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1.  Direct RNA-Based Detection and Differentiation of CTX-M-Type Extended-Spectrum β-Lactamases (ESBL) 
PLoS ONE  2013;8(11):e80079.
The current global spread of multi-resistant Gram-negatives, particularly extended spectrum β-lactamases expressing bacteria, increases the likelihood of inappropriate empiric treatment of critically ill patients with subsequently increased mortality. From a clinical perspective, fast detection of resistant pathogens would allow a pre-emptive correction of an initially inappropriate treatment. Here we present diagnostic amplification-sequencing approach as proof of principal based on the fast molecular detection and correct discrimination of CTX-M-β-lactamases, the most frequent ESBL family. The workflow consists of the isolation of total mRNA and CTX-M-specific reverse transcription (RT), amplification and pyrosequencing. Due to the high variability of the CTX-M-β-lactamase-genes, degenerated primers for RT, qRT as well as for pyrosequencing, were used and the suitability and discriminatory performance of two conserved positions within the CTX-M genes were analyzed, using one protocol for all isolates and positions, respectively. Using this approach, no information regarding the expected CTX-M variant is needed since all sequences are covered by these degenerated primers. The presented workflow can be conducted within eight hours and has the potential to be expanded to other β-lactamase families.
PMCID: PMC3818264  PMID: 24224038
2.  Drug-induced QT-interval shortening following antiepileptic treatment with oral rufinamide 
The arrhythmogenic potential of short QT intervals has recently been highlighted in patients with a short QT syndrome. Drug-induced QT-interval prolongation is a known risk factor for ventricular tachyarrhythmias. However, reports on drug-induced QT-interval shortening are rare and proarrhythmic effects remain unclear.
Recently, rufinamide, a new antiepileptic drug for the add-on treatment of Lennox-Gastaut syndrome, was approved in the European Union and the United States. Initial trials showed drug-induced QT-interval shortening. The aim of our study was to evaluate the effects of rufinamide on QT intervals in patients with difficult-to-treat epilepsies.
Nineteen consecutive patients with Lennox-Gastaut syndrome and other epilepsy syndromes were included (n = 12 men; mean age 41 ± 12 years). QRS, QT, and Tpeak-Tend intervals were analyzed before and during rufinamide treatment.
The mean QT interval shortened significantly following rufinamide administration (QT interval 349 ± 23 ms vs 327 ± 17 ms; corrected QT interval 402 ± 22 ms vs 382 ± 16 ms; P = .002). Tpeak-Tend intervals were 79 ± 17 ms before and 70 ± 20 ms on treatment (P = .07). The mean reduction of the corrected QT interval was 20 ± 18 ms. During follow-up (3.04 ± 1.09 years), no adverse events including symptomatic cardiac arrhythmias or sudden cardiac deaths were observed.
QTc-interval shortening following oral rufinamide administration in a small patient group was not associated with significant clinical adverse effects. These observations nothwithstanding, the ability of rufinamide to significantly shorten the QT interval portends a potential arrhythmogenic risk that may best be guarded against by periodic electrocardiographic recordings.
PMCID: PMC3482382  PMID: 22245794
Drug-induced QT-interval shortening; Short QT syndrome; SUDEP; sudden cardiac death; Proarrhythmia; Rufinamide
3.  Specialization for underwater hearing by the tympanic middle ear of the turtle, Trachemys scripta elegans 
Turtles, like other amphibious animals, face a trade-off between terrestrial and aquatic hearing. We used laser vibrometry and auditory brainstem responses to measure their sensitivity to vibration stimuli and to airborne versus underwater sound. Turtles are most sensitive to sound underwater, and their sensitivity depends on the large middle ear, which has a compliant tympanic disc attached to the columella. Behind the disc, the middle ear is a large air-filled cavity with a volume of approximately 0.5 ml and a resonance frequency of approximately 500 Hz underwater. Laser vibrometry measurements underwater showed peak vibrations at 500–600 Hz with a maximum of 300 µm s−1 Pa−1, approximately 100 times more than the surrounding water. In air, the auditory brainstem response audiogram showed a best sensitivity to sound of 300–500 Hz. Audiograms before and after removing the skin covering reveal that the cartilaginous tympanic disc shows unchanged sensitivity, indicating that the tympanic disc, and not the overlying skin, is the key sound receiver. If air and water thresholds are compared in terms of sound intensity, thresholds in water are approximately 20–30 dB lower than in air. Therefore, this tympanic ear is specialized for underwater hearing, most probably because sound-induced pulsations of the air in the middle ear cavity drive the tympanic disc.
PMCID: PMC3367789  PMID: 22438494
underwater sound; evolution; cochlea; auditory brainstem response
4.  Hearing in the African lungfish (Protopterus annectens): pre-adaptation to pressure hearing in tetrapods? 
Biology Letters  2010;7(1):139-141.
Lungfishes are the closest living relatives of the tetrapods, and the ear of recent lungfishes resembles the tetrapod ear more than the ear of ray-finned fishes and is therefore of interest for understanding the evolution of hearing in the early tetrapods. The water-to-land transition resulted in major changes in the tetrapod ear associated with the detection of air-borne sound pressure, as evidenced by the late and independent origins of tympanic ears in all of the major tetrapod groups. To investigate lungfish pressure and vibration detection, we measured the sensitivity and frequency responses of five West African lungfish (Protopterus annectens) using brainstem potentials evoked by calibrated sound and vibration stimuli in air and water. We find that the lungfish ear has good low-frequency vibration sensitivity, like recent amphibians, but poor sensitivity to air-borne sound. The skull shows measurable vibrations above 100 Hz when stimulated by air-borne sound, but the ear is apparently insensitive at these frequencies, suggesting that the lungfish ear is neither adapted nor pre-adapted for aerial hearing. Thus, if the lungfish ear is a model of the ear of early tetrapods, their auditory sensitivity was limited to very low frequencies on land, mostly mediated by substrate-borne vibrations.
PMCID: PMC3030901  PMID: 20826468
lungfish; hearing; vibration; tetrapod; sound; evolution
5.  Synchronized Bilateral Synaptic Inputs to Drosophila melanogaster Neuropeptidergic Rest/Arousal Neurons 
Neuropeptide Pigment-Dispersing Factor (PDF)-secreting large ventrolateral neurons (lLNvs) in the Drosophila brain regulate daily patterns of rest and arousal. These bilateral wake-promoting neurons are light-responsive and integrate information from the circadian system, sleep circuits, and light environment. In order to begin to dissect the synaptic circuitry of the circadian neural network, we performed simultaneous dual whole-cell patch clamp recordings of pairs of lLNvs. Both ipsilateral and contralateral pairs of lLNvs exhibit synchronous rhythmic membrane activity with a periodicity of about 5 to 10 seconds. This rhythmic lLNv activity is blocked by tetrodotoxin (TTX) voltage-gated sodium blocker, or α-bungarotoxin (α-BuTX) nicotinic acetylcholine receptor antagonist, indicating that action potential-dependent cholinergic synaptic connections are required for rhythmic lLNv activity. Since injecting current into one neuron of the pair had no effect on the membrane activity of the other neuron of the pair, this suggests that the synchrony is due to bilateral inputs and not coupling between the pairs of lLNvs. To further elucidate the nature of these synaptic inputs to lLNvs, we blocked or activated a variety of neurotransmitter receptors and measured effects on network activity and ionic conductances. These measurements indicate the lLNvs possess excitatory nicotinic ACh receptors, inhibitory ionotropic GABAA receptors, and inhibitory ionotropic glutamate-gated chloride (GluCl) receptors. We demonstrate that cholinergic input, but not GABAergic input, is required for synchronous membrane activity, while GABA can modulate firing patterns. We conclude that neuropeptidergic lLNvs that control rest and arousal receive synchronous synaptic inputs mediated by ACh.
PMCID: PMC3125135  PMID: 21632940
synaptic activity; clock neuron; nicotinic acetylcholine receptor; GABAA receptor; glutamate-gated Cl− channel; synchronous activity
6.  Bacteremia causes hippocampal apoptosis in experimental pneumococcal meningitis 
Bacteremia and systemic complications both play important roles in brain pathophysiological alterations and the outcome of pneumococcal meningitis. Their individual contributions to the development of brain damage, however, still remain to be defined.
Using an adult rat pneumococcal meningitis model, the impact of bacteremia accompanying meningitis on the development of hippocampal injury was studied. The study comprised of the three groups: I. Meningitis (n = 11), II. meningitis with attenuated bacteremia resulting from iv injection of serotype-specific pneumococcal antibodies (n = 14), and III. uninfected controls (n = 6).
Pneumococcal meningitis resulted in a significantly higher apoptosis score 0.22 (0.18-0.35) compared to uninfected controls (0.02 (0.00-0.02), Mann Whitney test, P = 0.0003). Also, meningitis with an attenuation of bacteremia by antibody treatment resulted in significantly reduced apoptosis (0.08 (0.02-0.20), P = 0.01) as compared to meningitis.
Our results demonstrate that bacteremia accompanying meningitis plays an important role in the development of hippocampal injury in pneumococcal meningitis.
PMCID: PMC2824701  PMID: 20044936
7.  Analysis of Respiratory Sounds: State of the Art 
This paper describes state of the art, scientific publications and ongoing research related to the methods of analysis of respiratory sounds.
Methods and material:
Review of the current medical and technological literature using Pubmed and personal experience.
The study includes a description of the various techniques that are being used to collect auscultation sounds, a physical description of known pathologic sounds for which automatic detection tools were developed. Modern tools are based on artificial intelligence and on technics such as artificial neural networks, fuzzy systems, and genetic algorithms…
The next step will consist in finding new markers so as to increase the efficiency of decision aid algorithms and tools.
PMCID: PMC2990233  PMID: 21157521
state of the art; auscultation; respiratory sounds; crackles; wheezes; respiratory phase detection; spectral analysis; wavelet; respiratory phase classification; signal processing; artificial neural networks; genetic algorithm; multilayer perceptron; fuzzy rule base identification system
8.  In vivo study of experimental pneumococcal meningitis using magnetic resonance imaging 
Magnetic Resonance Imaging (MRI) methods were evaluated as a tool for the study of experimental meningitis. The identification and characterisation of pathophysiological parameters that vary during the course of the disease could be used as markers for future studies of new treatment strategies.
Rats infected intracisternally with S. pneumoniae (n = 29) or saline (n = 13) were randomized for imaging at 6, 12, 24, 30, 36, 42 or 48 hours after infection. T1W, T2W, quantitative diffusion, and post contrast T1W images were acquired at 4.7 T. Dynamic MRI (dMRI) was used to evaluate blood-brain-barrier (BBB) permeability and to obtain a measure of cerebral and muscle perfusion. Clinical- and motor scores, bacterial counts in CSF and blood, and WBC counts in CSF were measured.
MR images and dMRI revealed the development of a highly significant increase in BBB permeability (P < 0.002) and ventricle size (P < 0.0001) among infected rats. Clinical disease severity was closely related to ventricle expansion (P = 0.024).
Changes in brain water distribution, assessed by ADC, and categorization of brain 'perfusion' by cortex ΔSI(bolus) were subject to increased inter-rat variation as the disease progressed, but without overall differences compared to uninfected rats (P > 0.05). Areas of well-'perfused' muscle decreased with the progression of infection indicative of septicaemia (P = 0.05).
The evolution of bacterial meningitis was successfully followed in-vivo with MRI. Increasing BBB-breakdown and ventricle size was observed in rats with meningitis whereas changes in brain water distribution were heterogeneous. MRI will be a valuable technique for future studies aiming at evaluating or optimizing adjunctive treatments
PMCID: PMC2253532  PMID: 18194516
9.  Danish Integrated Antimicrobial Resistance Monitoring and Research Program 
Emerging Infectious Diseases  2007;13(11):1633-1639.
This program has led to changes in the use of antimicrobial agents in Denmark and other countries.
Resistance to antimicrobial agents is an emerging problem worldwide. Awareness of the undesirable consequences of its widespread occurrence has led to the initiation of antimicrobial agent resistance monitoring programs in several countries. In 1995, Denmark was the first country to establish a systematic and continuous monitoring program of antimicrobial drug consumption and antimicrobial agent resistance in animals, food, and humans, the Danish Integrated Antimicrobial Resistance Monitoring and Research Program (DANMAP). Monitoring of antimicrobial drug resistance and a range of research activities related to DANMAP have contributed to restrictions or bans of use of several antimicrobial agents in food animals in Denmark and other European Union countries.
PMCID: PMC3375779  PMID: 18217544
Antibiotics; resistance; humans; epidemiology; DANMAP; perspective
10.  Influence of the blood bacterial load on the meningeal inflammatory response in Streptococcus pneumoniae meningitis 
Despite bacteraemia is present in the majority of patients with pneumococcal, little is known about the influence of the systemic infection on the meningeal inflammatory response.
To explore the role of systemic infection on the meningeal inflammation, experimental meningitis was induced by intracisternal injection of ~1 × 106 CFU Streptococcus pneumoniae, type 3, and the 26 rabbits were either provided with ~1 × 106 CFU S. pneumoniae intravenously at 0 hour ("bacteraemic" rabbits, n = 9), immunized with paraformaldehyde-killed S. pneumoniae for 5 weeks prior to the experiment ("immunized" rabbits", n = 8), or not treated further ("control" rabbits, n = 9). WBC and bacterial concentrations were determined in CSF and blood every second hour during a 16 hours study period together with CSF IL-8 and protein levels. We also studied CSF and blood WBC levels in 153 pneumococcal meningitis patients with and without presence of bacteraemia.
As designed, blood bacterial concentrations were significantly different among three experimental groups during the 16 hours study period (Kruskal Wallis test, P < 0.05), whereas no differences in CSF bacterial levels were observed (P > 0.05). Blood WBC decreased in bacteraemic rabbits between ~10–16 hours after the bacterial inoculation in contrast to an increase for both the immunized rabbits and controls (P < 0.05). The CSF pleocytosis was attenuated in bacteraemic rabbits as compared to the two other groups between 12–16 hours from time of infection (P < 0.017), despite accelerated CSF IL-8 levels in bacteraemic rabbits.
In patients with pneumococcal meningitis, no significant difference in CSF WBC was observed between patients with or without bacteraemia at admission (n = 103, 1740 cells/μL (123–4032) vs. n = 50, 1961 cells/μL (673–5182), respectively, P = 0.18), but there was a significant correlation between CSF and blood WBC (n = 127, Spearman rho = 0.234, P = 0.008).
Our results suggest that a decrease in peripheral WBC induced by enhanced bacteraemia in pneumococcal meningitis results in an attenuated CSF pleocytosis.
PMCID: PMC1475592  PMID: 16643642
11.  Attenuation of the Bacterial Load in Blood by Pretreatment with Granulocyte-Colony-Stimulating Factor Protects Rats from Fatal Outcome and Brain Damage during Streptococcus pneumoniae Meningitis  
Infection and Immunity  2004;72(8):4647-4653.
A model of pneumococcal meningitis in young adult rats receiving antibiotics once the infection was established was developed. The intent was to mimic clinical and histopathological features of pneumococcal meningitis in humans. The primary aim of the present study was to evaluate whether medical boosting of the peripheral neutrophil count affected the outcome of the meningitis. The risk of terminal illness over the first 7 days after infection was significantly reduced for rats who had elevated peripheral white blood cell counts after receiving granulocyte-colony-stimulating factor (G-CSF) prior to the infection compared to that for untreated rats (P = 0.039 by the log rank test). The improved outcome was associated with reduced signs of cerebral cortical damage (P = 0.008). Furthermore, the beneficial effects of G-CSF were associated with reduced bacterial loads in the cerebrospinal fluid (median, 1.1 × 105 versus 2.9 × 105 CFU/ml; P = 0.023) and in blood (median, 2.9 × 102 versus 6.3 × 102 CFU/ml; P = 0.024), as well as attenuated pleocytosis (median, 800 × 106 versus 1,231 × 106 cells/liter; P = 0.025), 24 h after the infection. Conversely, initiation of G-CSF therapy 28 h postinfection did not alter the clinical or histological outcome relative to that for non-G-CSF-treated rats. The magnitude of bacteremia and pretreatment with G-CSF were found to be prognostic factors for both outcome and brain damage. In summary, elevated neutrophil levels prior to the development of meningitis result in reduced risks of death and brain damage. This beneficial effect is most likely achieved through improved control of the systemic disease.
PMCID: PMC470620  PMID: 15271925
12.  CD4+ T-Cell-Mediated Antiviral Protection of the Upper Respiratory Tract in BALB/c Mice following Parenteral Immunization with a Recombinant Respiratory Syncytial Virus G Protein Fragment 
Journal of Virology  2000;74(8):3455-3463.
We analyzed the protective mechanisms induced against respiratory syncytial virus subgroup A (RSV-A) infection in the lower and upper respiratory tracts (LRT and URT) of BALB/c mice after intraperitoneal immunization with a recombinant fusion protein incorporating residues 130 to 230 of RSV-A G protein (BBG2Na). Mother-to-offspring antibody (Ab) transfer and adoptive transfer of BBG2Na-primed B cells into SCID mice demonstrated that Abs are important for LRT protection but have no effect on URT infection. In contrast, RSV-A clearance in the URT was achieved in a dose-dependent fashion after adoptive transfer of BBG2Na-primed T cells, while it was abolished in BBG2Na-immunized mice upon in vivo depletion of CD4+, but not CD8+, T cells. Furthermore, the conserved RSV-A G protein cysteines and residues 193 and 194, overlapping the recently identified T helper cell epitope on the G protein (P. W. Tebbey et al., J. Exp. Med. 188:1967–1972, 1998), were found to be essential for URT but not LRT protection. Taken together, these results demonstrate for the first time that CD4+ T cells induced upon parenteral immunization with an RSV G protein fragment play a critical role in URT protection of normal mice against RSV infection.
PMCID: PMC111852  PMID: 10729118

Results 1-12 (12)