Lack of sensitivity of an IVD/CE-labelled kit targeting the S gene for detection of SARS-CoV-2.

OBJECTIVES: New molecular tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are being rapidly launched in response to the coronavirus disease 2019 (COVID-19) pandemic. The aim of this study was to evaluate the analytical and clinical performance of the VIASURE SARS-CoV-2 S gene RT-PCR Kit on the BD Max™ system and to compare results with those obtained with the cobas® SARS-CoV-2 test on the cobas® 6800 system. METHODS: For testing the analytical performance, reference material was used. Clinical samples (n = 101) obtained from individuals with symptoms compatible with COVID-19 were studied. Oropharyngeal and nasopharyngeal swabs were collected by using either ESwab™ or UTM™ collection systems. RESULTS: When the analytical performance was evaluated, the sample containing the lowest SARS-CoV-2 concentration tested negative with the VIASURE test whereas results obtained with the cobas® test were found to be concordant with the results expected. Six out of the 101 clinical samples (5.9%) showed an inhibition with the VIASURE test. When analysing the remaining 95 clinical samples, 27 were found to be negative with both assays. Of 68 samples that were positive with the cobas® test, the VIASURE test missed 21 (30.9 %) samples. All of those 21 samples had shown Ct values ≥ 31 with the cobas® 6800 system. None of the samples tested positive with the VIASURE test and negative with the cobas® test. CONCLUSIONS: The VIASURE test was impaired by a lack of sensitivity and a relatively high number of invalid results. When using the VIASURE test for routine testing, a significant number of COVID-19-positive samples would have been missed.

Analytical determination of bisphenol A (BPA) and bisphenol analogues in paper products by GC-MS/MS.

Bisphenol A (BPA; 4-[2-(4-hydroxyphenyl)propan-2-yl]phenol), a suspected endocrine disruptor with a weak estrogenic activity, is used in a variety of consumer products, including food-contact materials made of paper and cardboard products. Due to restrictions on the use of BPA because of its potential health risks, BPA is gradually being replaced by other bisphenols because no limitations exist for these substances. This study presents a method for the simultaneous analysis of BPA, bisphenol AF (BPAF), bisphenol B (BPB), bisphenol E (BPE), bisphenol F (BPF) and bisphenol S (BPS) in paper and board products using gas chromatography-tandem mass spectrometry (GC-MS/MS). Paper samples were extracted by liquid extraction, as well as by Folch extraction, derivatised with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and the results compared. The developed method showed good linearity (R2 > 0.9965) and precision, yielding relative standard deviations (RSDs) of less than 16.6% for reproducibility and 19.8% for repeatability. The limits of detection and limits of quantification for the different bisphenols ranged from 0.23 to 2.70 µg kg-1 paper and from 0.78 to 9.10 µg kg-1 paper, respectively. Analysis of different paper products (recycled, virgin fibre) showed that all the analysed bisphenols were present in the samples, except for BPAF and BPB. A calculation of the 'worst-case' scenario assuming a maximum potential migration of 100% of the analytes into food showed that the analysed products can be assumed to be safe regarding the migration of bisphenols.

The effect of leptin replacement on sleep-disordered breathing in the leptin-deficient ob/ob mouse.

Obese leptin-deficient (ob/ob) mice demonstrate defects in upper airway structural and neuromuscular control. We hypothesized that these defects predispose to upper airway obstruction during sleep, and improve with leptin administration. High-fidelity polysomnographic recordings were conducted to characterize sleep and breathing patterns in conscious, unrestrained ob/ob mice (23 wk, 67.2 ± 4.1 g, n = 13). In a parallel-arm crossover study, we compared responses to subcutaneous leptin (1 µg/h) vs. vehicle on respiratory parameters during NREM and REM sleep. Upper airway obstruction was defined by the presence of inspiratory airflow limitation (IFL), as characterized by an early inspiratory plateau in airflow at a maximum level (V̇Imax) with increasing effort. The severity of upper airway obstruction (V̇Imax) was assessed along with minute ventilation (V̇E), tidal volume (VT), respiratory rate (RR), inspiratory duty cycle, and mean inspiratory flow at each time point. IFL occurred more frequently in REM sleep (37.6 ± 0.2% vs. 1.1 ± 0.0% in NREM sleep, P < 0.001), and leptin did not alter its frequency. V̇Imax (3.7 ± 1.1 vs. 2.7 ± 0.8 ml/s, P < 0.001) and V̇E increased (55.4 ± 22.0 vs. 39.8 ± 16.4 ml/min, P < 0.001) with leptin vs. vehicle administration. The increase in V̇E was due to a significant increase in VT (0.20 ± 0.06 vs. 0.16 ± 0.05 ml, P < 0.01) rather than RR. Increases in V̇E were attributable to increases in mean inspiratory flow (2.5 ± 0.8 vs. 1.8 ± 0.6 ml/s, P < 0.001) rather than inspiratory duty cycle. Similar increases in V̇E and its components were observed in non-flow-limited breaths during NREM and REM sleep. These responses suggest that leptin stabilized pharyngeal patency and increased drive to both the upper airway and diaphragm during sleep.

Comparing different gas chromatographic methods for the quantification of bisphenol A (BPA) trace levels in paper and cardboard products from the market.

Bisphenol A (BPA; 4,4'-(propane-2,2-diyl)diphenol), a suspected endocrine disruptor with weak estrogenic activity, is used in a variety of consumer products, including paper and cardboard products used as food contact materials. The present study compared four different gas chromatographic methods for the analysis of BPA in paper and cardboard food packages. Eighteen different food packages were extracted and BPA was determined using two different derivatisation reactions--trimethylsilylation with N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA) and halide alkylation with pentafluorobenzoyl chloride (PFBOCl)--and four different separation and detection techniques. The BSTFA derivatives were quantified with (1) GC-MS in single-ion monitoring (SIM) mode with electron ionisation (EI-GC-MS) and (2) GC-MS/MS in multiple reaction monitoring (MRM) mode using electron ionisation (EI-GC-MS/MS); while the PFBOCl derivatives were quantified with (3) GC-MS using electron ionisation (EI-GC-MS) as well as (4) GC-MS with negative chemical ionisation (NCI-GC-MS). All developed methods showed good linearity (R(2) > 0.9938), precision (CV < 4.5% for reproducibility; CV < 2.2% for repeatability) and sensitivity, with limits of detection (LODs) between 0.02 µg kg(-1) for the pentafluorobenzoyl derivatives measured with the NCI-GC-MS method and 6 µg kg(-1) for the pentafluorobenzoyl derivatives determined with EI-GC-MS. Levels of BPA in the samples were in agreement for all methods, ranging from values below the limit of quantitation (LOQ) to 11.9 mg kg(-1) paper. In a last step, the maximum potential migration into food products was calculated for all tested paper and cardboard samples, assuming a 'worst case' scenario of 100% migration.

Procalcitonin fails to predict bacteremia in SIRS patients: a cohort study.

BACKGROUND: Procalcitonin (PCT) has previously been proposed as useful marker to rule out bloodstream-infection (BSI). The objective of this study was to evaluate the sensitivity of different PCT cut-offs for prediction of BSI in patients with community (CA)- and hospital-acquired (HA)-BSI. METHODS: A total of 898 patients fulfilling systemic-inflammatory-response-syndrome (SIRS) criteria were enrolled in this prospective cohort study at the Medical University of Graz, Austria. Of those 666 patients had positive blood cultures (282 CA-BSI, 384 HA-BSI, enrolled between January 2011 and December 2012) and 232 negative blood cultures (enrolled between January 2011 and July 2011 at the emergency department). Blood samples for determination of laboratory infection markers (e.g. PCT) were collected simultaneously with blood cultures. RESULTS: Procalcitonin was significantly (p < 0.001) higher in SIRS patients with bacteremia/fungemia than in those without. Receiver operating characteristic curve analysis revealed an area under the curve (AUC) value of 0.675 for PCT (95% CI 0.636-0.714) for differentiating patients with BSI from those without. AUC for IL-6 was 0.558 (95% CI 0.515-0.600). However, even at the lowest cut-off evaluated (i.e. 0.1 ng/ml) PCT failed to predict BSI in 7% (n = 46) of patients. In the group of patients with SIRS and negative blood culture 79% (n = 185) had PCT levels > 0.1. CONCLUSION: Procalcitonin was significantly higher in patients with BSI than in those without and superior to IL-6 and CRP. The clinical importance of this is questionable, because a suitable PCT threshold for excluding BSI was not established. An approach where blood cultures are guided by PCT only can therefore not be recommended.

[Epidemics and pandemics in general practice. What can we learn from the swine flu (H1N1) and EHEC outbreak?]. / Epidemien und Pandemien in der hausärztlichen Praxis. Was können wir aus dem Schweinegrippe (H1N1)- und EHEC-Ausbruch lernen?

BACKGROUND: As primary care givers with a coordinating function, general practitioners (GP) play a key role in dealing with epidemics and pandemics. As of yet, there are no studies in Germany describing the difficulties experienced by GPs in patient care during epidemics/pandemics. OBJECTIVES: This study aimed at identifying the problem areas in GPs' patient care during the H1N1 and EHEC (enterohemorrhagic strain of Escherichia coli) outbreaks. With this information, recommendations for guaranteeing proper patient care during future epidemics/pandemics can be derived. MATERIALS AND METHODS: In all, 12 qualitative, semi-structured, open guideline interviews with GPs in Hamburg and Lübeck were conducted, transcribed, and evaluated with qualitative content analysis. RESULTS: Five areas in ambulatory patient care were identified in which changes are needed from the primary care perspective: provision of information for GPs, workload, financing of epidemic-related measures, organization of the practices, care of those taken ill. CONCLUSIONS: The workload of GPs in particular can and should be reduced through successful, centralized information distribution during epidemics/pandemics. The GP's function as a coordinator should be supported and consolidated, in order to relieve the in-patient sector in cases of an epidemic/pandemic. Secured financing of epidemic-associated measures can help ensure patient care.

Soluble urokinase plasminogen activator receptor predicts mortality in patients with systemic inflammatory response syndrome.

OBJECTIVE: The soluble urokinase plasminogen activator receptor (suPAR) reflects inflammation. However, the prognostic value of suPAR measurements, particularly at the very early onset of systemic inflammatory response syndrome (SIRS), is less well defined. METHODS: The prognostic potential of suPAR levels in patients with SIRS was evaluated. From November 2010 until April 2013, 902 adult patients presenting with SIRS were investigated. Blood samples for laboratory testing of inflammation markers were collected simultaneously with initial blood cultures. suPAR testing was performed using suPARnostic(©) assay. RESULTS: Analyses of receiver operating characteristics curves revealed areas under the curve (AUCs) of 0.818 for predicting overall mortality within 48 h (36/902 patients died), 0.739 for 30-day mortality (117/902 died) and 0.706 for predicting 90-day mortality (151/902 died). AUCs for procalcitonin (0.777, 0.671 and 0.638), interleukin-6 (0.709, 0.593 and 0.569) and C-reactive protein (0.66, 0.594 and 0.586) as well as renal function and age were markedly lower. Using multivariable regression analyses, suPAR levels (P < 0.001) remained significant predictors of 48-h mortality, whereas suPAR levels (P < 0.001) and bacteraemia (P = 0.002 and P = 0.001, respectively) remained significant predictors of 30- and 90-day mortality. Using Kaplan-Meier survival plots, patients with suPAR <9.15 ng mL(-1) at SIRS onset had a clear benefit. CONCLUSION: suPAR plasma level determined at early SIRS is predictive for mortality.

PAK-dependent STAT5 serine phosphorylation is required for BCR-ABL-induced leukemogenesis.

The transcription factor STAT5 (signal transducer and activator of transcription 5) is frequently activated in hematological malignancies and represents an essential signaling node downstream of the BCR-ABL oncogene. STAT5 can be phosphorylated at three positions, on a tyrosine and on the two serines S725 and S779. We have investigated the importance of STAT5 serine phosphorylation for BCR-ABL-induced leukemogenesis. In cultured bone marrow cells, expression of a STAT5 mutant lacking the S725 and S779 phosphorylation sites (STAT5(SASA)) prohibits transformation and induces apoptosis. Accordingly, STAT5(SASA) BCR-ABL(+) cells display a strongly reduced leukemic potential in vivo, predominantly caused by loss of S779 phosphorylation that prevents the nuclear translocation of STAT5. Three distinct lines of evidence indicate that S779 is phosphorylated by group I p21-activated kinase (PAK). We show further that PAK-dependent serine phosphorylation of STAT5 is unaffected by BCR-ABL tyrosine kinase inhibitor treatment. Interfering with STAT5 phosphorylation could thus be a novel therapeutic approach to target BCR-ABL-induced malignancies.

Comparison of extended-spectrum-ß-lactamase (ESBL) carrying Escherichia coli from sewage sludge and human urinary tract infection.

For many years, extended-spectrum-beta-lactamase (ESBL) producing bacteria were a problem mainly located in medical facilities. Within the last decade however, ESBL-producing bacteria have started spreading into the community and the environment. In this study, ESBL-producing Escherichia coli from sewage sludge were collected, analysed and compared to ESBL-E. coli from human urinary tract infections (UTIs). The dominant ESBL-gene-family in both sample groups was bla(CTX-M), which is the most prevalent ESBL-gene-family in human infection. Still, the distribution of ESBL genes and the frequency of additional antibiotic resistances differed in the two sample sets. Nevertheless, phenotyping did not divide isolates of the two sources into separate groups, suggesting similar strains in both sample sets. We speculate that an exchange is taking place between the ESBL E. coli populations in infected humans and sewage sludge, most likely by the entry of ESBL E. coli from UTIs into the sewage system.

Virulence and antimicrobial resistance genes in human MRSA ST398 isolates in Austria.

This study determined the genetic background of virulence and resistance genes of MRSA ST398 in Austria. From 2004 up to 2008 a total of 41 human isolates of MRSA ST398 were investigated for virulence and resistance gene patterns using DNA microarray chip analysis. Highly similar virulence gene profiles were found in 29 (70·7%) of the isolates but genes encoding Panton-Valentine leukocidin, enterotoxins, or toxic shock syndrome toxin were not detected. Genes conferring resistance to tetracycline and erythromycin-lincosamide were common as all but one of the isolates exhibited tetM and/or tetK, which are involved in tetracycline resistance, and 12 (29·9%) were positive for ermC, conferring resistance to erythromycin/lincosamide. SplitsTree analysis showed that 40 isolates were closely related. Changes in virulence and resistance gene patterns were minimal over the observed time period.

Emergence of carbapenem-resistant Enterobacteriaceae in Austria, 2001-2010.

We report the emergence of carbapenem-resistant Enterobacteriaceae in Austria. Over a 10-year period, carbapenem-resistant Enterobacteriaceae isolates were obtained from 13 hospitalized patients, with the first isolation in the year 2005 and a remarkable increase in the number of involved patients in 2010. Carbapenem-resistant Enterobacteriaceae comprise eight Klebsiella pneumoniae isolates, four Klebsiella oxytoca isolates, and one Escherichia coli isolate. The detected carbapenemases were the metallo-ß-lactamases New Delhi ß-lactamase, VIM and IMP, and the serin-ß-lactamase Klebsiella pneumoniae carbapenemase.

Enzymatic single-chain antibody tagging: a universal approach to targeted molecular imaging and cell homing in cardiovascular disease.

RATIONALE: Antibody-targeted delivery of imaging agents can enhance the sensitivity and accuracy of current imaging techniques. Similarly, homing of effector cells to disease sites increases the efficacy of regenerative cell therapy while reducing the number of cells required. Currently, targeting can be achieved via chemical conjugation to specific antibodies, which typically results in the loss of antibody functionality and in severe cell damage. An ideal conjugation technique should ensure retention of antigen-binding activity and functionality of the targeted biological component. OBJECTIVE: To develop a biochemically robust, highly reproducible, and site-specific coupling method using the Staphylococcus aureus sortase A enzyme for the conjugation of a single-chain antibody (scFv) to nanoparticles and cells for molecular imaging and cell homing in cardiovascular diseases. This scFv specifically binds to activated platelets, which play a pivotal role in thrombosis, atherosclerosis, and inflammation. METHODS AND RESULTS: The conjugation procedure involves chemical and enzyme-mediated coupling steps. The scFv was successfully conjugated to iron oxide particles (contrast agents for magnetic resonance imaging) and to model cells. Conjugation efficiency ranged between 50% and 70%, and bioactivity of the scFv after coupling was preserved. The targeting of scFv-coupled cells and nanoparticles to activated platelets was strong and specific as demonstrated in in vitro static adhesion assays, in a flow chamber system, in mouse intravital microscopy, and in in vivo magnetic resonance imaging of mouse carotid arteries. CONCLUSIONS: This unique biotechnological approach provides a versatile and broadly applicable tool for procuring targeted regenerative cell therapy and targeted molecular imaging in cardiovascular and inflammatory diseases and beyond.

Opposing effects of monomeric and pentameric C-reactive protein on endothelial progenitor cells.

C-reactive protein (CRP) has been linked to the pathogenesis of atherosclerosis. The dissociation of native, pentameric (p)CRP to monomeric (m)CRP on the cell membrane of activated platelets has recently been demonstrated. The dissociation of pCRP to mCRP may explain local pro-inflammatory reactions at the site of developing atherosclerotic plaques. As a biomarker, pCRP predicts cardiovascular adverse events and so do reduced levels and function of circulating endothelial progenitor cells (EPCs). We hypothesised that mCRP and pCRP exert a differential effect on EPC function and differentiation. EPCs were treated with mCRP or pCRP for 72 h, respectively. Phenotypical characterisation was done by flow cytometry and immunofluorescence microscopy, while the effect of mCRP and pCRP on gene expression was examined by whole-genome gene expression analysis. The functional capacity of EPCs was determined by colony forming unit (CFU) assay and endothelial tube formation assay. Double staining for acetylated LDL and ulex lectin significantly decreased in cells treated with pCRP. The length of tubuli in a matrigel assay with HUVECs decreased significantly in response to pCRP, but not to mCRP. The number of CFUs increased after pCRP treatment. RNA expression profiling demonstrated that mCRP and pCRP cause highly contradictory gene regulation. Interferon-responsive genes (IFI44L, IFI44, IFI27, IFI 6, MX1, OAS2) were among the highly up-regulated genes after mCRP, but not after pCRP treatment. In conclusion, EPC phenotype, genotype and function were differentially affected by mCRP and pCRP, strongly arguing for differential roles of these two CRP conformations. The up-regulation of interferon-inducible genes in response to mCRP may constitute a mechanism for the local regulation of EPC function.

Resistance trends and in vitro activity of tigecycline and 17 other antimicrobial agents against Gram-positive and Gram-negative organisms, including multidrug-resistant pathogens, in Germany.

To document the development of resistance to tigecycline in comparison with 17 other antimicrobials, the susceptibilities of 2,741 isolates comprising 16 bacterial species recovered from hospitalised patients in 15 German centres in 2009 were assessed. The results were compared with those of previous trials (German Tigecycline Evaluation Surveillance Trial, G-TEST I and II, performed in 2005 and 2007, respectively) conducted prior to and shortly after the introduction of tigecycline in Germany. Moreover, the in vitro activities of tigecycline against the subset of multidrug-resistant (MDR) pathogens recovered within all three sampling periods (n = 4,988) were evaluated in comparison to the corresponding non-MDR isolates. All susceptibility tests were performed by broth microdilution. Between 2005 and 2009, tigecycline retained its high activity against Gram-positive and Gram-negative organisms, including MDR pathogens. By contrast, an in part marked increase in resistance to broad-spectrum beta-lactams and fluoroquinolones was observed for many Enterobacteriaceae and for non-fermenting Gram-negative bacteria. Against a background of a steadily increasing number of multiresistant pathogens, the activity of tigecycline remained unaltered. With the exception of Acinetobacter isolates with decreased susceptibility to carbapenems, tigecycline's activity profile was not notably affected by organisms resistant to other drug classes and, thus, holds promise as an important therapeutic agent, particularly for situations in which MDR organisms are suspected.

Antimicrobial resistance of Streptococcus pneumoniae in Southeast Austria, 1997-2008.

Antibiotic resistance in Streptococcus pneumoniae has increased worldwide but varies within geographical regions. We conducted a retrospective analysis of resistance in S. pneumoniae over a 12-year period to assess local and temporal trends in antibacterial resistance. From 1997 to 2008, a total of 1814 non-duplicate S. pneumoniae isolates were identified at the Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, Austria. Antibiotic resistance was determined by the Clinical and Laboratory Standards Institute (CLSI) disk diffusion test. For penicillin, the minimum inhibitory concentration was determined by Etest. Susceptibility was defined according to CLSI interpretive criteria. For penicillin, resistance rates were consistently low at 0.2% over the 12-year study period. An increase in resistance was remarkable for erythromycin (3.5% in 1997; 14.7% in 2008), clindamycin (1.8% in 1997; 10.6% in 2008) and tetracycline (1.8% in 2000; 11.0% in 2008). For trimethoprim/sulfamethoxazole, resistance increased slightly to 9.2% in 2008. Quinolones showed a low resistance rate of 0.2% that persisted over the whole study period. In contrast to previously published national data, resistance to penicillin was observed to remain at a remarkably low and constant level. Although international surveillance programmes have set up sustainable and interlinked data networks, our results suggest that regional surveillance may still be needed as decision support for appropriate empirical antibiotic therapy in the local health setting.

Use of automated repetitive-sequence-based PCR for rapid laboratory confirmation of nosocomial outbreaks.

OBJECTIVE: Rapid and reliable diagnosis of genetic relatedness of clinical isolates in microbiologic laboratory is essential in case of nosocomial outbreak investigation. Most molecular techniques used to type microorganisms are technically demanding and time consuming. Currently repetitive-sequence-based PCR (rep-PCR) technique has been adapted to an automated format on the DiversiLab system (bioMérieux, Marcy l'Etoile, France). Aim of this study was to compare the performance of the DiversiLab system to that of pulsed-field gel electrophoresis (PFGE) in nosocomial outbreaks. METHODS: 122 clinical isolates (28 Methicillin-resistant Staphylococcus aureus (MRSA), 26 Acinetobacter baumannii, 45 extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae and 13 ESBL-producing Klebsiella oxytoca) were investigated. 70 isolates originated from six well-documented outbreaks, 52 were non-outbreak isolates. RESULTS: Concordant results for identification of outbreak and non-outbreak MRSA, A. baumannii and ESBL-producing K. pneumoniae strains were achieved with both methods. In the outbreak of ESBL-producing K. oxytoca automated rep-PCR was slightly more discriminatory than PFGE. Rep-PCR identified investigated ESBL-producing K. oxytoca outbreak-strains as indistinguishable or closely related, showing similarity of >90%, while PFGE identified these strains as indistinguishable. CONCLUSION: Automated rep-PCR assays on the DiversiLab system were used for MRSA, A. baumannii and for the first time ESBL-producing Klebsiella spp. and proved as a rapid and reliable method for molecular analysis of nosocomial outbreaks.

Evaluation of the safety and immunogenicity of Plasmodium falciparum apical membrane antigen 1, merozoite surface protein 1 or RTS,S vaccines with adjuvant system AS02A administered alone or concurrently in rhesus monkeys.

In an effort to broaden the immune response induced by the RTS,S/AS02(A),vaccine, we have evaluated the immunogenicity of the RTS,S antigen when combined with MSP1(42) and with AMA1, antigens derived from the asexual blood stage. The objectives of this study were (i) to determine whether MSP1(42) and AMA1 vaccines formulated with the AS02(A) Adjuvant System were safe and immunogenic in the rhesus monkey model; (ii) to investigate whether MSP1(42) or AMA1 induced immune interference to each other, or to RTS,S, when added singly or in combinations at a single injection site; (iii) in the event of immune interference, to determine if this could be reduced when antigens were administered at separate sites. We found that MSP1(42) and AMA1 were safe and immunogenic, eliciting antibodies, and Th1 and Th2 responses using IFN-gamma and IL-5 as markers. When malaria antigens were delivered together in one formulation, MSP1(42) and RTS,S reduced AMA1-specific antibody responses as measured by ELISA however, only MSP1(42) lowered parasite growth inhibitory activity of anti-AMA1 antibodies as measured by in vitro growth inhibition assay. Unlike RTS,S, MSP1(42) significantly reduced AMA1 IFN-gamma and IL-5 responses. MSP1(42) suppression of AMA1 IFN-gamma responses was not seen in animals receiving RTS,S+AMA1+MSP1(42) suggesting that RTS,S restored IFN-gamma responses. Conversely, AMA1 had no effect on MSP1(42) antibody and IFN-gamma and IL-5 responses. Neither AMA1 alone or combined with MSP1(42) affected RTS,S antibody or IFN-gamma and IL-5 responses. Immune interference by MSP1(42) on AMA1 antibody responses was also evident when AMA1, MSP1(42) and RTS,S were administered concurrently at separate sites. These results suggest that immune interference may be complex and should be considered for the design of multi-antigen, multi-stage vaccines against malaria.

Susceptibility of clinical isolates of frequently encountered bacterial species to tigecycline one year after the introduction of this new class of antibiotics: results of the second multicentre surveillance trial in Germany (G-TEST II, 2007).

Tigecycline, a broad-spectrum antibiotic for parenteral use, was introduced in Germany in May 2006. In the G-TEST-II trial, the susceptibility of isolates, recovered in 2007 from hospitalised patients in 15 centres, was assessed against tigecycline and comparators. Susceptibility tests were performed by the microdilution procedure. This study reports on the susceptibility of the isolates of 16 bacterial species and compares the results with those of a trial (G-TEST I) conducted prior to the introduction of tigecycline. Between 2005 and 2007, tigecycline retained activity against Gram-positive and Gram-negative organisms. By contrast, the rate of vancomycin-resistant strains among Enterococcus faecium isolates almost doubled. Moreover, an increase in resistance to broad-spectrum beta-lactams and fluoroquinolones was observed for members of the family Enterobacteriaceae. Against a background of a steadily rising number of pathogens that are resistant to various antibiotic classes, tigecycline represents an important treatment option.

Lipid analysis of mitochondrial membranes from the yeast Pichia pastoris.

Here we describe for the first time isolation and biochemical characterization of highly purified mitochondrial inner and outer membranes from Pichia pastoris and systematic lipid analysis of submitochondrial fractions. Mitochondria of this yeast are best developed during growth on glycerol or sorbitol, but also on methanol or fatty acids. To obtain organelle membranes at high quality, methods of isolation and subfractionation of mitochondria originally developed for Saccharomyces cerevisiae were adapted and employed. A characteristic feature of the outer mitochondrial membrane of P. pastoris is the higher phospholipid to protein ratio and the lower ergosterol to phospholipid ratio compared to the inner membrane. Another marked difference between the two mitochondrial membranes is the phospholipid composition. Phosphatidylcholine and phosphatidylethanolamine are major phospholipids of both membranes, but the inner membrane is enriched in cardiolipin, whereas the outer membrane contains a high amount of phosphatidylinositol. The fatty acid composition of both mitochondrial membranes is similar. Variation of the carbon source, however, leads to marked changes of the fatty acid pattern both in total and mitochondrial membranes. In summary, our data are the first step to understand the P. pastoris lipidome which will be prerequisite to manipulate membrane components of this yeast for biotechnological purposes.

Susceptibility of common aerobic pathogens to tigecycline: results of a surveillance study in Germany.

Tigecycline is a novel antimicrobial agent for parenteral use encompassing a broad spectrum of bacterial pathogens, including multi-resistant organisms. Here, we report the results of the first nationwide surveillance trial that was conducted in order to evaluate the susceptibility of bacterial isolates to tigecycline in a European country prior to its clinical use. A total of 2,610 Gram-positive and Gram-negative organisms recovered from hospitalized patients were tested. Minimum inhibitory concentrations (MICs) were determined using the microdilution method. All enterococci, staphylococci (including methicillin-resistant Staphylococcus aureus; MRSA), and streptococci tested were tigecycline-susceptible, except one isolate of Staphylococcus haemolyticus. Among the Gram-negative bacteria, 100% of the Escherichia coli isolates (including extended spectrum beta-lactamase [ESBL]-producers) were tigecycline-susceptible, while about 10% of the Enterobacter cloacae and Klebsiella pneumoniae isolates were resistant. Based on the results of this surveillance study, tigecycline may represent a suitable option most notably for the empiric treatment of bacterial mixed infections, including in clinical situations in which multi-resistant organisms are suspected.