Evaluation of MRSA-Screen, a simple anti-PBP 2a slide latex agglutination kit, for rapid detection of methicillin resistance in Staphylococcus aureus.

The MRSA-Screen test (Denka Seiken Co., Ltd., Tokyo, Japan), consisting of a slide latex agglutination kit that detects PBP 2a with a monoclonal antibody, was blindly compared to the oxacillin disk diffusion test, the oxacillin-salt agar screen, and PCR of the mecA gene for the detection of methicillin resistance in Staphylococcus aureus. A total of 120 methicillin-susceptible S. aureus (MSSA) and 80 methicillin-resistant S. aureus (MRSA) isolates, defined by the absence or presence of the mecA gene, respectively, were tested. The MRSA-Screen test, the oxacillin disk diffusion test, and the oxacillin-salt agar screening test showed sensitivities of 100, 61.3, and 82.5% and specificities of 99.2, 96.7, and 98.3%, respectively. We conclude that the MRSA-Screen is a very accurate, reliable, and fast test (15 min) for differentiation of MRSA from MSSA colonies on agar plates.

Schwangerschaftsassoziierte Lebererkrankungen [Pregnancy-associated liver diseases]

Lebererkrankungen treten in der Schwangerschaft selten auf, können jedoch für Mutter und Kind dramatische Folgen haben, wenn sie nicht rechtzeitig erkannt werden. Prinzipiell unterscheidet man schwangerschaftsspezifische Lebererkrankungen von interkurrierenden Lebererkrankungen während der Schwangerschaft. Zu ersteren gehören die hepatischen Manifestationen der Hyperemesis gravidarum, die intrahepatische Schwangerschaftscholestase, die Leberbeteiligung bei Präeklampsie bzw. Eklampsie inkl. HELLP-Syndrom und die akute Schwangerschaftsfettleber. Die Differentialdiagnose schwangerschaftsassoziierter Lebererkrankungen basiert auf der Anamnese (Stadium der Schwangerschaft), der Klinik, wenigen Laboruntersuchungen und einer Ultrasonographie als primärem bildgebendem Verfahren. Die Behandlung der intrahepatischen Schwangerschaftscholestase mit Ursodeoxycholsäure verbessert den Pruritus und die mütterlichen Leberwerte. Eine engmaschige Überwachung der Schwangerschaft bleibt jedoch unabdingbar. Beim HELLP-Syndrom und der akuten Schwangerschaftsfettleber ist die rasche Entbindung anzustreben. Vorbestehende Lebererkrankungen bedürfen in der Schwangerschaft einer intensivierten Kontrolle. While liver diseases are a rare occurrence in pregnancy, they may have dramatic implications for mother and child if not detected in good time. A distinction is drawn between pregnancy-specific liver diseases and intercurrent liver diseases during pregnancy. The former include hepatic manifestations of hyperemesis gravidarum, intrahepatic cholestasis of pregnancy, hepatic involvement in preeclampsia or eclampsia, including the HELLP syndrome, and acute fatty liver of pregnancy. Differential diagnosis of pregnancy-associated liver disorders is based on history (stage of pregnancy), clinical findings, a few laboratory tests and ultrasound as the primary imaging technique. Treatment of intrahepatic cholestasis of pregnancy with ursodeoxycholic acid improves pruritus and maternal liver tests. Close monitoring of pregnancy remains however indispensable. In HELLP syndrome and acute fatty liver of pregnancy the aim should be rapid delivery. Preexisting liver diseases require intensified monitoring during pregnancy.

Highly pathogenic adapted HIV-1 strains limit host immunity and dictate rapid disease progression.

OBJECTIVE:: The study of HIV-1 rapid progressors has been limited to specific case reports. Nevertheless, identification and characterization of the viral and host factors involved in rapid progression are crucial when attempting to uncover the correlates of rapid disease outcome. DESIGN:: We carried out comparative functional analyses in rapid progressors (n = 46) and standard progressors (n = 46) early after HIV-1 seroconversion (≤1 year). The viral traits tested were viral replicative capacity, co-receptor usage, and genomic variation. Host CD8 T-cell responses, humoral activity, and HLA immunogenetic markers were also determined. RESULTS:: Our data demonstrate an unusual convergence of highly pathogenic HIV-1 strains in rapid progressors. Compared with standard progressors, rapid progressor viral strains show higher in-vitro replicative capacity (81.5 vs. 67.9%; P = 0.025) and greater X4/DM co-receptor usage (26.3 vs. 2.8%; P = 0.006) in early infection. Limited or absent functional HIV-1 CD8 T-cell responses and neutralizing activity were measured in rapid progressors. Moreover, the increase in common HLA allele-restricted CD8 T-cell escape mutations in rapid progressors acts as a signature of uncontrolled HIV-1 replication and early impairment of adaptive cellular responses. CONCLUSION:: Our data support a dominant role for viral factors in rapid progressors. Robust HIV-1 replication and intrinsic viral properties limit host adaptive immune responses, thus driving rapid disease progression.

Nanoparticle usage and protection measures in the manufacturing industry : a representative survey

Addressing the risks of nanoparticles requires knowledge about release into the environment and occupational exposure. However, such information currently is not systematically collected; therefore, this risk assessment lacks quantitative data. The goal was to evaluate the current level of nanoparticle usage in Swiss industry as well as health, safety, and environmental measures, and the number of potentially exposed workers. A representative, stratified mail survey was conducted among 1626 clients of the Swiss National Accident Insurance Fund (SUVA), which insures 80,000 manufacturing firms, representing 84% of all Swiss manufacturing companies (947 companies answered the survey for a 58.3% response rate). The extrapolation to all Swiss manufacturing companies results in 1309 workers (95% confidence interval [CI]: 1073 to 1545) potentially exposed to nanoparticles in 586 companies (95% CI: 145 to 1027). This corresponds to 0.08% of workers (95% CI: 0.06% to 0.09%) and to 0.6% of companies (95% CI: 0.2% to 1.1%). The industrial chemistry sector showed the highest percentage of companies using nanoparticles (21.2%). Other important sectors also reported nanoparticles. Personal protection equipment was the predominant protection strategy. Only a few applied specific environmental protection measures. This is the first nationwide representative study on nanoparticle use in the manufacturing sector. The information gained can be used for quantitative risk assessment. It can also help policymakers design strategies to support companies developing a safer use of nanomaterial. Notingthe current low use of nanoparticles, there is still time to proactively introduce protective methods. If the predicted "nano-revolution" comes true, now is the time to take action. [Supplementary materials are available for this article. Go to the publisher's online edition of Journal of occupational and Environmental Hygiene for the following free supplemental resource: a pdf file containing a detailed description of the approach to statistical analyses, English translation of the questionnaire, additional information for Figure 1, and additional information for the SUVA-code.] [Authors]

Natural evidence for rapid abiogenic hydrothermal generation of CH4

Although recent hydrothermal experiments imply that abiogenic methane (CH4) generation from hydrothermal reduction of CO2 can occur, evidence from natural systems was still lacking. Based on the chemical and isotopic equilibrium signatures of low-temperature fumarolic gas discharges, we are able to provide hard evidence for its natural occurrence, namely in three subduction-related bi-phase hydrothermal systems of the Mediterranean, whose temperatures range from 260 to 470 degrees C. The attainment of equilibrium and the time spans of recent volcanic dormancy allowed us to calculate minimum rates for chemical and isotopic equilibration. These are significantly higher than those previously reported and might be due to the presence of a saturated water vapor phase in the investigated systems. The fact that nature provides conditions enabling relatively fast production of hydrocarbons from CO2 strongly supports the concerns that were recently raised from laboratory experiments. These address the use of the carbon isotope composition of reduced carbon in Archean sediments as a tracer of early life and the occurrence of CH4 on extraterrestrial planets as a bioindicator. In view of the potential role of abiogenic CH4 as a precursor of life, we also present an estimate of abiogenic hydrothermal CH4 fluxes throughout the Archean. It is not expected that these fluxes exceeded 80 Mt/yr during the past 4.0 Ga. This, however, would have been enough to facilitate HCN production on the prebiotic Earth. (C) 2007 Elsevier Ltd. All rights reserved.

Infections cutanées causées par des mycobactéries à croissance rapide [Skin infections due to rapid-growing mycobacteria].

Rapid-growing mycobacteria (e.g. M. abscessus and M. chelonae) are emerging pathogens with various clinical manifestations. Among immunocompetent individuals, rapid-growing mycobacteria may be responsible of pulmonary, cutaneous, osteoarticular and postoperative infections, as well as lymphadenitis and catheter-associated infections. Among immunocompromised patients, disseminated infections are also observed. Diagnosis relies on specific microbiological investigations to confirm etiology and guide antibiotic treatment. The treatment requires a multi-disciplinary approach that includes specific long-term antibiotic treatment, surgical debridement and reduction of immunosuppression whenever possible.

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.

Manufacturing and characterization of a recombinant adeno-associated virus type 8 reference standard material.

Gene therapy approaches using recombinant adeno-associated virus serotype 2 (rAAV2) and serotype 8 (rAAV8) have achieved significant clinical benefits. The generation of rAAV Reference Standard Materials (RSM) is key to providing points of reference for particle titer, vector genome titer, and infectious titer for gene transfer vectors. Following the example of the rAAV2RSM, here we have generated and characterized a novel RSM based on rAAV serotype 8. The rAAV8RSM was produced using transient transfection, and the purification was based on density gradient ultracentrifugation. The rAAV8RSM was distributed for characterization along with standard assay protocols to 16 laboratories worldwide. Mean titers and 95% confidence intervals were determined for capsid particles (mean, 5.50×10(11) pt/ml; CI, 4.26×10(11) to 6.75×10(11) pt/ml), vector genomes (mean, 5.75×10(11) vg/ml; CI, 3.05×10(11) to 1.09×10(12) vg/ml), and infectious units (mean, 1.26×10(9) IU/ml; CI, 6.46×10(8) to 2.51×10(9) IU/ml). Notably, there was a significant degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This outcome emphasizes the need to use RSMs to calibrate the titers of rAAV vectors in preclinical and clinical studies at a time when the field is maturing rapidly. The rAAV8RSM has been deposited at the American Type Culture Collection (VR-1816) and is available to the scientific community.

Rapid Site-Directed Mutagenesis Using Two-PCR-Generated DNA Fragments Reproducing the Plasmid Template.

We describe a new rapid and efficient polymerase chain reaction (PCR)-based site-directed mutagenesis method. This procedure is effective with any plasmid and it employs four oligonucleotide primers. One primer contains the desired mutation, the second is oriented in the opposite direction (one of these two primers should be phosphorylated), and the third and fourth should be coding in complementary fashion for a unique restriction site to be introduced in a nonessential region. The method consists of two simultaneous PCR reactions; the PCR products are digested with the enzyme that recognizes the newly introduced unique restriction site and then ligased and used to transform competent bacteria. Additionally, the use of Dpn I facilitates the elimination of template DNA. The newly introduced restriction site is essential for ligation in the correct orientation of the two-PCR products and is further used for mutant screening. Resulting plasmids carry both the new restriction site and the desired mutation. Using this method, more than 20 mutants have already been generated (using two different kinds of templates); all these mutants were sequenced for the desired mutation and transfected into AtT-20 cells and the expressed mutant proteins encoded by the vector were assayed.

Situation des personnes transgenres par rapport au VIH/IST en Suisse : rapid assessment

Ce rapport permet d'identifier le sous-groupe des personnes transgenres pratiquant ou ayant pratiqué le travail du sexe comme une population clairement exposée à un risque notoirement élevé d'infection par le VIH et les IST. L'ampleur rapportée du phénomène justifie pleinement l'inclusion de cette population dans le dispositif de surveillance comportementale du VIH et des autres IST, mais également la réalisation urgente d'actions de prévention communautaire. Par contre, il ne nous permet pas d'arriver à un consensus fort en ce qui concerne la situation des personnes transgenres non travailleuses du sexe par rapport au VIH et aux autres IST en Suisse. Les données internationales sont cependant suffisamment préoccupantes pour justifier la réalisation d'une recherche sur la santé sexuelle au sein de cette population. Cette recherche devra prendre en compte les contextes de vie souvent hostiles auxquels sont confrontées les personnes transgenres. Par ailleurs, les personnes transgenres devraient pouvoir être reconnues en tant que telles et enregistrées de manière systématique dans les systèmes de déclaration du VIH et des autres IST, dans les outils de suivi statistique des centres de dépistage et de conseil VIH/IST, ainsi que dans l'enquête suisse sur la santé.

Rapid detection of MRSA in screening specimens during a hospital outbreak.

Objectives: To compare the results of rapid PCR screening for MRSA using the GeneXpert system with those of cultures in an outbreak setting. Methods: GeneXpert was used for screening MRSA in nose, throat, groin, and other clinical samples during a 6-month period. Samples were performed using a double-swab transystem. When >1 sample was found positive in a screening set, all second swabs of the set were analysed by culture. Results: From June to October 2009, 7568 rapid tests were performed, among which 432 (5.7%) were positive (nose: 149/2090, 7.1%; throat: 98/2078, 4.7%; groin: 152/2080, 7.3%; urine: 14/1090, 1.3%; wounds: 18/150, 12%; and others:1/27, 3.7%), and 84 (1.1%) were invalid. A total of 1517 samples were analyzed by both rapid PCR and culture. Rapid tests had a sensitivity of 0.896 compared to cultures, a specificity of 0.769, a PPV of 0.763, and a NPV of 0.899. The rapid test was found to be less sensitive in throat samples (0.81) than in nose or inguinal samples (0.93 for both). In 32/192 (16%) patients a positive rapid PCR result was not confirmed by culture, despite several subsequent screening samples in some patients. Cycle threshold (Ct) for SCCmec of these PCR positive reactions were all >30. Conclusions: GeneXpert MRSA was found to be suitable for the rapid detection in nose, inguinal, and throat samples, however with a lower sensitivity in the later. Negative cultures in 16% of our PCR-positive patients raised the question of false positivity or higher sensitivity of GeneXpert. Further work is needed to investigate these cases.

A rapid assay for the coupled cell free generation of oxylipins

We developed a rapid and simple assay for the coupled in vitro synthesis of oxylipins using free unsaturated fatty acids as substrates. Reactions were catalysed with extracts expressed from living plant tissues. Preliminary experiments involving the cell free transformation of fatty acid hydroperoxides revealed that storage or pretreatment of the plant extract rapidly altered its capacity to catalyse the generation of oxidised fatty acid derivatives. This could reflect changes in oxylipin generation that might take place in situ in damaged plant cells during herbivory. All subsequent experiments were performed without dilution, titration or any other modification of the plant extract prior to its addition to the assay system. The assays were used to study, for the first time, tissue-specific differences in fatty acid transformation to divinyl ethers. Root tissues from tomato efficiently catalysed the formation of corneleic and colnelenic acids from linoleic acid and linolenic acids, respectively, whereas leaf, hypocotyl and cotyledon extracts did not promote the formation of these compounds. We observed the efficient generation of 9-oxo-nonanoic acid from the substrate linolenic acid and speculate that this aldehyde could arise either from the action of hydroperoxide lyase on 9-hydroperoxylinolenic acid or by a novel route involving cleavage of colnelenic acid which was also present among the products of the reaction. A potential role of divinyl ethers as substrates for the generation of toxic aldehydes is discussed