Evaluation of the NucliSENS EasyQ KPC assay for detection of Klebsiella pneumoniae carbapenemase-producing Enterobacteriaceae.

The NucliSENS EasyQ KPC assay (bioMérieux SA, Marcy l'Etoile, France) was compared with a routinely used phenotypic method for detection of Enterobacteriaceae producing Klebsiella pneumoniae carbapenemase (KPC)-type carbapenemases, using 806 stool samples and rectal swabs. Compared with the phenotypic method, the EasyQ KPC assay had a sensitivity and specificity of 93.3% and 99.0%, respectively, in this setting, with diverse KPC producers not limited to ST258 Klebsiella pneumoniae.

Digital gene expression analysis might aid in the diagnosis of thyroid cancer.

Background: Thyroid cancer represents approximately 90% of endocrine cancers. Difficulties in diagnosis and low inter-observer agreement are sometimes encountered, especially in the distinction between the follicular variant of papillary thyroid carcinoma (fvptc) and other follicular-patterned lesions, and can present significant challenges. In the present proof-of-concept study, we report a gene-expression assay using NanoString nCounter technology (NanoString Technologies, Seattle, WA, U.S.A.) that might aid in the differential diagnosis of thyroid neoplasms based on gene-expression signatures. Methods: Our cohort included 29 patients with classical papillary thyroid carcinoma (ptc), 13 patients with fvptc, 14 patients with follicular thyroid carcinoma (ftc), 14 patients with follicular adenoma (fa), and 14 patients without any abnormality. We developed a 3-step classifier that shows good correlation with the pathologic diagnosis of various thyroid neoplasms. Step 1 differentiates normal from abnormal thyroid tissue; step 2 differentiates benign from malignant lesions; and step 3 differentiates the common malignant entities ptc, ftc, and fvptc. Results: Using our 3-step classifier approach based on selected genes, we developed an algorithm that attempts to differentiate thyroid lesions with varying levels of sensitivity and specificity. Three genes-namely SDC4, PLCD3, and NECTIN4/PVRL4-were the most informative in distinguishing normal from abnormal tissue with a sensitivity and a specificity of 100%. One gene, SDC4, was important for differentiating benign from malignant lesions with a sensitivity of 89% and a specificity of 92%. Various combinations of genes were required to classify specific thyroid neoplasms. Conclusions: This preliminary proof-of-concept study suggests a role for nCounter technology, a digital gene expression analysis technique, as an adjunct assay for the molecular diagnosis of thyroid neoplasms.

[Antibiotics susceptibility of Streptococcus and Enterococcus: data of Onerba network]. / Sensibilité aux antibiotiques chez les streptocoques (hors pneumocoque) et les entérocoques: données Onerba.

This work was aimed to analyze trends in susceptibility to antibiotics among the main species of beta-hemolytic streptococci involved in community-acquired infections in human (Streptococcus pyogenes and Streptococcus agalactiae), or in animals (Streptococcus suis and Streptococcus uberis) and also among the main enterocci species, Enterococcus faecalis and Enterococcus faecium. Data were recorded since 1996 through the Onerba networks. S. pyogenes, as the other beta-hemolytic streptococci studied remained fully susceptible to beta-lactam antibiotics. However, susceptibility to macrolides is clearly decreasing in S. pyogenes. In 2002, only 62 to 65% of the strains according to the network considered, were susceptible to erythromycin. A similar trend was observed for S. agalactiae with only 75% of erythromycin susceptibility in 2002, and for both species isolated from animals S. suis and S. uberis, with respectively 35 and 76% of strains susceptible to erythromycin. In enterococci, susceptibility to beta-lactams remained stable between 2000 and 2004. Indeed, the susceptibility to aminopenicillins remained high in E. faecalis (about 98%), whereas the proportion of E. faecium isolates susceptible to these antibiotics were lower than 60%. From 1999 to 2004, various studies conducted in French hospitals showed that the vancomycin resistance among enterococci accounted for less than 2%. However, the recent emergence of glycopeptide resistant enterococci clusters in French hospitals is a matter of concern and emphasizes the need for an ongoing surveillance. Such trend in macrolide resistance among S. pyogenes or S. agalactiae should consequently lead to propose other alternatives in case of beta-lactam allergy, and for pharyngitis, to rethink the place of the culture for susceptibility testing.

Brain activity sustaining the modulation of pain by empathetic comments.

Empathetic verbal feedback from others has been shown to alleviate the intensity of experimental pain. To investigate the brain changes associated with this effect, we conducted 3T-fMRI measurements in 30 healthy subjects who received painful thermal stimuli on their left hand while overhearing empathetic, neutral or unempathetic comments, supposedly made by experimenters, via headsets. Only the empathetic comments significantly reduced pain intensity ratings. A whole-brain BOLD analysis revealed that both Empathetic and Unempathetic conditions significantly increased the activation of the right anterior insular and posterior parietal cortices to pain stimuli, while activations in the posterior cingulate cortex and precuneus (PCC/Prec) were significantly stronger during Empathetic compared to Unempathetic condition. BOLD activity increased in the DLPFC in the Empathetic condition and decreased in the PCC/Prec and vmPFC in the Unempathetic condition. In the Empathetic condition only, functional connectivity increased significantly between the vmPFC and the insular cortex. These results suggest that modulation of pain perception by empathetic feedback involves a set of high-order brain regions associated with autobiographical memories and self-awareness, and relies on interactions between such supra-modal structures and key nodes of the pain system.

Branched-polymer separations using comprehensive two-dimensional molecular-topology fractionation x size-exclusion chromatography.

Branching has a strong influence on the processability and properties of polymers. However, the accurate characterization of branched polymers is genuinely difficult. Branched molecules of a certain molecular weight exhibit the same hydrodynamic volumes as linear molecules of substantially lower weights. Therefore, separation by size-exclusion chromatography (SEC), will result in the co-elution of molecules with different molecular weights and branching characteristics. Chromatographic separation of the polymer molecules in sub-microm channels, known as molecular-topology fractionation (MTF), may provide a better separation based on topological differences among sample molecules. MTF elution volumes depend on both the topology and molar mass. Therefore co-elution of branched molecules with linear molecules of lower molar mass may also occur in this separation. Because SEC and MTF exhibit significantly different selectivity, the best and clearest separations can be achieved by combining the two techniques in a comprehensive two-dimensional (MTFxSEC) separation system. In this work such a system has been used to demonstrate branching-selective separations of star branched polymers and of randomly long-chain-branched polymers. Star-shaped polymers were separated from linear polymers above a column-dependent molecular weight or size.

[Impact of nociceptive stimuli on cervical kinesthesia]. / Influence de stimulations nociceptives sur le sens de repositionnement céphalique.

UNLABELLED: The goal of this study was to evaluate the impact of nociceptive stimuli upon the cervical proprioception ability. METHOD: Thirty healthy young subjects performed a cervicocephalic relocation test (CRT) in two random conditions: the first one was based on a nociceptive electric stimulation called condition "pain", whereas the second one was targeting a painless electric condition called condition "control". The CRT consisted of repositioning the head on the trunk, after an active transversal movement of the head in the transverse field with closed eyes. The pointing was recorded at the beginning and at the end of each rotation using a custom video acquisition system. RESULTS: The average mean of error repositioning was worth 3.98+/-0.99 degrees (average mean, standard deviation) in the condition "pain", and 1.75+/-0.37 degrees in the condition "control" (p<0.01). CONCLUSION: Acute pain provokes a disturbance of the cervical proprioception ability without damaging the anatomic structure. This observation suggests the interest of an early follow-up of the pain to avoid sensory disturbances, as well as the establishment of a cervical proprioceptive rehabilitation program after an algic event.

First outbreak of colonization by linezolid- and glycopeptide-resistant Enterococcus faecium harbouring the cfr gene in a UK nephrology unit.

AIM: To describe an outbreak of colonization by linezolid- and glycopeptide-resistant Enterococcus faecium harbouring the cfr gene in a UK nephrology unit. METHODS: Isolates of linezolid-resistant E. faecium were typed by pulsed-field gel electrophoresis (PFGE), and examined by polymerase chain reaction (PCR) and sequencing for the transmissible cfr gene that confers resistance to linezolid. Enhanced environmental cleaning, initial and weekly screening of all patients, and monitoring of adherence to standard infection control precautions were implemented. FINDINGS: Five patients with pre-existing renal disease were found to have rectal colonization with linezolid-resistant E. faecium over a two-week period. The index case was a 57-year-old male from India who had travelled to the UK. One patient also had a linezolid-resistant E. faecium of a different PFGE profile isolated from a heel wound. All isolates were confirmed to harbour the cfr gene by PCR and Sanger sequencing, and all were resistant to glycopeptides (VanA phenotype). CONCLUSIONS: This article describes the first UK outbreak with a single strain of linezolid- and glycopeptide-resistant E. faecium harbouring the cfr gene, affecting five patients in a nephrology unit. Following the implementation of aggressive infection control measures, no further cases were detected beyond a two-week period.

Paclitaxel induces release of cytochrome c from mitochondria isolated from human neuroblastoma cells'.

Paclitaxel is an antimicrotubule agent that induces mitotic block and apoptosis. We show for the first time that paclitaxel acts directly or mitochondria isolated from human cancer cells. In isolated yeast mito chondria, paclitaxel (15 microM) induced an 18% increase in the respiration rate, with no concomitant release of cytochrome c. In isolated neuroblas toma mitochondria, paclitaxel (10-100 microM) induced a 27-72% release o cytochrome c. Release was prevented by cyclosporin A, suggesting the involvement of the permeability transition pore. Doxorubicin did no induce cytochrome c release, whereas vinorelbine, another antimicrotu bule agent, did. Thus, antimicrotubule agents can directly affect mito chondria to induce apoptosis.

Carbapenemase-producing Enterobacteriaceae in hospital wastewater: a reservoir that may be unrelated to clinical isolates.

BACKGROUND: Carbapenemase-producing Enterobacteriaceae (CPE) are an emerging infection control problem in hospitals worldwide. Identifying carriers may help reduce potential spread and infections. AIM: To assess whether testing hospital wastewater for CPE can supplement patient-based screening for infection prevention purposes in a hospital without a recognized endemic CPE problem. METHODS: Wastewater collected from hospital pipework on 16 occasions during February to March 2014 was screened for CPE using chromID(®) CARBA agar and chromID(®) CPS agar with a 10µg ertapenem disc and combination disc testing. Minimum inhibitory concentrations were determined using British Society for Antimicrobial Chemotherapy methodology and carbapenemase genes detected by polymerase chain reaction or whole-genome sequencing. Selected isolates were typed by pulsed-field gel electrophoresis. FINDINGS: Suspected CPE were recovered from all 16 wastewater samples. Of 17 isolates sent to the Antimicrobial Resistance and Healthcare Associated Infections Reference Unit, six (four Citrobacter freundii and two Enterobacter cloacae complex) were New Delhi metallo-ß-lactamase (NDM) producers and the remaining 11 (six Klebsiella oxytoca and five Enterobacter cloacae complex) were Guiana-Extended-Spectrum-5 (GES-5) producers, the first to be described among Enterobacteriaceae in the UK. The four NDM-producing C. freundii, two NDM-producing E. cloacae complex, and four out of five GES-5-producing E. cloacae complex were each indistinguishable isolates of the same three strains, whereas the six GES-5-producing K. oxytoca overall shared 79% similarity. CONCLUSION: CPE are readily isolated from hospital wastewater using simple culture methods. There are either undetected carriers of CPE excreting into the wastewater, or these CPE represent colonization of the pipework from other sources. Surveillance of hospital wastewater for CPE does not appear helpful for infection control purposes within acute hospitals.

Analysis of suppressor mutation reveals long distance interactions in the bc(1) complex of Saccharomyces cerevisiae.

Four totally conserved glycines are involved in the packing of the two cytochrome b hemes, b(L) and b(H), of the bc(1) complex. The conserved glycine 131 is involved in the packing of heme b(L) and is separated by only 3 A from this heme in the bc(1) complex structure. The cytochrome b respiratory deficient mutant G131S is affected in the assembly of the bc(1) complex. An intragenic suppressor mutation was obtained at position 260, in the ef loop, where a glycine was replaced by an alanine. This respiratory competent revertant exhibited a low bc(1) complex activity and was affected in the electron transfer at the Q(P) site. The k(min) for the substrate DBH(2) was diminished by an order of magnitude and EPR spectra showed a partially empty Q(P) site. However, the binding of the Q(P) site inhibitors stigmatellin and myxothiazol remained unchanged in the suppressor strain. Optical spectroscopy revealed that heme b(L) is red shifted by 0.8 nm and that the E(m) of heme b(L) was slightly increased (+20 mV) in the revertant strain as compared to wild type strain values. Addition of a methyl group at position 260 is thus sufficient to allow the assembly of the bc(1) complex and the insertion of heme b(L) despite the presence of the serine at position 131. Surprisingly, reversion at position 260 was located 13 A away from the original mutation and revealed a long distance interaction in the yeast bc(1) complex.

Apparent redundancy of electron transfer pathways via bc(1) complexes and terminal oxidases in the extremophilic chemolithoautotrophic Acidithiobacillus ferrooxidans.

Acidithiobacillus ferrooxidans is an acidophilic chemolithoautotrophic bacterium that can grow in the presence of either the weak reductant Fe(2+), or reducing sulfur compounds that provide more energy for growth than Fe(2+). We have previously shown that the uphill electron transfer pathway between Fe(2+) and NAD(+) involved a bc(1) complex that functions only in the reverse direction [J. Bacteriol. 182, (2000) 3602]. In the present work, we demonstrate both the existence of a bc(1) complex functioning in the forward direction, expressed when the cells are grown on sulfur, and the presence of two terminal oxidases, a bd and a ba(3) type oxidase expressed more in sulfur than in iron-grown cells, besides the cytochrome aa(3) that was found to be expressed only in iron-grown cells. Sulfur-grown cells exhibit a branching point for electron flow at the level of the quinol pool leading on the one hand to a bd type oxidase, and on the other hand to a bc(1)-->ba(3) pathway. We have also demonstrated the presence in the genome of transcriptionally active genes potentially encoding the subunits of a bo(3) type oxidase. A scheme for the electron transfer chains has been established that shows the existence of multiple respiratory routes to a single electron acceptor O(2). Possible reasons for these apparently redundant pathways are discussed.

Early evolution of cytochrome bc complexes.

Primary structures, functional characteristics and phylogenetic relationships of subunits of cytochrome bc complexes from phylogenetically diverse bacterial and archaeal species were analysed. A single case of lateral gene transfer, i.e. the import of an epsilon-proteobacterial cytochrome bc(1) complex into Aquificales, was identified. For the enzyme in the remainder of the species studied, the obtained phylogenies were globally in line with small subunit rRNA trees. The distribution of a few key phylogenetic markers, such as contiguousness of cytochrome b, nature of the c-type subunit or spacing between b-heme ligands, are discussed. A localised modification of previous tree topologies is proposed on the basis of the obtained data. The comparison of extant enzymes furthermore allowed us to define the minimal functional and evolutionary core of the enzyme. The data furthermore suggest that the ancestral enzyme was put together from subunits that previously had played a role in other electron transfer chains.

Assessment of a disease-specific muscular impairment rating scale in myotonic dystrophy.

OBJECTIVE: To document the intra/interrater reliability and the construct validity of the Muscular Impairment Rating Scale (MIRS) in assessing patients with myotonic dystrophy type 1 (DM1). The MIRS is a ordinal five-point rating scale, established in accordance with the clinically recognized distal to proximal progression of the muscular involvement in DM1, based partly on a manual muscle testing (MMT) of 11 muscle groups. METHODS: To assess the reliability of the MIRS, 55 patients with DM1 were examined by three different observers, one of them evaluating each patient twice. Intra- and interobserver reliability of the MIRS was measured using Cohen's weighted kappa. To assess the construct validity of the MIRS, correlations were made with the Functional Status Index (FSI) and eight timed functional tasks. RESULTS: The intraobserver reliability of the MIRS was excellent (weighted kappa = 0.84), and the interobserver reliability was interpreted as a substantial agreement (weighted kappa = 0.77 to 0.79). The correlation coefficients between MMT scores and MIRS grades were all highly significant (r(s) = -0.81 to -0.88, p < 0.001). The FSI showed a significant progressive increase of the total median dependence score in activities of daily living from 0 in MIRS grade 1 to 39 in MIRS grade 5 (p < 0.001). The time needed to perform the eight functional tasks was also found to significantly increase in relation with the progression of the MIRS grades. CONCLUSION: The MIRS is a quick, simple, and reliable measurement of muscular impairment in DM1. The FSI questionnaire and the timed motor activities supported its construct validity. The MIRS is useful to monitor major stages of DM1 progression, to study the natural history of the disease, and to identify homogeneous groups of patients for clinical trials.

Studies on the CoQH2-cytochrome c reductase segment of the respiratory chain of yeast mitochondria, using mutants of the cytochrome b split gene.

Our work relating to the role of cytochrome b in the CoQH2-cytochrome c reductase segment of the respiratory chain of S. cerevisiae mitochondria is reviewed here and new results are reported. The results concerning the structure-function relationship of cytochrome b in this complex, analyzed within the framework of the eight transmembrane alpha helice cytochrome b folding model, agree with the following features of the proton motive Q cycle (or SQ cycle): i) the antimycin A and myxothiazol binding domains are located on opposite sides of the inner mitochondrial membrane; and ii) the antimycin A binding domain is associated with the b562 domain, the myxothiazol domain with the b565 domain. These results were obtained from structural data derived from amino-acid sequence studies on mit- mutants and from biochemical studies of these mutants. However, functional studies are reported here that are not in agreement with the following features of the above models: i) the serial arrangement of the two hemes of cytochrome b and ii) the isolation of cytochrome b from redox changes with the couple fumarate/succinate in the presence of antimycin A and myxothiazol.

Diarrheal morbidity and mortality in children in the Central African Republic.

Diarrheal morbidity and mortality in children less than 5 years old were studied in Bangui, Central African Republic, by a cluster survey. We found a high prevalence of diarrheal disease with an estimated annual incidence of 7 episodes of diarrhea per child per year. The estimated annual mortality rate for children less than 5 years old was 28.6 per 1,000 and 85.8 per 1,000 for infants; 51.6% of deaths were reported to be associated with diarrhea. During the survey, stool specimens were collected from 133 children with current diarrhea and 117 control children to study the etiologic agents of diarrheal disease in the community. An enteric pathogen was identified in 58% of diarrheal children's stools and 48% of stools of well children. A statistically significant association between diarrhea and rotavirus was found, with it being isolated from 8 of 33 (24%) of stools of infants with diarrhea compared to 0 of 25 (0%) of control infants. Isolation rates for Campylobacter jejuni, Entamoeba histolytica, pathogenic Escherichia coli, and other bacterial enteropathogens did not differ significantly between children with diarrhea and control children.

PIP: Using a cluster sample survey, researchers studied diarrheal morbidity and mortality in children 5 years old in Bangui, Central African Republic in November 1983. They collected fecal samples from all children who had diarrhea the day of the investigation. They also took stools from children not ill with diarrhea at the time and who had 3 stools/day for the past week. Researchers looked for an age matched control in the same cluster, but not in the same home, for each child with diarrhea. They were unable to explain at least 42% of the diarrhea cases and much more when they considered the high isolation rates among the nondiarrheic children. This highlights the need for further research to better understand the carrier state. 57.8% of the diarrheic children's stools had 1 or more enteric pathogens, while 47.8% of the nondiarrheic children's tools did. In children 1 year old, rotavirus was the most frequent enteropathogen associated with diarrhea (p.05). Parasitic organisms were found more frequently as the age of the child increased. A high carrier state of different enteric pathogens existed, including Campylobacter jejuni, Entamoeba histolytica, Giardia intestinalis, and enteropathogenic Escherichia coli. The annual child and infant mortality rates were very high (28.6/1000 and 85.8/1000 respectively). Death was related to diarrhea in 19.1% of the cases and associated with diarrhea in 50% of the cases. The annual attack rate stood at 7 episodes/child which is greater than is usually reported.

Identification of membrane-bound c-type cytochromes in an acidophilic ferrous ion oxidizing bacterium Thiobacillus ferrooxidans.

Three membrane-bound acid-stable cytochromes c with molecular masses of 46, 30 and 21 kDa were characterized from a new Thiobacillus ferrooxidans strain. They were solubilized with high concentrations of dodecylmaltoside at pH 8. The 30 kDa cytochrome c was purified to a homogeneous state as established by SDS-PAGE analysis. It showed an absorption peak at 410 nm in the oxidized form and at 418, 523 and 552 nm in the reduced form. The 46 kDa cytochrome c co-purified with a non-heme protein of 36 kDa. The amino acid composition and the N-terminal amino acid sequence of the 46 kDa cytochrome c were determined and compared with those of the soluble 14 kDa and the membrane-bound 21, 22.3 and 68 kDa cytochromes c isolated from two different strains. The results clearly show that this cytochrome is distinct from both the 22.3, 21 and 14 kDa cytochrome species, and exhibits some similarities with the 68 kDa cytochrome c as regards its amino acid composition.

A seven-year survey of susceptibility to marbofloxacin of pathogenic strains isolated from pets.

This study, Vetoquinol S.A. epidemiosurveillance, was conducted from 1994 to 2001 in order to determine the susceptibility (by MIC determination) to marbofloxacin (a third generation fluoroquinolone used only in individual administration for animals). Strains from infected pets originated from six European countries. Isolates were collected from urinary infections (Escherichia coli), respiratory infections (Pasteurella multocida), dermatological infections (Staphylococcus intermedius, Pseudomonas aeruginosa) and otitis (S. intermedius, P. aeruginosa). The MIC distribution for each species was the same both before and after the launch of marbofloxacin in 1995. In E. coli, a resistant population was present before the use of marbofloxacin; this resistance was induced by co- or cross-resistance to other antibiotics used previously. Over this period, there was no significant evolution of MIC(90) for any bacterial species studied and no development of resistance was observed. Marbofloxacin was the most active antibiotic against P. multocida isolates and had the lowest MIC. No difference in MIC distribution was seen between the S. intermedius (unimodal distribution) isolated from dermatological infections and those from otitis. This was also true for P. aeruginosa. The use of marbofloxacin was not found to have induced a significant increase or spread of resistant bacteria.

Seven years survey of susceptibility to marbofloxacin of bovine pathogenic strains from eight European countries.

This study was conducted from 1994 to 2001 to determine the susceptibility of bovine pathogenic bacteria to marbofloxacin (a third generation fluoroquinolone used only in individual administration for animals). Strains originated in bovine diseases from eight European countries. They were isolated from gut infections (Escherichia coli, salmonellae), mastitis (E. coli, Staphylococcus aureus, Streptococcus uberis, S. agalactiae, S. dysgalactiae) and respiratory diseases (Mannheimia haemolytica, Pasteurella multocida, Haemophilus somnus). There was no change in the MIC distributions for each species after the launch of marbofloxacin in 1997. In E. coli, a resistant population was present before the use of marbofloxacin having been induced by co- or cross-resistance to other antibiotics used previously. Over this period the only a significant change seen was an increase in MIC(90) of E. coli from the gut (1.275 microg/ml in 1994/1995 to 5.098 microg/ml in 2001). All the salmonellae were susceptible to marbofloxacin with a MIC(90) = 0.073 microg/ml in 2001 without development of high level resistance. The use of marbofloxacin seems not to have favoured a significant increase and spreading of resistant bacteria.

Antibodies to Lassa and Lassa-like viruses in man and mammals in the Central African Republic.

715 human sera from different locations in the Central African Republic were tested by indirect immunofluorescence against the three African arenaviruses, Lassa, Mobala and Mopeia. Four were positive for Lassa, seven for Mobala, and one for both Lassa and Mopeia. The epidemiology of African arenaviruses is discussed.

Number-average molecular mass determination of polymeric material by pyrolysis-gas chromatography.

The number-average molecular mass of a polymeric material has been determined by pyrolysis-gas chromatography (Py-GC) via end-group analysis. The major advantage of this technique is that no sample preparation is required. The sample is not required to be in the dilute solution form, and the amount of sample needed is approximately 0.5 mg. Phenyl group-terminated polybutadiene systems have been studied as an example. The application of Py-GC to obtain the end-group concentration, the number-average molecular mass and the limitations of this method are discussed in detail. The success of this development elevates the role of Py-GC as an important technique for end-group analysis for the determination of number-average molecular mass.