated in a clinical microbiology laboratory.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently been introduced in diagnostic microbiology laboratories for the identification of bacterial and yeast strains isolated from clinical samples. In the present study, we prospectively compared MALDI-TOF MS to the conventional phenotypic method for the identification of routine isolates. Colonies were analyzed by MALDI-TOF MS either by direct deposition on the target plate or after a formic acid-acetonitrile extraction step if no valid result was initially obtained. Among 1,371 isolates identified by conventional methods, 1,278 (93.2%) were putatively identified to the species level by MALDI-TOF MS and 73 (5.3%) were identified to the genus level, but no reliable identification was obtained for 20 (1.5%). Among the 1,278 isolates identified to the species level by MALDI-TOF MS, 63 (4.9%) discordant results were initially identified. Most discordant results (42/63) were due to systematic database-related taxonomical differences, 14 were explained by poor discrimination of the MALDI-TOF MS spectra obtained, and 7 were due to errors in the initial conventional identification. An extraction step was required to obtain a valid MALDI-TOF MS identification for 25.6% of the 1,278 valid isolates. In conclusion, our results show that MALDI-TOF MS is a fast and reliable technique which has the potential to replace conventional phenotypic identification for most bacterial strains routinely isolated in clinical microbiology laboratories.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry for direct bacterial identification from positive blood culture pellets.

An ammonium chloride erythrocyte-lysing procedure was used to prepare a bacterial pellet from positive blood cultures for direct matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectrometry analysis. Identification was obtained for 78.7% of the pellets tested. Moreover, 99% of the MALDI-TOF identifications were congruent at the species level when considering valid scores. This fast and accurate method is promising.

Mechanical external work and recovery at preferred walking speed in obese subjects

PURPOSE: The aim of this study was to compare the mechanical external work (per kg) and pendular energy transduction at preferred walking speed (PWS) in obese versus normal body mass subjects to investigate whether obese adults adopt energy conserving gait mechanics. METHODS: The mechanical external work (Wext) and the fraction of mechanical energy recovered by the pendular mechanism (Rstep) were computed using kinematic data acquired by an optoelectronic system and were compared in 30 obese (OG; body mass index [BMI] = 39.6 +/- 0.6 kg m(-2); 29.5 +/- 1.3 yr) and 19 normal body mass adults (NG; BMI = 21.4 +/- 0.5 kg m(-2); 31.2 +/- 1.2 yr) walking at PWS. RESULTS: PWS was significantly lower in OG (1.18 +/- 0.02 m s(-1)) than in NG (1.33 +/- 0.02 m s(-1); P <or= 0.001). There was no significant difference in Wext per unit mass between groups (OG: 0.36 +/- 0.03 J kg(-1) m(-1); NG: 0.31 +/- 0.02 J kg(-1) m(-1); P = 0.12). Rstep was significantly lower in OG (68.4% +/- 2.0%) compared with NG (74.4% +/- 1.0%; P = 0.01). In OG only, Wext per unit mass was positively correlated with PWS (r = 0.57; P < 0.001). CONCLUSION: Obese adults do not appear to alter their gait to improve pendular energy transduction and may select slower PWS to reduce mechanical and metabolic work.

Sedimentary record of the northward flight of India and its collision with Eurasia (Ladakh Himalaya, India)

Stratigraphic and petrographic analysis of the Cretaceous to Eocene Tibetan sedimentary succession has allowed us to reinterpret in detail the sequence of events which led to closure of Neotethys and continental collision in the NW Himalaya. During the Early Cretaceous, the Indian passive margin recorded basaltic magmatic activity. Albian volcanic arenites, probably related to a major extensional tectonic event, are unconformably overlain by an Upper Cretaceous to Paleocene carbonate sequence, with a major quartzarenite episode triggered by the global eustatic sea-level fall at the Cretaceous/Tertiary boundary. At the same time, Neotethyan oceanic crust was being subducted beneath Asia, as testified by calc-alkalic volcanism and forearc basin sedimentation in the Transhimalayan belt. Onset of collision and obduction of the Asian accretionary wedge onto the Indian continental rise was recorded by shoaling of the outer shelf at the Paleocene/Eocene boundary, related to flexural uplift of the passive margin. A few My later, foreland basin volcanic arenites derived from the uplifted Asian subduction complex onlapped onto the Indian continental terrace. All along the Himalaya, marine facies were rapidly replaced by continental redbeds in collisional basins on both sides of the ophiolitic suture. Next, foreland basin sedimentation was interrupted by fold-thrust deformation and final ophiolite emplacement. The observed sequence of events compares favourably with theoretical models of rifted margin to overthrust belt transition and shows that initial phases of continental collision and obduction were completed within 10 to 15 My, with formation of a proto-Himalayan chain by the end of the middle Eocene.

Ultra-high pressure liquid chromatography-mass spectrometry for plant metabolomics: a systematic comparison of high-resolution quadrupole-time-of-flight and single stage Orbitrap mass spectrometers.

The response of Arabidopsis to stress caused by mechanical wounding was chosen as a model to compare the performances of high resolution quadrupole-time-of-flight (Q-TOF) and single stage Orbitrap (Exactive Plus) mass spectrometers in untargeted metabolomics. Both instruments were coupled to ultra-high pressure liquid chromatography (UHPLC) systems set under identical conditions. The experiment was divided in two steps: the first analyses involved sixteen unwounded plants, half of which were spiked with pure standards that are not present in Arabidopsis. The second analyses compared the metabolomes of mechanically wounded plants to unwounded plants. Data from both systems were extracted using the same feature detection software and submitted to unsupervised and supervised multivariate analysis methods. Both mass spectrometers were compared in terms of number and identity of detected features, capacity to discriminate between samples, repeatability and sensitivity. Although analytical variability was lower for the UHPLC-Q-TOF, generally the results for the two detectors were quite similar, both of them proving to be highly efficient at detecting even subtle differences between plant groups. Overall, sensitivity was found to be comparable, although the Exactive Plus Orbitrap provided slightly lower detection limits for specific compounds. Finally, to evaluate the potential of the two mass spectrometers for the identification of unknown markers, mass and spectral accuracies were calculated on selected identified compounds. While both instruments showed excellent mass accuracy (<2.5ppm for all measured compounds), better spectral accuracy was recorded on the Q-TOF. Taken together, our results demonstrate that comparable performances can be obtained at acquisition frequencies compatible with UHPLC on Q-TOF and Exactive Plus MS, which may thus be equivalently used for plant metabolomics.

Fast analysis of doping agents in urine by ultra-high-pressure liquid chromatography-quadrupole time-of-flight mass spectrometry. II: Confirmatory analysis.

For doping control, analyses of samples are generally achieved in two steps: a rapid screening and, in the case of a positive result, a confirmatory analysis. A two-step methodology based on ultra-high-pressure liquid chromatography coupled to a quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) was developed to screen and confirm 103 doping agents from various classes (e.g., beta-blockers, stimulants, diuretics, and narcotics). The screening method was presented in a previous article as part I (i.e., Fast analysis of doping agents in urine by ultra-high-pressure liquid chromatography-quadrupole time-of-flight mass spectrometry. Part I: screening analysis). For the confirmatory method, basic, neutral and acidic compounds were extracted by a dedicated solid-phase extraction (SPE) in a 96-well plate format and detected by MS in the tandem mode to obtain precursor and characteristic product ions. The mass accuracy and the elemental composition of precursor and product ions were used for compound identification. After validation including matrix effect determination, the method was considered reliable to confirm suspect results without ambiguity according to the positivity criteria established by the World Anti-Doping Agency (WADA). Moreover, an isocratic method was developed to separate ephedrine from its isomer pseudoephedrine and cathine from phenylpropanolamine in a single run, what allowed their direct quantification in urine.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and PCR-based rapid diagnosis of Staphylococcus aureus bacteraemia.

Effective empirical treatment is of paramount importance to improve the outcome of patients with Staphylococcus aureus bacteraemia. We aimed to evaluate a PCR-based rapid diagnosis of methicillin resistance (GeneXpert MRSA) after early detection of S. aureus bacteraemia using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Patients with a first episode of S. aureus bacteraemia identified using MALDI-TOF MS were randomized in a prospective interventional open study between October 2010 and August 2012. In the control group, antibiotic susceptibility testing was performed after MALDI-TOF MS identification on blood culture pellets. In the intervention group, a GeneXpert MRSA was performed after S. aureus identification. The primary outcome was the performance of GeneXpert MRSA directly on blood cultures. We then assessed the impact of early diagnosis of methicillin resistance on the empirical treatment. In all, 197 episodes of S. aureus bacteraemia were included in the study, of which 106 were included in the intervention group. Median time from MALDI-TOF MS identification to GeneXpert MRSA result was 97 min (range 25-250). Detection of methicillin resistance using GeneXpert MRSA had a sensitivity of 99% and a specificity of 100%. There was less unnecessary coverage of MRSA in the intervention group (17.1% versus 29.2%, p 0.09). GeneXpert MRSA was highly reliable in diagnosing methicillin resistance when performed directly on positive blood cultures. This could help to avoid unnecessary prescriptions of anti-MRSA agents and promote the introduction of earlier adequate coverage in unsuspected cases.

Determination of Vasopressin and Desmopressin in urine by means of liquid chromatography coupled to quadrupole time-of-flight mass spectrometry for doping control purposes.

The anti-diuretic neurohypophysial hormone Vasopressin (Vp) and its synthetic analogue Desmopressin (Dp, 1-desamino-vasopressin) have received considerable attention from doping control authorities due to their impact on physiological blood parameters. Accordingly, the illicit use of Desmopressin in elite sport is sanctioned by the World Anti-Doping Agency (WADA) and the drug is classified as masking agent. Vp and Dp are small (8-9 amino acids) peptides administered orally as well as intranasally. Within the present study a method to determine Dp and Vp in urinary doping control samples by means of liquid chromatography coupled to quadrupole high resolution time-of-flight mass spectrometry was developed. After addition of Lys-Vasopressin as internal standard and efficient sample clean up with a mixed mode solid phase extraction (weak cation exchange), the samples were directly injected into the LC-MS system. The method was validated considering the parameters specificity, linearity, recovery (80-100%), accuracy, robustness, limit of detection/quantification (20/50 pg mL(-1)), precision (inter/intra-day<10%), ion suppression and stability. The analysis of administration study urine samples collected after a single intranasal or oral application of Dp yielded in detection windows for the unchanged target analyte for up to 20 h at concentrations between 50 and 600 pg mL(-1). Endogenous Vp was detected in concentrations of approximately 20-200 pg mL(-1) in spontaneous urine samples obtained from healthy volunteers. The general requirements of the developed method provide the characteristics for an easy transfer to other anti-doping laboratories and support closing another potential gap for cheating athletes.

Effect of obesity onset on pendular energy transduction at spontaneous walking speed: Prader-Willi versus nonsyndromal obese individuals.

OBJECTIVE: To compare the mechanical external work (Wext ) and pendular energy transduction (Rstep ) at spontaneous walking speed (Ss ) in individuals with Prader-Willi syndrome (PWS) versus subjects with nonsyndromal obesity (OB) to investigate whether the early onset of obesity allows PWS subjects to adopt energy conserving gait mechanics. DESIGN AND METHODS: Wext and Rstep were computed using kinematic data acquired by an optoelectronic system and compared in 15 PWS (BMI = 39.5 ± 1.8 kg m(-2) ; 26.7 ± 1.5 year) and 15 OB (BMI = 39.3 ± 1.0 kg m(-2) ; 28.7 ± 1.9 year) adults matched for gender, age and BMI and walking at Ss . RESULTS: Ss was significantly lower in PWS (0.98 ± 0.03 m s(-1) ) than in OB (1.20 ± 0.02 m s(-1) ; P < 0.001). There were no significant differences in Wext per kilogram between groups (PWS: 0.37 ± 0.04 J kg(-1) m(-1) ; OB: 0.40 ± 0.05 J kg(-1) m(-1) ; P = 0.66) and in Rstep (PWS: 69.9 ± 2.9%; OB: 67.7 ± 2.4%; P = 0.56). However, Rstep normalized to Froude number (Rstep /Fr) was significantly greater in PWS (6.0 ± 0.6) than in OB (3.8 ± 0.2; P = 0.001). Moreover, Rstep /Fr was inversely correlated with age of obesity onset (r = -0.49; P = 0.006) and positively correlated with obesity duration (r = 0.38; P = 0.036). CONCLUSION: Individuals with PWS seem to alter their gait to improve pendular energy transduction as a result of precocious and chronic adaptation to loading.

Assessment of speed of human locomotion using a differential satellite global positioning system.

PURPOSE: The objective was to explore whether a satellite-based navigation system, global positioning system used in differential mode (DGPS), could accurately assess the speed of running in humans. METHODS: A subject was equipped with a portable GPS receptor coupled to a receiver for differential corrections, while running outdoors on a straight asphalt road at 27 different speeds. Actual speed (reference method) was assessed by chronometry. RESULTS: The accuracy of speed prediction had a standard deviation (SD) of 0.08 km x h(-1) for walking, 0.11 km x h(-1) for running, yielding a coefficient of variation (SD/mean) of 1.38% and 0.82%, respectively. There was a highly significant linear relationship between actual and DGPS speed assessment (r2 = 0.999) with little bias in the prediction equation, because the slope of the regression line was close to unity (0.997). CONCLUSION: the DGPS technique appears to be a valid and inconspicuous tool for "on line" monitoring of the speed of displacement of individuals located on any field on earth, for prolonged periods of time and unlimited distance, but only in specific environmental conditions ("open sky"). Furthermore, the accuracy of speed assessment using the differential GPS mode was improved by a factor of 10 as compared to non-differential GPS.