A simple, robust and rapid approach to detect carbapenemases in Gram-negative isolates by MALDI-TOF mass spectrometry: validation with triple quadripole tandem mass spectrometry, microarray and PCR.

Carbapenemases should be accurately and rapidly detected, given their possible epidemiological spread and their impact on treatment options. Here, we developed a simple, easy and rapid matrix-assisted laser desorption ionization-time of flight (MALDI-TOF)-based assay to detect carbapenemases and compared this innovative test with four other diagnostic approaches on 47 clinical isolates. Tandem mass spectrometry (MS-MS) was also used to determine accurately the amount of antibiotic present in the supernatant after 1 h of incubation and both MALDI-TOF and MS-MS approaches exhibited a 100% sensitivity and a 100% specificity. By comparison, molecular genetic techniques (Check-MDR Carba PCR and Check-MDR CT103 microarray) showed a 90.5% sensitivity and a 100% specificity, as two strains of Aeromonas were not detected because their chromosomal carbapenemase is not targeted by probes used in both kits. Altogether, this innovative MALDI-TOF-based approach that uses a stable 10-μg disk of ertapenem was highly efficient in detecting carbapenemase, with a sensitivity higher than that of PCR and microarray.

A comparison of rapid bioassessment protocols used in 2 regions with Mediterranean climates, the Iberian Peninsula and South Africa

Abstract. The ability of 2 Rapid Bioassessment Protocols (RBPs) to assess stream water quality was compared in 2 Mediterranean-climate regions. The most commonly used RBPs in South Africa (SAprotocol) and the Iberian Peninsula (IB-protocol) are both multihabitat, field-based methods that use macroinvertebrates. Both methods use preassigned sensitivity weightings to calculate metrics and biotic indices. The SA- and IB-protocols differ with respect to sampling equipment (mesh size: 1000 lm vs 250 300 lm, respectively), segregation of habitats (substrate vs flow-type), and sampling and sorting procedures (variable time and intensity). Sampling was undertaken at 6 sites in South Africa and 5 sites in the Iberian Peninsula. Forty-four and 51 macroinvertebrate families were recorded in South Africa and the Iberian Peninsula, respectively; 77.3% of South African families and 74.5% of Iberian Peninsula families were found using both protocols. Estimates of community similarity compared between the 2 protocols were .60% similar among sites in South Africa and .54% similar among sites in the Iberian Peninsula (BrayCurtis similarity), and no significant differences were found between protocols (Multiresponse Permutation Procedure). Ordination based on Non-metric Multidimensional Scaling grouped macroinvertebrate samples on the basis of site rather than protocol. Biotic indices generated with the 2 protocols at each site did not differ. Thus, both RBPs produced equivalent results, and both were able to distinguish between biotic communities (mountain streams vs foothills) and detect water-quality impairment, regardless of differences in sampling equipment, segregation of habitats, and sampling and sorting procedures. Our results indicate that sampling a single habitat may be sufficient for assessing water quality, but a multihabitat approach to sampling is recommended where intrinsic variability of macroinvertebrate assemblages is high (e.g., in undisturbed sites in regions with Mediterranean climates). The RBP of choice should depend on whether the objective is routine biomonitoring of water quality or autecological or faunistic studies.

Rapid detection of Staphylococcus aureus strains with reduced susceptibility to vancomycin by isothermal microcalorimetry.

Methicillin-resistant Staphylococcus aureus (MRSA) usually harbors a vancomycin-susceptible phenotype (VSSA) but can exhibit reduced vancomycin susceptibility phenotypes that can be heterogeneous-intermediate (hVISA), intermediate (VISA), or fully resistant (VRSA). Current detection techniques (e.g., Etest and population analysis profiles [PAPs]) are slow and time-consuming. We investigated the potential of microcalorimetry to detect reduced susceptibilities to vancomycin in MRSA strains. Representative MSSA, VSSA, hVISA, VISA, and VRSA reference strains, as well as clinical isolates, were used. PAPs were performed by standard methods. Microcalorimetry was performed by inoculating 5 × 10(7) CFU of overnight cultures into 3-ml vials of brain heart infusion broth supplemented with increasing concentrations of vancomycin, and growth-related heat production was measured at 37°C. For the reference strains, no heat production was detected in the VSSA isolates at vancomycin concentrations of >3 μg/ml during the 72 h of incubation. The hVISA and VISA strains showed heat production with concentration-proportional delays of up to 6 μg/ml in 48 h and up to 12 μg/ml in 72 h, respectively. The VRSA strain showed heat production at concentrations up to 16 μg/ml in 12 h. The testing of clinical strains indicated an excellent negative predictive value, allowing us to rule out a decreased vancomycin susceptibility phenotype in <8 h of incubation. Sequential isolates from a patient undergoing vancomycin therapy showed evolving microcalorimetric profiles up to a VISA phenotype. Microcalorimetry was able to detect strains with reduced susceptibilities to vancomycin in <8 h. The measurement of bacterial heat production might represent a simple and rapid method for the detection of reduced susceptibilities to vancomycin in MRSA strains.

Rapid and sensitive hormonal profiling of complex plant samples by liquid chromatography coupled to electrospray ionization tandem mass spectrometry

Background Plant hormones play a pivotal role in several physiological processes during a plant's life cycle, from germination to senescence, and the determination of endogenous concentrations of hormones is essential to elucidate the role of a particular hormone in any physiological process. Availability of a sensitive and rapid method to quantify multiple classes of hormones simultaneously will greatly facilitate the investigation of signaling networks in controlling specific developmental pathways and physiological responses. Due to the presence of hormones at very low concentrations in plant tissues (10-9 M to 10-6 M) and their different chemistries, the development of a high-throughput and comprehensive method for the determination of hormones is challenging. Results The present work reports a rapid, specific and sensitive method using ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem spectrometry (UPLC/ESI-MS/MS) to analyze quantitatively the major hormones found in plant tissues within six minutes, including auxins, cytokinins, gibberellins, abscisic acid, 1-amino-cyclopropane-1-carboxyic acid (the ethylene precursor), jasmonic acid and salicylic acid. Sample preparation, extraction procedures and UPLC-MS/MS conditions were optimized for the determination of all plant hormones and are summarized in a schematic extraction diagram for the analysis of small amounts of plant material without time-consuming additional steps such as purification, sample drying or re-suspension. Conclusions This new method is applicable to the analysis of dynamic changes in endogenous concentrations of hormones to study plant developmental processes or plant responses to biotic and abiotic stresses in complex tissues. An example is shown in which a hormone profiling is obtained from leaves of plants exposed to salt stress in the aromatic plant, Rosmarinus officinalis.

Volcanic arcs fed by rapid pulsed fluid flow through subducting slabs

At subduction zones, oceanic lithosphere that has interacted with sea water is returned to the mantle, heats up during descent and releases fluids by devolatilization of hydrous minerals. Models for the formation of magmas feeding volcanoes above subduction zones require largescale transport of these fluids into overlying mantle wedges(1-3). Fluid flow also seems to be linked to seismicity in subducting slabs. However, the spatial and temporal scales of this fluid flow remain largely unknown, with suggested timescales ranging from tens to tens of thousands of years(3-5). Here we use the Li-Ca-Sr isotope systems to consider fluid sources and quantitatively constrain the duration of subduction-zone fluid release at similar to 70 km depth within subducting oceanic lithosphere, now exhumed in the Chinese Tianshan Mountains. Using lithium-diffusion modelling, we find that the wall-rock porosity adjacent to the flowpath of the fluids increased ten times above the background level. We show that fluids released by devolatilization travelled through the slab along major conduits in pulses with durations of about similar to 200 years. Thus, although the overall slab dehydration process is continuous over millions of years and over a wide range of pressures and temperatures, we conclude that the fluids produced by dehydration in subducting slabs are mobilized in short-lived, channelized fluid-flow events.

Rapid IL-4 production by Leishmania homolog of mammalian RACK1-reactive CD4(+) T cells in resistant mice treated once with anti-IL-12 or -IFN-gamma antibodies at the onset of infection with Leishmania major instructs Th2 cell development, resulting in nonhealing lesions.

Rapid production of IL-4 by Leishmania homolog of mammalian RACK1 (LACK)-reactive CD4(+) T cells expressing the V beta 4-V alpha 8 TCR chains has been shown to drive aberrant Th2 cell development and susceptibility to Leishmania major in BALB/c mice. In contrast, mice from resistant strains fail to express this early IL-4 response. However, administration of either anti-IL-12 or -IFN-gamma at the initiation of infection allows the expression of this early IL-4 response in resistant mice. In this work we show that Leishmania homolog of mammalian RACK1-reactive CD4(+) T cells also expressing the V beta 4-V alpha 8 TCR chains are the source of the early IL-4 response to L. major in resistant mice given anti-IL-12 or -IFN-gamma Abs only at the onset of infection. Strikingly, these cells were found to be required for the reversal of the natural resistance of C57BL/6 mice following a single administration of anti-IL-12 or -IFN-gamma Abs. Together these results suggest that a deficiency in mechanisms capable of down-regulating the early IL-4 response to L. major contributes to the exquisite susceptibility of BALB/c mice to L. major.

Randomized Trial of prophylactic granulocyte colony-stimulating factor during rapid COJEC induction in pediatric patients with high-risk neuroblastoma: the European HR-NBL1/SIOPEN study.

Purpose To reduce the incidence of febrile neutropenia during rapid COJEC (cisplatin, vincristine, carboplatin, etoposide, and cyclophosphamide given in a rapid delivery schedule) induction. In the High-Risk Neuroblastoma-1 (HR-NBL1) trial, the International Society of Paediatric Oncology European Neuroblastoma Group (SIOPEN) randomly assigned patients to primary prophylactic (PP) versus symptom-triggered granulocyte colony-stimulating factor (GCSF; filgrastim). Patients and Methods From May 2002 to November 2005, 239 patients in 16 countries were randomly assigned to receive or not receive PPGCSF. There were 144 boys with a median age of 3.1 years (range, 1 to 17 years) of whom 217 had International Neuroblastoma Staging System (INSS) stage 4 and 22 had stage 2 or 3 MYCN-amplified disease. The prophylactic arm received a single daily dose of 5 μg/kg GCSF, starting after each of the eight COJEC chemotherapy cycles and stopping 24 hours before the next cycle. Chemotherapy was administered every 10 days regardless of hematologic recovery, provided that infection was controlled. Results The PPGCSF arm had significantly fewer febrile neutropenic episodes (P = .002), days with fever (P = .004), hospital days (P = .017), and antibiotic days (P = .001). Reported Common Toxicity Criteria (CTC) graded toxicity was also significantly reduced: infections per cycle (P = .002), fever (P < .001), severe leucopenia (P < .001), neutropenia (P < .001), mucositis (P = .002), nausea/vomiting (P = .045), and constipation (P = .008). Severe weight loss was reduced significantly by 50% (P = .013). Protocol compliance with the rapid induction schedule was also significantly better in the PPGCSF arm shown by shorter time to completion (P = .005). PPGCSF did not adversely affect response rates or success of peripheral-blood stem-cell harvest. Following these results, PPG-GSF was advised for all patients on rapid COJEC induction.

Rapid sample pre-treatment prior to GC-MS and GC-MS/MS urinary toxicological screening.

Drug screening is an important issue in clinical and forensic toxicology. Gas chromatography coupled to mass spectrometry (GC-MS) remains the gold standard technique for the screening of unknown compounds in urine samples. However, this technique requires substantial sample preparation, which is time consuming. Moreover, some common drugs such as cannabis cannot be easily detected in urine using general procedures. In this work, a sample preparation protocol for treating 200 μL of urine in less than 30 min is described. The enzymatic hydrolysis of glucuro-conjugates was performed in 5 min thanks to the use of microwaves. The use of a deconvolution software allowed reducing the GC-MS run to 10 min, without impairing the quality of the compound identifications. Comparing the results from 139 authentic urine samples to those obtained using the current routine analysis indicated this method performed well. Moreover, additional 5-min GC-MS/MS programs are described, enabling a very sensitive target screening of 54 drugs, including THC-COOH or buprenorphine, without further sample preparation. These methods appeared as an interesting alternative to immuno-assays based screening. The analytical strategy presented in this article proved to be a promising approach for systematic toxicological analysis (STA) of drugs in urine.

Which anatomical sites should be sampled for screening of methicillin-resistant Staphylococcus aureus carriage by culture or by rapid PCR test?

The nose is the anatomical site usually recommended for methicillin-resistant Staphylococcus aureus (MRSA) screening. Other sites are also recommended, but are more controversial. We showed that the sensitivities of MRSA detection from nasal swabs alone were 48% and 62% by culture or by rapid PCR test, respectively. These percentages increased to 79% and 92% with the addition of groin swabs, and to 96% and 99% with the addition of groin and throat swabs. In conclusion, neither by culture nor by rapid PCR test is nose sampling alone sufficient for MRSA detection. Additional anatomical sites should include at least the groin and throat.

Rapid Improvement in Health-Related Quality of Life (HRQoL) in Gouty Arthritis Patients Treated with Canakinumab (ACZ885) Compared to Triamcinolone Acetonide

Background: Gouty arthritis is a painful inflammatory disease with a significant impact on patients' HRQoL. In gouty arthritis, the inflammatory response is initiated by interleukin-1b (IL-1b) release, due to activation of the NALP3 inflammasome by MSU crystals. Canakinumab, a fully human anti-IL-1b antibody has a long half-life and has been shown to control inflammation in gouty arthritis. This study evaluated changes in HRQoL in gouty arthritis patients following treatment with canakinumab or triamcinolone acetonide (TA).Methods: This was an 8-week, dose-ranging, multi-center, active controlled, single-blind study. Patients (>=18 to <=80 years) experiencing an acute gouty arthritis flare, refractory to or contraindicated to NSAlDs and/or colchicine, were randomized to canakinumab 10, 25, 50, 90, 150 mg sc or TA 40 mg im. HRQoL was assessed as an exploratory endpoint at baseline and different pre-specified time-points using patient reported outcomes evaluating general mental and physical component summary scores and subscale scores of SF-36® (acute version 2) and functional disability (HAQ-DI©). We report HRQoL results for canakinumab 150 mg, the dose that was selected for the Phase III studies.Results: Baseline assessments showed a major impact on the HRQoL during acute gouty arthritis. Compared to TA, canakinumab 150 mg showed greater improvements in SF-36® physical and mental component summary and subscale scores at 7 days post-dose.In the canakinumab 150 mg group, the most severe impairment at baseline was reported for physical functioning and bodily pain; levels of 41.5 and 36.0, respectively, which improved within 7 days to 80.0 and 72.2 (mean increases of 39.0 and 35.6) approaching levels of the general US population (84.2 and 75.2). 8 weeks post-dose patients reached levels of 86.1 and 86.6 (mean increases of 44.6 and 50.6 for physical functioning and bodily pain, respectively) and these were higher than levels seen in the general US population. This was in contrast to patients treated with TA, who showed less improvement within 7 days (mean increases of 23.3 and 21.3 for physical function and bodily pain, respectively). None of the scores reached levels of the general US population 8 weeks post-dose. Functional disability scores, as measured by the HAQ-DI© decreased in both treatment groupsConclusions: All canakinumab doses showed a rapid improvement in physical and mental well-being of gouty arthritis patients based on SF-36® scores, in particular the 150 mg dose. In contrast to the TA group, patients treated with canakinumab showed improvement within 7 days in physical function and bodily pain approaching levels of the general population. The 150 mg dose of canakinumab was selected for further development in Phase III studies.

Refinement of Rapid PCC Coarse Aggregate Testing Program, HR-1071, 1999

The characterization and categorization of coarse aggregates for use in portland cement concrete (PCC) pavements is a highly refined process at the Iowa Department of Transportation. Over the past 10 to 15 years, much effort has been directed at pursuing direct testing schemes to supplement or replace existing physical testing schemes. Direct testing refers to the process of directly measuring the chemical and mineralogical properties of an aggregate and then attempting to correlate those measured properties to historical performance information (i.e., field service record). This is in contrast to indirect measurement techniques, which generally attempt to extrapolate the performance of laboratory test specimens to expected field performance. The purpose of this research project was to investigate and refine the use of direct testing methods, such as X-ray analysis techniques and thermal analysis techniques, to categorize carbonate aggregates for use in portland cement concrete. The results of this study indicated that the general testing methods that are currently used to obtain data for estimating service life tend to be very reliable and have good to excellent repeatability. Several changes in the current techniques were recommended to enhance the long-term reliability of the carbonate database. These changes can be summarized as follows: (a) Limits that are more stringent need to be set on the maximum particle size in the samples subjected to testing. This should help to improve the reliability of all three of the test methods studied during this project. (b) X-ray diffraction testing needs to be refined to incorporate the use of an internal standard. This will help to minimize the influence of sample positioning errors and it will also allow for the calculation of the concentration of the various minerals present in the samples. (c) Thermal analysis data needs to be corrected for moisture content and clay content prior to calculating the carbonate content of the sample.

Rapid, simple and high yield production of recombinant proteins in mammalian cells using a versatile episomal system.

Many research projects in life sciences require purified biologically active recombinant protein. In addition, different formats of a given protein may be needed at different steps of experimental studies. Thus, the number of protein variants to be expressed and purified in short periods of time can expand very quickly. We have therefore developed a rapid and flexible expression system based on described episomal vector replication to generate semi-stable cell pools that secrete recombinant proteins. We cultured these pools in serum-containing medium to avoid time-consuming adaptation of cells to serum-free conditions, maintain cell viability and reuse the cultures for multiple rounds of protein production. As such, an efficient single step affinity process to purify recombinant proteins from serum-containing medium was optimized. Furthermore, a series of multi-cistronic vectors were designed to enable simultaneous expression of proteins and their biotinylation in vivo as well as fast selection of protein-expressing cell pools. Combining these improved procedures and innovative steps, exemplified with seven cytokines and cytokine receptors, we were able to produce biologically active recombinant endotoxin free protein at the milligram scale in 4-6weeks from molecular cloning to protein purification.

Tests diagnostiques rapides (TDR): la panacée pour le praticien [Rapid diagnostic tests (RDT): the cure-all for the practitioner?].

Many rapid diagnostic tests (RDT) for the diagnosis of infectious diseases have been developed over the last 20 years. These allow (1) administering a treatment immediately in case of a potentially fatal disease, (2) prescribing a specific rather than presumptive treatment, (3) quickly introducing measures aimed at interrupting the transmission of the disease, (4) avoiding useless antibiotic treatments and (5) implementing a sequential diagnostic strategy to avoid extensive investigations. Using the example of malaria, a new strategy that includes a RDT as first-line emergency diagnostic tool and, when negative, delayed microscopy at the laboratory opening time is implemented in Lausanne since 1999. This strategy has been shown to be safe. Each TDR has its own characteristics that imperatively need to be known by the practitioner if he/she wants to use it in a rational way.