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.

Statistical evaluation of the reproducibility and the influence of paper on the analysis of black gel pen ink using laser desorption ionisation mass spectrometry

Laser desorption ionisation mass spectrometry (LDI-MS) has demonstrated to be an excellent analytical method for the forensic analysis of inks on a questioned document. The ink can be analysed directly on its substrate (paper) and hence offers a fast method of analysis as sample preparation is kept to a minimum and more importantly, damage to the document is minimised. LDI-MS has also previously been reported to provide a high power of discrimination in the statistical comparison of ink samples and has the potential to be introduced as part of routine ink analysis. This paper looks into the methodology further and evaluates statistically the reproducibility and the influence of paper on black gel pen ink LDI-MS spectra; by comparing spectra of three different black gel pen inks on three different paper substrates. Although generally minimal, the influences of sample homogeneity and paper type were found to be sample dependent. This should be taken into account to avoid the risk of false differentiation of black gel pen ink samples. Other statistical approaches such as principal component analysis (PCA) proved to be a good alternative to correlation coefficients for the comparison of whole mass spectra.

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.

Dispersion of natural arsenic in the Malcantone watershed, Southern Switzerland: field evidence for repeated sorption-desorption and oxidation-reduction processes

In recent years, elevated arsenic concentrations have been found in waters and soils of many, countries, often resulting in a health threat for the local population. Switzerland is not an exception and this paper deals with the release and subsequent fate of arsenic in a 200-km(2) mountainous watershed, characterized by crystalline silicate rocks (gneisses, schists, amphibolites) that contain abundant As-bearing sulfide ore deposits, some of which have been mined for iron and gold in the past. Using analytical methods common for mineralogical, ground water and soil studies (XRD, XRF, XAS-XANES and -EXAFS, electron microprobe, extraction, ICP, AAS with hydride generator, ion chromatography), seven different field situations and related dispersion processes of natural arsenic have been studied: (1) release by rock weathering, (2) transport and deposition by water and ice; (3) release of As to the ground and surface water due to increasing pH; (4) accumulation in humic soil horizons; (5) remobilization by reduction in water-saturated soils and stagnant ground waters; (6) remobilization by using P-rich fertilizers or dung and (7) oxidation, precipitation and dilution in surface waters. Comparison of the results with experimental adsorption studies and speciation diagrams from the literature allows us to reconstruct and identify the typical behavior of arsenic in a natural environment under temperate climatic conditions. The main parameters identified are: (a) once liberated from the primary minerals, sorption processes on Fe-oxy-hydroxides dominate over Al-phases, such as Al-hydroxides or clay minerals and limit the As concentrations in the spring and well waters between 20 and 300 mug/l. (b) Precipitation as secondary minerals is limited to the weathering domain, where the As concentrations are still high and not yet too diluted by rain and soils waters. (c) Although neutral and alkaline pH conditions clearly increase the mobility of As, the main factor to mobilize As is a low redox potential (Eh close or below 0 mV), which favors the dissolution of the Fe-oxy-hydroxides on which the As is sorbed. (d) X-ray absorption spectroscopy (XAS) of As in water-logged humic forest soils indicates that the reduction to As III only occurs at the solid-water interface and that the solid contains As as As V (e) A and Bh horizons of humic cambisols can effectively capture As when As-rich waters flow through them. Complex spatial and temporal variation of the various parameters in a watershed results in repeated mobilization and immobilization of As, which continuously transports As from the upper to the lower part of a watershed and ultimately to the ocean. (C) 2004 Elsevier B.V. All rights reserved.

Statistical discrimination of black gel pen inks analysed by laser desorption/ionization mass spectrometry

Pearson correlation coefficients were applied for the objective comparison of 30 black gel pen inks analysed by laser desorption ionization mass spectrometry (LDI-MS). The mass spectra were obtained for ink lines directly on paper using positive and negative ion modes at several laser intensities. This methodology has the advantage of taking into account the reproducibility of the results as well as the variability between spectra of different pens. A differentiation threshold could thus be selected in order to avoid the risk of false differentiation. Combining results from positive and negative mode yielded a discriminating power up to 85%, which was better than the one obtained previously with other optical comparison methodologies. The technique also allowed discriminating between pens from the same brand.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, a revolution in clinical microbial identification.

Until recently, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) techniques for the identification of microorganisms remained confined to research laboratories. In the last 2 years, the availability of relatively simple to use MALDI-TOF MS devices, which can be utilized in clinical microbiology laboratories, has changed the laboratory workflows for the identification of pathogens. Recently, the first prospective studies regarding the performance in routine bacterial identification showed that MALDI-TOF MS is a fast, reliable and cost-effective technique that has the potential to replace and/or complement conventional phenotypic identification for most bacterial strains isolated in clinical microbiology laboratories. For routine bacterial isolates, correct identification by MALDI-TOF MS at the species level was obtained in 84.1-93.6% of instances. In one of these studies, a protein extraction step clearly improved the overall valid identification yield, from 70.3% to 93.2%. This review focuses on the current state of use of MALDI-TOF MS for the identification of routine bacterial isolates and on the main difficulties that may lead to erroneous or doubtful identifications.

A statistical methodology for the comparison of blue gel pen inks analyzed by laser desorption/ionization mass spectrometry

A statistical methodology for the objective comparison of LDI-MS mass spectra of blue gel pen inks was evaluated. Thirty-three blue gel pen inks previously studied by RAMAN were analyzed directly on the paper using both positive and negative mode. The obtained mass spectra were first compared using relative areas of selected peaks using the Pearson correlation coefficient and the Euclidean distance. Intra-variability among results from one ink and inter-variability between results from different inks were compared in order to choose a differentiation threshold minimizing the rate of false negative (i.e. avoiding false differentiation of the inks). This yielded a discriminating power of up to 77% for analysis made in the negative mode. The whole mass spectra were then compared using the same methodology, allowing for a better DP in the negative mode of 92% using the Pearson correlation on standardized data. The positive mode results generally yielded a lower differential power (DP) than the negative mode due to a higher intra-variability compared to the inter-variability in the mass spectra of the ink samples.

Impact of Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry on the Clinical Management of Patients With Gram-negative Bacteremia: A Prospective Observational Study.

Background. Early identification of pathogens from blood cultures using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry may optimize the choice of empirical antibiotic therapy in the setting of bloodstream infections. We aimed to assess the impact of this new technology on the use of antibiotic treatment in patients with gram-negative bacteremia. Methods. We conducted a prospective observational study from January to December 2010 to evaluate the sequential and separate impacts of Gram stain reporting and MALDI-TOF bacterial identification performed on blood culture pellets in patients with gram-negative bacteremia. The primary outcome was the impact of MALDI-TOF on empirical antibiotic choice. Results. Among 202 episodes of gram-negative bacteremia, Gram stain reporting had an impact in 42 cases (20.8%). MALDI-TOF identification led to a modification of empirical therapy in 71 of all 202 cases (35.1%), and in 16 of 27 cases (59.3%) of monomicrobial bacteremia caused by AmpC-producing Enterobacteriaceae. The most frequently observed impact was an early appropriate broadening of the antibiotic spectrum in 31 of 71 cases (43.7%). In total, 143 of 165 episodes (86.7%) of monomicrobial bacteremia were correctly identified at genus level by MALDI-TOF. Conclusions. In a low prevalence area for extended spectrum betalactamases (ESBL) and multiresistant gram-negative bacteria, MALDI-TOF performed on blood culture pellets had an impact on the clinical management of 35.1% of all gram-negative bacteremia cases, demonstrating a greater impact than Gram stain reporting. Thus, MALDI-TOF could become a vital second step beside Gram stain in guiding the empirical treatment of patients with bloodstream infection.

Silver-assisted laser desorption ionization for high spatial resolution imaging mass spectrometry of olefins from thin tissue sections.

Silver has been demonstrated to be a powerful cationization agent in mass spectrometry (MS) for various olefinic species such as cholesterol and fatty acids. This work explores the utility of metallic silver sputtering on tissue sections for high resolution imaging mass spectrometry (IMS) of olefins by laser desorption ionization (LDI). For this purpose, sputtered silver coating thickness was optimized on an assorted selection of mouse and rat tissues including brain, kidney, liver, and testis. For mouse brain tissue section, the thickness was adjusted to 23 ± 2 nm of silver to prevent ion suppression effects associated with a higher cholesterol and lipid content. On all other tissues, a thickness of at 16 ± 2 nm provided the best desorption/ionization efficiency. Characterization of the species by MS/MS showed a wide variety of olefinic compounds allowing the IMS of different lipid classes including cholesterol, arachidonic acid, docosahexaenoic acid, and triacylglyceride 52:3. A range of spatial resolutions for IMS were investigated from 150 μm down to the high resolution cellular range at 5 μm. The applicability of direct on-tissue silver sputtering to LDI-IMS of cholesterol and other olefinic compounds presents a novel approach to improve the amount of information that can be obtained from tissue sections. This IMS strategy is thus of interest for providing new biological insights on the role of cholesterol and other olefins in physiological pathways or disease.

On-line desorption of dried blood spot: A novel approach for the direct LC/MS analysis of micro-whole blood samples.

The aim of this work is to present a new concept, called on-line desorption of dried blood spots (on-line DBS), allowing the direct analysis of a dried blood spot coupled to liquid chromatography mass spectrometry device (LC/MS). The system is based on an inox cell which can receive a blood sample (10 microL) previously spotted on a filter paper. The cell is then integrated into LC/MS system where the analytes are desorbed out of the paper towards a column switching system ensuring the purification and separation of the compounds before their detection on a single quadrupole MS coupled to atmospheric pressure chemical ionisation (APCI) source. The described procedure implies that no pretreatment is necessary in spite the analysis is based on whole blood sample. To ensure the applicability of the concept, saquinavir, imipramine, and verapamil were chosen. Despite the use of a small sampling volume and a single quadrupole detector, on-line DBS allowed the analyses of these three compounds over their therapeutic concentrations from 50 to 500 ng/mL for imipramine and verapamil and from 100 to 1000 ng/mL for saquinavir. Moreover, the method showed good repeatability with relative standard deviation (RSD) lower than 15% based on two levels of concentration (low and high). Function responses were found to be linear over the therapeutic concentration for each compound and were used to determine the concentrations of real patient samples for saquinavir. Comparison of the founded values with those of a validated method used routinely in a reference laboratory showed a good correlation between the two methods. Moreover, good selectivity was observed ensuring that no endogenous or chemical components interfered with the quantitation of the analytes. This work demonstrates the feasibility and applicability of the on-line DBS procedure for bioanalysis.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry as an alternative to 16S rRNA gene sequencing for identification of difficult-to-identify bacterial strains.

Conventional methods are sometimes insufficient to identify human bacterial pathogens, and alternative techniques, often molecular, are required. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identified with a valid score 45.9% of 410 clinical isolates from 207 different difficult-to-identify species having required 16S rRNA gene sequencing. MALDI-TOF MS might represent an alternative to 16S rRNA gene sequencing.

Ink dating using thermal desorption and gas chromatography / mass spectrometry: comparison of results obtained in two laboratories

Recent ink dating methods focused mainly on changes in solvent amounts occurring over time. A promising method was developed at the Landeskriminalamt of Munich using thermal desorption (TD) followed by gas chromatography / mass spectrometry (GC/MS) analysis. Sequential extractions of the phenoxyethanol present in ballpoint pen ink entries were carried out at two different temperatures. This method is applied in forensic practice and is currently implemented in several laboratories participating to the InCID group (International Collaboration on Ink Dating). However, harmonization of the method between the laboratories proved to be a particularly sensitive and time consuming task. The main aim of this work was therefore to implement the TD-GC/MS method at the Bundeskriminalamt (Wiesbaden, Germany) in order to evaluate if results were comparable to those obtained in Munich. At first validation criteria such as limits of reliable measurements, linearity and repeatability were determined. Samples were prepared in three different laboratories using the same inks and analyzed using two TDS-GC/MS instruments (one in Munich and one in Wiesbaden). The inter- and intra-laboratory variability of the ageing parameter was determined and ageing curves were compared. While inks stored in similar conditions yielded comparable ageing curves, it was observed that significantly different storage conditions had an influence on the resulting ageing curves. Finally, interpretation models, such as thresholds and trend tests, were evaluated and discussed in view of the obtained results. Trend tests were considered more suitable than threshold models. As both approaches showed limitations, an alternative model, based on the slopes of the ageing curves, was also proposed.

On-line desorption of dried blood spots coupled to hydrophilic interaction/reversed-phase LC/MS/MS system for the simultaneous analysis of drugs and their polar metabolites.

An assay for the simultaneous analysis of pharmaceutical compounds and their metabolites from micro-whole blood samples (i.e. 5 microL) was developed using an on-line dried blood spot (on-line DBS) device coupled with hydrophilic interaction/reversed-phase (HILIC/RP) LC/MS/MS. Filter paper is directly integrated to the LC device using a homemade inox desorption cell. Without any sample pretreatment, analytes are desorbed from the paper towards an automated system of valves linking a zwitterionic-HILIC column to an RP C18 column. In the same run, the polar fraction is separated by the zwitterionic-HILIC column while the non-polar fraction is eluted on the RP C18. Both fractions are detected by IT-MS operating in full scan mode for the survey scan and in product ion mode for the dependant scan using an ESI source. The procedure was evaluated by the simultaneous qualitative analysis of four probes and their relative phase I and II metabolites spiked in whole blood. In addition, the method was successfully applied to the in vivo monitoring of buprenorphine metabolism after the administration of an intraperitoneal injection of 30 mg/kg on adult female Wistar rat.

Graphitized carbon black in quartz tubes for the sampling of indoor air nicotine and analysis by microwave thermal desorption-capillary gas chromatography

Nicotine in a smoky indoor air environment can be determined using graphitized carbon black as a solid sorbent in quartz tubes. The temperature stability, high purity, and heat absorption characteristics of the sorbent, as well as the permeability of the quartz tubes to microwaves, enable the thermal desorption by means of microwaves after active sampling. Permeation and dynamic dilution procedures for the generation of nicotine in the vapor phase at low and high concentrations are used to evaluate the performances of the sampler. Tube preparation is described and the microwave desorption temperature is measured. Breakthrough volume is determined to allow sampling at 0.1-1 L/min for definite periods of time. The procedure is tested for the determination of gas and paticulate phase nicotine in sidestream smoke produced in an experimental chamber.