Assessing viral metagenomics for the diagnosis of acute undifferentiated fever in returned travellers: a multicenter cohort study.

BACKGROUND: Up to 45% of febrile returning travellers remain undiagnosed after a thorough diagnostic work-up, even at referral centres. Although metagenomic next-generation sequencing (mNGS) has emerged as a promising tool, evidence of its usefulness in imported fever is very limited. METHODS: Travellers returning with fever were prospectively recruited in three referral clinics from November 2017 to November 2019. Unbiased mNGS optimised for virus detection was performed on serum samples of participants with acute undifferentiated febrile illness (AUFI), and results were compared to those obtained by reference diagnostic methods (RDM). RESULTS: Among 507 returned febrile travellers, 433(85.4%) presented with AUFI. Dengue virus (n = 86) and Plasmodium spp. (n = 83) were the most common causes of fever. 103/433(23.8%) AUFI remained undiagnosed at the end of the follow-up.Metagenomic next-generation sequencing unveiled potentially pathogenic microorganisms in 196/433(38.7%) AUFI. mNGS identifications were more common in patients with a shorter duration of fever (42.3% in ≤5 days vs 28.7% in >5 days, P = 0.005). Potential causes of fever were revealed in 25/103(24.2%) undiagnosed AUFI and 5/23(21.7%) travellers with severe undiagnosed AUFI. Missed severe aetiologies included eight bacterial identifications and one co-infection of B19 parvovirus and Aspergillus spp.Additional identifications indicating possible co-infections occurred in 29/316(9.2%) travellers with AUFI, and in 11/128(8.6%) travellers with severe AUFI, who had received a diagnosis through RDM. The most common co-infections detected in severe AUFI were caused by Gram-negative bacteria. Serum mNGS was unable to detect >50% of infectious diagnoses achieved by RDM and also yielded 607 non-pathogenic identifications. DISCUSSION: mNGS of serum can be a valuable diagnostic tool for selected travellers with undiagnosed AUFI or severe disease in addition to reference diagnostic techniques, especially during the first days of symptoms. Nevertheless, mNGS results interpretation presents a great challenge. Further studies evaluating the performance of mNGS using different sample types and protocols tailored to non-viral agents are needed.

Persistent Postpartum Pain - A Somatic and Psychologic Perfect Storm.

Persistent postpartum pain is common and has a complex etiology. It has both somatic and psychosocial provoking factors and has both functional and psychological ramifications following childbirth. Pain that limits the functional capacity of a person who has the daunting task to take care of all the demands of managing a growing newborn and infant can have debilitating consequences for several people simultaneously. We will review the incidence of persistent postpartum pain, analyze the risk factors, and discuss obstetric, anesthetic, and psychological tools for prevention and management. Based on the current knowledge, early antenatal screening and management is described as the most likely measure to identify patients at risk for persistent postpartum pain. Such antenatal management should be based on the close collaboration between obstetricians, anesthesiologists, and psychologists to tailor peripartum pain management and psychological support-based individual needs.

INNOVATIVE TECHNIQUES AND NEW INSIGHTS: Studying cardiac ionic currents and action potentials in physiologically relevant conditions.

Cardiac arrhythmias are associated with various forms of heart diseases. Ventricular arrhythmias present a significant risk for sudden cardiac death. Atrial fibrillations predispose to blood clots leading to stroke and heart attack. Scientists have been developing patch-clamp technology to study ion channels and action potentials (APs) underlying cardiac excitation and arrhythmias. Beyond the traditional patch-clamp techniques, innovative new techniques were developed for studying complex arrhythmia mechanisms. Here we review the recent development of methods including AP-Clamp, Dynamic Clamp, AP-Clamp Sequential Dissection, and Patch-Clamp-in-Gel. These methods provide powerful tools for researchers to decipher how the dynamic systems in excitation-Ca2+ signaling-contraction feedforward and feedback to control cardiac function and how their dysregulations lead to heart diseases.

Las arritmias cardiacas están asociadas a diferentes tipos de enfermedad cardiaca. Las arritmias ventriculares constituyen un alto riesgo de muerte súbita. La fibrilación auricular predispone a coágulos sanguíneos que pueden producir accidentes cerebrovasculares e infarto miocárdico. Los científicos han desarrollado la técnica de patch-clamp para estudiar los canales iónicos y los potenciales de acción (PAs), que constituyen la base de la excitación y las arritmias cardiacas. Además de las clásicas técnicas de patch-clamp, se desarrollaron técnicas innovativas para estudiar los mecanismos complejos de las arritmias. En este trabajo, describimos diferentes métodos recientemente desarrollados tales como AP-clamp ("clampeo" del PA), Dynamic Clamp ("clampeo" dinámico), AP-Clamp Sequential Dissection, (disección secuencial del "clampeo" del AP), y Patch-Clamp-in-Gel (Patch clamp en gel). Estos métodos constituyen herramientas poderosas para descifrar cómo los sistemas dinámicos que constituyen la excitación-las señales de Ca2+ y la contracción, se retroalimentan para controlar la función cardiaca y cómo sus alteraciones llevan a la enfermedad cardiaca.

A randomized controlled simulation trial comparing video-assisted with telephone-assisted and unassisted cardiopulmonary resuscitation performed by non-healthcare university students.

Our randomized controlled simulation study aimed to compare the CPR quality, time-related factors, attitude and self-assessment of non-healthcare university students (aged 18-25) compared video-assisted (V-CPR, n = 50) with telephone-assisted (T-CPR, n = 49) and unassisted (U-CPR, n = 48) CPR in a simulation setting. Regarding to chest compression depth, no difference was found between the three groups (p = 0.065): 41.8 mm, SD = 9.9 in the V-CPR; 35.9 mm, SD = 11.6 in the T-CPR; and 39.4 mm, SD = 15.6 in the U-CPR group. The mean chest compression rate was the best in the V-CPR group (100.9 min-1, SD = 17.1) which was superior to the T-CPR (82.4 min-1, SD = 35.4; p = 0.005), and the U-CPR (84.2 min-1, SD = 30.6; p = 0.013) groups. The overall proportion of correct hand position was the highest in the V-CPR group (48, 96%), compared to the T-CPR (28, 57.1%; p = 0.001), and the U-CPR (34, 70.8%; p = 0.001) groups. V-CPR led to a delay in the time to the first chest compression compared with the U-CPR group (77.5 s, SD = 19.2 vs. 31.3 s, SD = 13.3, p < 0.001). Although V-CPR technology holds the potential to improve overall CPR quality, the importance of appropriate chest compression depth should be emphasized in training for laypeople and dispatchers, as well. Our study was registered at ClinicalTrials.gov (NCT05639868, 06/12/2022).

Selective Inhibition of Cardiac Late Na+ Current Is Based on Fast Offset Kinetics of the Inhibitor.

The present study was designed to test the hypothesis that the selectivity of blocking the late Na+ current (INaL) over the peak Na+ current (INaP) is related to the fast offset kinetics of the Na+ channel inhibitor. Therefore, the effects of 1 µM GS967 (INaL inhibitor), 20 µM mexiletine (I/B antiarrhythmic) and 10 µM quinidine (I/A antiarrhythmic) on INaL and INaP were compared in canine ventricular myocardium. INaP was estimated as the maximum velocity of action potential upstroke (V+max). Equal amounts of INaL were dissected by the applied drug concentrations under APVC conditions. The inhibition of INaL by mexiletine and quinidine was comparable under a conventional voltage clamp, while both were smaller than the inhibitory effect of GS967. Under steady-state conditions, the V+max block at the physiological cycle length of 700 ms was 2.3% for GS967, 11.4% for mexiletine and 26.2% for quinidine. The respective offset time constants were 110 ± 6 ms, 456 ± 284 ms and 7.2 ± 0.9 s. These results reveal an inverse relationship between the offset time constant and the selectivity of INaL over INaP inhibition without any influence of the onset rate constant. It is concluded that the selective inhibition of INaL over INaP is related to the fast offset kinetics of the Na+ channel inhibitor.

ABT-333 (Dasabuvir) Increases Action Potential Duration and Provokes Early Afterdepolarizations in Canine Left Ventricular Cells via Inhibition of IKr.

ABT-333 (dasabuvir) is an antiviral agent used in hepatitis C treatment. The molecule, similarly to some inhibitors of hERG channels, responsible for the delayed rectifier potassium current (IKr), contains the methanesulfonamide group. Reduced IKr current leads to long QT syndrome and early afterdepolarizations (EADs), therefore potentially causing life-threatening arrhythmias and sudden cardiac death. Our goal was to investigate the acute effects of ABT-333 in enzymatically isolated canine left ventricular myocardial cells. Action potentials (APs) and ion currents were recorded with a sharp microelectrode technique and whole-cell patch clamp, respectively. Application of 1 µM ABT-333 prolonged the AP in a reversible manner. The maximal rates of phases 0 and 1 were irreversibly decreased. Higher ABT-333 concentrations caused larger AP prolongation, elevation of the early plateau potential, and reduction of maximal rates of phases 0, 1, and 3. EADs occurred in some cells in 3-30 µM ABT-333 concentrations. The 10 µM ABT-333-sensitive current, recorded with AP voltage clamp, contained a late outward component corresponding to IKr and an early outward one corresponding to transient outward potassium current (Ito). ABT-333 reduced hERG-channel-mediated ion current in a concentration-dependent, partially reversible manner with a half-inhibitory concentration of 3.2 µM. As the therapeutic plasma concentration of ABT-333 is 1 nM, the arrhythmic risk of ABT-333 is very low, even in the case of drug overdose.

Conductance Changes of Na+ Channels during the Late Na+ Current Flowing under Action Potential Voltage Clamp Conditions in Canine, Rabbit, and Guinea Pig Ventricular Myocytes.

Late sodium current (INa,late) is an important inward current contributing to the plateau phase of the action potential (AP) in the mammalian heart. Although INa,late is considered as a possible target for antiarrhythmic agents, several aspects of this current remained hidden. In this work, the profile of INa,late, together with the respective conductance changes (GNa,late), were studied and compared in rabbit, canine, and guinea pig ventricular myocytes using the action potential voltage clamp (APVC) technique. In canine and rabbit myocytes, the density of INa,late was relatively stable during the plateau and decreased only along terminal repolarization of the AP, while GNa,late decreased monotonically. In contrast, INa,late increased monotonically, while GNa,late remained largely unchanged during the AP in guinea pig. The estimated slow inactivation of Na+ channels was much slower in guinea pig than in canine or rabbit myocytes. The characteristics of canine INa,late and GNa,late were not altered by using command APs recorded from rabbit or guinea pig myocytes, indicating that the different shapes of the current profiles are related to genuine interspecies differences in the gating of INa,late. Both INa,late and GNa,late decreased in canine myocytes when the intracellular Ca2+ concentration was reduced either by the extracellular application of 1 µM nisoldipine or by the intracellular application of BAPTA. Finally, a comparison of the INa,late and GNa,late profiles induced by the toxin of Anemonia sulcata (ATX-II) in canine and guinea pig myocytes revealed profound differences between the two species: in dog, the ATX-II induced INa,late and GNa,late showed kinetics similar to those observed with the native current, while in guinea pig, the ATX-II induced GNa,late increased during the AP. Our results show that there are notable interspecies differences in the gating kinetics of INa,late that cannot be explained by differences in AP morphology. These differences must be considered when interpreting the INa,late results obtained in guinea pig.

Omecamtiv mecarbil augments cardiomyocyte contractile activity both at resting and systolic Ca2+ levels.

AIMS: Heart failure with reduced ejection fraction (HFrEF) is a disease with high mortality and morbidity. Recent positive inotropic drug developments focused on cardiac myofilaments, that is, direct activators of the myosin molecule and Ca2+ sensitizers for patients with advanced HFrEF. Omecamtiv mecarbil (OM) is the first direct myosin activator with promising results in clinical studies. Here, we aimed to elucidate the cellular mechanisms of the positive inotropic effect of OM in a comparative in vitro investigation where Ca2+ -sensitizing positive inotropic agents with distinct mechanisms of action [EMD 53998 (EMD), which also docks on the myosin molecule, and levosimendan (Levo), which binds to troponin C] were included. METHODS: Enzymatically isolated canine cardiomyocytes with intact cell membranes were loaded with Fura-2AM, a Ca2+ -sensitive, ratiometric, fluorescent dye. Changes in sarcomere length (SL) and intracellular Ca2+ concentration were recorded in parallel at room temperature, whereas cardiomyocyte contractions were evoked by field stimulation at 0.1 Hz in the presence of different OM, EMD, or Levo concentrations. RESULTS: SL was reduced by about 23% or 9% in the presence of 1 µM OM or 1 µM EMD in the absence of electrical stimulation, whereas 1 µM Levo had no effect on resting SL. Fractional sarcomere shortening was increased by 1 µM EMD or 1 µM Levo to about 152%, but only to about 128% in the presence of 0.03 µM OM. At higher OM concentrations, no significant increase in fractional sarcomere shortening could be recorded. Contraction durations largely increased, whereas the kinetics of contractions and relaxations decreased with increasing OM concentrations. One-micromole EMD or 1 µM Levo had no effects on contraction durations. One-micromole Levo, but not 1 µM EMD, accelerated the kinetics of cardiomyocyte contractions and relaxations. Ca2+ transient amplitudes were unaffected by all treatments. CONCLUSIONS: Our data revealed major distinctions between the cellular effects of myofilament targeted agents (OM, EMD, or Levo) depending on their target proteins and binding sites, although they were compatible with the involvement of Ca2+ -sensitizing mechanisms for all three drugs. Significant part of the cardiotonic effect of OM relates to the prolongation of systolic contraction in combination with its Ca2+ -sensitizing effect.

Simplified coronary flow reserve calculations based on three-dimensional coronary reconstruction and intracoronary pressure data.

BACKGROUND: Measurements of fractional flow reserve (FFR) and/or coronary flow reserve (CFR) are widely used for hemodynamic characterization of coronary lesions. The frequent combination of the epicardial and microvascular disease may indicate a need for complex hemodynamic evaluation of coronary lesions. This study aims at validating the calculation of CFR based on a simple hemodynamic model to detailed computational fluid dynamics (CFD) analysis. METHODS: Three-dimensional (3D) morphological data and pressure values from FFR measurements were used to calculate the target vessel. Nine patients with one intermediate stenosis each, measured by pressure wire, were included in this study. RESULTS: A correlation was found between the determined CFR from simple equations and from a steady flow simulation (r = 0.984, p < 10-5). There was a significant correlation between the CFR values calculated by transient and steady flow simulations (r = 0.94, p < 10-3). CONCLUSIONS: Feasibility was demonstrated of a simple hemodynamic calculation of CFR based on 3D-angiography and intracoronary pressure measurements. A simultaneous determination of both the FFR and CFR values provides the capability to diagnose microvascular dysfunction: the CFR/FFR ratio characterizes the microvascular reserve.

How effective are chest compressions when wearing mask? A randomised simulation study among first-year health care students during the COVID-19 pandemic.

BACKGROUND: The resuscitation guidelines provided for the COVID-19 pandemic strongly recommended wearing personal protective equipment. The current study aimed to evaluate and compare the effectiveness of chest compressions and the level of fatigue while wearing two different types of mask (surgical vs. cloth). METHODS: A randomized, non-inferiority, simulation study was conducted. Participants were randomised into two groups: surgical mask group (n = 108) and cloth mask group (n = 108). The effectiveness (depth and rate) of chest compressions was measured within a 2-min continuous chest-compression-only CPR session. Data were collected through an AMBU CPR Software, a questionnaire, recording vital parameters, and using Borg-scale related to fatigue (before and after the simulation). For further analysis the 2-min session was segmented into 30-s intervals. RESULTS: Two hundred sixteen first-year health care students participated in our study. No significant difference was measured between the surgical mask and cloth mask groups in chest compression depth (44.49 ± 10.03 mm vs. 45.77 ± 10.77 mm), rate (113.34 ± 17.76/min vs. 111.23 ± 17.51/min), and the level of fatigue (5.72 ± 1.69 vs. 5.56 ± 1.67) (p > 0.05 in every cases). Significant decrease was found in chest compression depth between the first 30-s interval and the second, third, and fourth intervals (p < 0.01). CONCLUSION: The effectiveness of chest compressions (depth and rate) was non-inferior when wearing cloth mask compared to wearing surgical mask. However, the effectiveness of chest compressions decreased significantly in both groups during the 2-min chest-compression-only CPR session and did not reach the appropriate chest compression depth range recommended by the ERC.

Exploring the Coordination of Cardiac Ion Channels With Action Potential Clamp Technique.

The patch clamp technique underwent continual advancement and developed numerous variants in cardiac electrophysiology since its introduction in the late 1970s. In the beginning, the capability of the technique was limited to recording one single current from one cell stimulated with a rectangular command pulse. Since that time, the technique has been extended to record multiple currents under various command pulses including action potential. The current review summarizes the development of the patch clamp technique in cardiac electrophysiology with special focus on the potential applications in integrative physiology.

Late Sodium Current of the Heart: Where Do We Stand and Where Are We Going?

Late sodium current has long been linked to dysrhythmia and contractile malfunction in the heart. Despite the increasing body of accumulating information on the subject, our understanding of its role in normal or pathologic states is not complete. Even though the role of late sodium current in shaping action potential under physiologic circumstances is debated, it's unquestioned role in arrhythmogenesis keeps it in the focus of research. Transgenic mouse models and isoform-specific pharmacological tools have proved useful in understanding the mechanism of late sodium current in health and disease. This review will outline the mechanism and function of cardiac late sodium current with special focus on the recent advances of the area.

Real-time amino acid and glucose monitoring system for the automatic control of nutrient feeding in CHO cell culture using Raman spectroscopy.

An innovative, Raman spectroscopy-based monitoring and control system is introduced in this paper for designing dynamic feeding strategies that allow the maintenance of key cellular nutrients at an ideal level in Chinese hamster ovary cell culture. The Partial Least Squares calibration models built for glucose, lactate and 16 (out of 20) individual amino acids had very good predictive power with low root mean square errors values and high square correlation coefficients. The developed models used for real-time measurement of nutrient and by-product concentrations allowed us to gain better insight into the metabolic behavior and nutritional consumption of cells. To establish a more beneficial nutritional environment for the cells, two types of dynamic feeding strategies were used to control the delivery of two-part multi-component feed media according to the prediction of Raman models (glucose or arginine). As a result, instead of high fluctuations, the nutrients (glucose together with amino acids) were maintained at the desired level providing a more balanced environment for the cells. Moreover, the use of amino acid-based feeding control enabled to prevent the excessive nutrient replenishment and was economically beneficial by significantly reducing the amount of supplied feed medium compared to the glucose-based dynamic fed culture.

Pharmacological Modulation and (Patho)Physiological Roles of TRPM4 Channel-Part 1: Modulation of TRPM4.

Transient receptor potential melastatin 4 is a unique member of the TRPM protein family and, similarly to TRPM5, is Ca2+-sensitive and permeable to monovalent but not divalent cations. It is widely expressed in many organs and is involved in several functions by regulating the membrane potential and Ca2+ homeostasis in both excitable and non-excitable cells. This part of the review discusses the pharmacological modulation of TRPM4 by listing, comparing, and describing both endogenous and exogenous activators and inhibitors of the ion channel. Moreover, other strategies used to study TRPM4 functions are listed and described. These strategies include siRNA-mediated silencing of TRPM4, dominant-negative TRPM4 variants, and anti-TRPM4 antibodies. TRPM4 is receiving more and more attention and is likely to be the topic of research in the future.

The Novel Cardiac Myosin Activator Danicamtiv Improves Cardiac Systolic Function at the Expense of Diastolic Dysfunction In Vitro and In Vivo: Implications for Clinical Applications.

Recent cardiotropic drug developments have focused on cardiac myofilaments. Danicamtiv, the second direct myosin activator, has achieved encouraging results in preclinical and clinical studies, thus implicating its potential applicability in the treatment of heart failure with reduced ejection fraction (HFrEF). Here, we analyzed the inotropic effects of danicamtiv in detail. To this end, changes in sarcomere length and intracellular Ca2+ levels were monitored in parallel, in enzymatically isolated canine cardiomyocytes, and detailed echocardiographic examinations were performed in anesthetized rats in the absence or presence of danicamtiv. The systolic and diastolic sarcomere lengths decreased; contraction and relaxation kinetics slowed down with increasing danicamtiv concentrations without changes in intracellular Ca2+ transients in vitro. Danicamtiv evoked remarkable increases in left ventricular ejection fraction and fractional shortening, also reflected by changes in systolic strain. Nevertheless, the systolic ejection time was significantly prolonged, the ratio of diastolic to systolic duration was reduced, and signs of diastolic dysfunction were also observed upon danicamtiv treatment in vivo. Taken together, danicamtiv improves cardiac systolic function, but it can also limit diastolic performance, especially at high drug concentrations.

Extracorporeal Cytokine Removal in Critically Ill COVID-19 Patients: A Case Series.

Introduction: Extracorporeal hemoadsorption (HA) is a potential adjunctive therapy in severe cases of COVID-19 associated pneumonia. In this retrospective study we report data from critically ill patients treated with HA during the first and second wave of the pandemic. Patients and Methods: All patients, who received HA therapy with CytoSorb within the first 96 h of intensive care unit (ICU) admission without hospital-acquired bacterial superinfection, were included. Clinical and laboratory data were collected: on admission, before (TB) and after (TA) HA therapy. Results: Out of the 367 COVID-19 cases, 13 patients were treated with CytoSorb, also requiring mechanical ventilation and renal replacement therapy. All patients were alive at the end of HA, but only 3 survived hospital stay. From TB-TA there was a tendency of decreasing norepinephrine requirement: 193.7 [IQR: 34.8-270.4] to 50.2 [6.5-243.5] ug/kg/day and increasing PaO2/FiO2 ratio 127.8 (95% CI: 96.0-159.6) to 155.0 (115.3-194.6) mmHg but they did not reach statistical significance (p = 0.14 and 0.58, respectively). Treatment related adverse events were not reported. Conclusion: The treatment was well-tolerated, and there was a tendency toward an improvement in vasopressor need and oxygenation during the course of HA. These observations render the need for prospective randomized trials.

Late Na+ Current Is [Ca2+]i-Dependent in Canine Ventricular Myocytes.

Enhancement of the late sodium current (INaL) increases arrhythmia propensity in the heart, whereas suppression of the current is antiarrhythmic. In the present study, we investigated INaL in canine ventricular cardiomyocytes under action potential voltage-clamp conditions using the selective Na+ channel inhibitors GS967 and tetrodotoxin. Both 1 µM GS967 and 10 µM tetrodotoxin dissected largely similar inward currents. The amplitude and integral of the GS967-sensitive current was significantly smaller after the reduction of intracellular Ca2+ concentration ([Ca2+]i) either by superfusion of the cells with 1 µM nisoldipine or by intracellular application of 10 mM BAPTA. Inhibiting calcium/calmodulin-dependent protein kinase II (CaMKII) by KN-93 or the autocamtide-2-related inhibitor peptide similarly reduced the amplitude and integral of INaL. Action potential duration was shortened in a reverse rate-dependent manner and the plateau potential was depressed by GS967. This GS967-induced depression of plateau was reduced by pretreatment of the cells with BAPTA-AM. We conclude that (1) INaL depends on the magnitude of [Ca2+]i in canine ventricular cells, (2) this [Ca2+]i-dependence of INaL is mediated by the Ca2+-dependent activation of CaMKII, and (3) INaL is augmented by the baseline CaMKII activity.