New Developments in Hemodynamic Monitoring.

Hemodynamic monitoring is an essential part of the perioperative management of the cardiovascular patient. It helps to detect hemodynamic alterations, diagnose their underlying causes, and optimize oxygen delivery to the tissues. Furthermore, hemodynamic monitoring is necessary to evaluate the adequacy of therapeutic interventions such as volume expansion or vasoactive medications. Recent developments include the move from static to dynamic variables to assess conditions such as cardiac preload and fluid responsiveness and the transition to less-invasive or even noninvasive monitoring techniques, at least in the perioperative setting. This review describes the available techniques that currently are being used in the care of the cardiovascular patient and discusses their strengths and limitations. Even though the thermodilution method remains the gold standard for measuring cardiac output (CO), the use of the pulmonary artery catheter has declined over the last decades, even in the setting of cardiovascular anesthesia. The transpulmonary thermodilution method, in addition to accurately measuring CO, provides the user with some additional helpful variables, of which extravascular lung water is probably the most interesting. Less-invasive monitoring techniques use, for example, pulse contour analysis to originate flow-derived variables such as stroke volume and CO from the arterial pressure signal, or they may measure the velocity-time integral in the descending aorta to estimate the stroke volume, using, for example, the esophageal Doppler. Completely noninvasive methods such as the volume clamp method use finger cuffs to reconstruct the arterial pressure waveform, from which stroke volume and CO are calculated. All of these less-invasive CO monitoring devices have percentage errors around 40% compared with reference methods (thermodilution), meaning that the values are not interchangeable.

International Liver Transplant Society Practice Guidelines: Diagnosis and Management of Hepatopulmonary Syndrome and Portopulmonary Hypertension.

Two distinct pulmonary vascular disorders, hepatopulmonary syndrome (HPS) and portopulmonary hypertension (POPH) may occur as a consequence of hepatic parenchymal or vascular abnormalities. HPS and POPH have major clinical implications for liver transplantation. A European Respiratory Society Task Force on Pulmonary-Hepatic Disorders convened in 2002 to standardize the diagnosis and guide management of these disorders. These International Liver Transplant Society diagnostic and management guidelines are based on that task force consensus and should continue to evolve as clinical experience dictates. Based on a review of over 1000 published HPS and POPH articles identified via a MEDLINE search (1985-2015), clinical guidelines were based on, selected single care reports, small series, registries, databases, and expert opinion. The paucity of randomized, controlled trials in either of these disorders was noted. Guidelines are presented in 5 parts; I. Definitions/Diagnostic criteria; II. Hepatopulmonary syndrome; III. Portopulmonary hypertension; IV. Implications for liver transplantation; and V. Suggestions for future clinical research.

An image-based algorithm for precise and accurate high throughput assessment of drug activity against the human parasite Trypanosoma cruzi.

We present a customized high content (image-based) and high throughput screening algorithm for the quantification of Trypanosoma cruzi infection in host cells. Based solely on DNA staining and single-channel images, the algorithm precisely segments and identifies the nuclei and cytoplasm of mammalian host cells as well as the intracellular parasites infecting the cells. The algorithm outputs statistical parameters including the total number of cells, number of infected cells and the total number of parasites per image, the average number of parasites per infected cell, and the infection ratio (defined as the number of infected cells divided by the total number of cells). Accurate and precise estimation of these parameters allow for both quantification of compound activity against parasites, as well as the compound cytotoxicity, thus eliminating the need for an additional toxicity-assay, hereby reducing screening costs significantly. We validate the performance of the algorithm using two known drugs against T.cruzi: Benznidazole and Nifurtimox. Also, we have checked the performance of the cell detection with manual inspection of the images. Finally, from the titration of the two compounds, we confirm that the algorithm provides the expected half maximal effective concentration (EC50) of the anti-T. cruzi activity.

Dexmedetomidine infusion for analgesia up to 48 hours after lung surgery performed by lateral thoracotomy.

Patients undergoing a lateral thoracotomy for pulmonary resection have moderate to severe pain postoperatively that is often treated with opioids. Opioid side effects such as respiratory depression can be devastating in patients with already compromised respiratory function. This prospective double-blinded clinical trial examined the analgesic effects and safety of a dexmedetomidine infusion for postthoracotomy patients when administered on a telemetry nursing floor, 24 to 48 hours after surgery, to determine if the drug's known early opioid-sparing properties were maintained. Thirty-eight thoracotomy patients were administered dexmedetomidine intraoperatively and overnight postoperatively and then randomized to receive placebo or dexmedetomidine titrated from 0.1 to 0.5 µg·kg·h(-1) the day following surgery for up to 24 hours on a telemetry floor. Opioids via a patient-controlled analgesia pump were available for both groups, and vital signs including transcutaneous carbon dioxide, pulse oximetry, respiratory rate, and pain and sedation scores were monitored. The dexmedetomidine group used 41% less opioids but achieved pain scores equal to those of the placebo group. The mean heart rate and systolic blood pressure were lower in the dexmedetomidine group but sedation scores were better. The mean respiratory rate and oxygen saturation were similar in the two groups. Mild hypercarbia occurred in both groups, but periods of significant respiratory depression were noted only in the placebo group. Significant hypotension was noted in one patient in the dexmedetomidine group in conjunction with concomitant administration of a beta-blocker agent. The placebo group reported a higher number of opioid-related adverse events. In conclusion, the known opioid-sparing properties of dexmedetomidine in the immediate postoperative period are maintained over 48 hours.

Sedation levels during propofol administration for outpatient colonoscopies.

The levels of sedation required for patients to comfortably undergo colonoscopy with propofol were examined. One hundred patients undergoing colonoscopy with propofol were enrolled. In addition to standard-of-care monitoring, sedation level was monitored with the Patient State Index (PSI) obtained from a brain function monitor, transcutaneous carbon dioxide (tcpCO2) was monitored with the TCM TOSCA monitor, and end-tidal carbon dioxide was monitored via nasal cannula. The Ramsay Sedation Score (RSS) was also assessed and recorded. After baseline data were obtained from the first 40 consecutive patients enrolled in the study, the remaining 60 patients were randomized into two groups. In one group the PSI value was blinded from the anesthesiologist and in the second group the PSI was visible and the impact of this information on the management of the sedation was analyzed. Overall 96% of patients reached levels of deep sedation and 89% reached levels of general anesthesia. When comparing the blinded to PSI versus unblinded groups, the blinded group had a significantly lower PSI and higher RSS and tcpCO2, indicating the blinded group was maintained at a deeper sedation level with more respiratory compromise than the unblinded group. Patients undergoing colonoscopy under propofol sedation delivered by a bolus technique are frequently taken to levels of general anesthesia and are at risk for respiratory depression, airway obstruction, and hemodynamic compromise.

Identification of novel compounds inhibiting chikungunya virus-induced cell death by high throughput screening of a kinase inhibitor library.

Chikungunya virus (CHIKV) is a mosquito-borne arthrogenic alphavirus that causes acute febrile illness in humans accompanied by joint pains and in many cases, persistent arthralgia lasting weeks to years. The re-emergence of CHIKV has resulted in numerous outbreaks in the eastern hemisphere, and threatens to expand in the foreseeable future. Unfortunately, no effective treatment is currently available. The present study reports the use of resazurin in a cell-based high-throughput assay, and an image-based high-content assay to identify and characterize inhibitors of CHIKV-infection in vitro. CHIKV is a highly cytopathic virus that rapidly kills infected cells. Thus, cell viability of HuH-7 cells infected with CHIKV in the presence of compounds was determined by measuring metabolic reduction of resazurin to identify inhibitors of CHIKV-associated cell death. A kinase inhibitor library of 4,000 compounds was screened against CHIKV infection of HuH-7 cells using the resazurin reduction assay, and the cell toxicity was also measured in non-infected cells. Seventy-two compounds showing ≥50% inhibition property against CHIKV at 10 µM were selected as primary hits. Four compounds having a benzofuran core scaffold (CND0335, CND0364, CND0366 and CND0415), one pyrrolopyridine (CND0545) and one thiazol-carboxamide (CND3514) inhibited CHIKV-associated cell death in a dose-dependent manner, with EC50 values between 2.2 µM and 7.1 µM. Based on image analysis, these 6 hit compounds did not inhibit CHIKV replication in the host cell. However, CHIKV-infected cells manifested less prominent apoptotic blebs typical of CHIKV cytopathic effect compared with the control infection. Moreover, treatment with these compounds reduced viral titers in the medium of CHIKV-infected cells by up to 100-fold. In conclusion, this cell-based high-throughput screening assay using resazurin, combined with the image-based high content assay approach identified compounds against CHIKV having a novel antiviral activity--inhibition of virus-induced CPE--likely by targeting kinases involved in apoptosis.

Incidence and severity of respiratory insufficiency detected by transcutaneous carbon dioxide monitoring after cardiac surgery and intensive care unit discharge.

Patients undergoing coronary artery bypass surgery and/or heart valve surgery using a median sternotomy approach coupled with the use of cardiopulmonary bypass often experience pulmonary complications in the postoperative period. These patients are initially monitored in an intensive care unit (ICU) but after discharge from this unit to the ward they may still have compromised pulmonary function. This dysfunction may progress to significant respiratory failure that will cause the patient to return to the ICU. To investigate the severity and incidence of respiratory insufficiency once the patient has been discharged from the ICU to the ward, this study used transcutaneous carbon dioxide monitoring to determine the incidence of unrecognized inadequate ventilation in 39 patients undergoing the current standard of care. The incidence and severity of hypercarbia, hypoxia, and tachycardia in post-cardiac surgery patients during the first 24 hours after ICU discharge were found to be high, with severe episodes of each found in 38%, 79%, and 44% of patients, respectively.

Chylopericardium following orthotopic lung transplantation.

Chylopericardium is an uncommon condition, reported to occur following routine cardiac surgery, orthotopic heart transplantation, cardiac trauma, intrathoracic tumors, or infection. It has not, to date, been reported following uncomplicated orthotopic lung transplantation. This article describes chylopericardium following bilateral orthotopic lung transplantation.

Development of a multiplex phenotypic cell-based high throughput screening assay to identify novel hepatitis C virus antivirals.

Hepatitis C virus (HCV) infection is a global health concern with chronic liver damage threatening 3% of the world's population. To date, the standard of care is a combination of pegylated interferon-alpha with ribavirin, and recently two direct acting antivirals have entered the clinics. However, because of side effects, drug resistance and viral genotype-specific differences in efficacy current and potentially also future therapies have their limitations. Here, we describe the development of a phenotypic high-throughput assay to identify new cross-genotype inhibitors with novel mechanism of action, by combining a genotype (gt) 1 replicon with the infectious HCV gt2 cell culture system. To develop this phenotypic multiplex assay, HCV reporter cells expressing RFP-NLS-IPS and gt1b replicon cells expressing NS5A-GFP were co-plated and treated with compounds followed by inoculation with gt2a HCV. At 72h post treatment, RFP translocation as a marker for HCV infection and GFP fluorescence intensity as a marker for gt1 RNA replication were measured. Additionally, the total cell number, which serves as an indicator of cytotoxicity, was determined. This phenotypic strategy supports multi-parameter data acquisition from a single well to access cross-genotypic activity, provides an indication of the stage of the viral life cycle targeted, and also assesses compound cytotoxicity. Taken together, this multiplex phenotypic platform facilitates the identification of novel compounds for drug development and chemical probes for continuing efforts to understand the HCV life cycle.

The accuracy, precision and reliability of measuring ventilatory rate and detecting ventilatory pause by rainbow acoustic monitoring and capnometry.

BACKGROUND: Current methods for monitoring ventilatory rate have limitations including poor accuracy and precision and low patient tolerance. In this study, we evaluated a new acoustic ventilatory rate monitoring technology for accuracy, precision, reliability, and the ability to detect pauses in ventilation, relative to capnometry and a reference method in postsurgical patients. METHODS: Adult patients presenting to the postanesthesia care unit were connected to a Pulse CO-Oximeter with acoustic monitoring technology (Rad-87, version 7804, Masimo, Irvine, CA) through an adhesive bioacoustic sensor (RAS-125, rev C) applied to the neck. Each subject also wore a nasal cannula connected to a bedside capnometer (Capnostream20, version 4.5, Oridion, Needham, MA). The acoustic monitor and capnometer were connected to a computer for continuous acoustic and expiratory carbon dioxide waveform recordings. Recordings were retrospectively analyzed by a trained technician in a setting that allowed for the simultaneous viewing of both waveforms while listening to the breathing sounds from the acoustic signal to determine inspiration and expiration reference markers within the ventilatory cycle without using the acoustic monitor- or capnometer-calculated ventilatory rate. This allowed the automatic calculation of a reference ventilatory rate for each device through a software program (TagEditor, Masimo). Accuracy (relative to the respective reference) and precision of each device were estimated and compared with each other. Sensitivity for detection of pauses in ventilation, defined as no inspiration or expiration activity in the reference ventilatory cycle for ≥30 seconds, was also determined. The devices were also evaluated for their reliability, i.e., the percentage of the time when each displayed a value and did not drop a measurement. RESULTS: Thirty-three adults (73% female) with age of 45 ± 14 years and weight 117 ± 42 kg were enrolled. A total of 3712 minutes of monitoring time (average 112 minutes per subject) were analyzed across the 2 devices, reference ventilatory rates ranged from 1.9 to 49.1 bpm. Acoustic monitoring showed significantly greater accuracy (P = 0.0056) and precision (P- = 0.0024) for respiratory rate as compared with capnometry. On average, both devices displayed data over 97% of the monitored time. The (0.95, 0.95) lower tolerance limits for the acoustic monitor and capnometer were 94% and 84%, respectively. Acoustic monitoring was marginally more sensitive (P = 0.0461) to pauses in ventilation (81% vs 62%) in 21 apneic events. CONCLUSIONS: In this study of a population of postsurgical patients, the acoustic monitor and capnometer both reliably monitored ventilatory rate. The acoustic monitor was statistically more accurate and more precise than the capnometer, but differences in performance were modest. It is not known whether the observed differences are clinically significant. The acoustic monitor was more sensitive to detecting pauses in ventilation. Acoustic monitoring may provide an effective and convenient means of monitoring ventilatory rate in postsurgical patients.

An image analysis algorithm for malaria parasite stage classification and viability quantification.

With more than 40% of the world's population at risk, 200-300 million infections each year, and an estimated 1.2 million deaths annually, malaria remains one of the most important public health problems of mankind today. With the propensity of malaria parasites to rapidly develop resistance to newly developed therapies, and the recent failures of artemisinin-based drugs in Southeast Asia, there is an urgent need for new antimalarial compounds with novel mechanisms of action to be developed against multidrug resistant malaria. We present here a novel image analysis algorithm for the quantitative detection and classification of Plasmodium lifecycle stages in culture as well as discriminating between viable and dead parasites in drug-treated samples. This new algorithm reliably estimates the number of red blood cells (isolated or clustered) per fluorescence image field, and accurately identifies parasitized erythrocytes on the basis of high intensity DAPI-stained parasite nuclei spots and Mitotracker-stained mitochondrial in viable parasites. We validated the performance of the algorithm by manual counting of the infected and non-infected red blood cells in multiple image fields, and the quantitative analyses of the different parasite stages (early rings, rings, trophozoites, schizonts) at various time-point post-merozoite invasion, in tightly synchronized cultures. Additionally, the developed algorithm provided parasitological effective concentration 50 (EC50) values for both chloroquine and artemisinin, that were similar to known growth inhibitory EC50 values for these compounds as determined using conventional SYBR Green I and lactate dehydrogenase-based assays.

Transcription sites are developmentally regulated during the asexual cycle of Plasmodium falciparum.

Increasing evidence shows that the spatial organization of transcription is an important epigenetic factor in eukaryotic gene regulation. The malaria parasite Plasmodium falciparum shows a remarkably complex pattern of gene expression during the erythrocytic cycle, paradoxically contrasting with the relatively low number of putative transcription factors encoded by its genome. The spatial organization of nuclear subcompartments has been correlated with the regulation of virulence genes. Here, we investigate the nuclear architecture of transcription during the asexual cycle of malaria parasites. As in mammals, transcription is organized into discrete nucleoplasmic sites in P. falciparum, but in a strikingly lower number of foci. An automated analysis of 3D images shows that the number and intensity of transcription sites vary significantly between rings and trophozoites, although the nuclear volume remains constant. Transcription sites are spatially reorganized during the asexual cycle, with a higher proportion of foci located in the outermost nuclear region in rings, whereas in trophozoites, foci are evenly distributed throughout the nucleoplasm. As in higher eukaryotes, transcription sites are predominantly found in areas of low chromatin density. Immunofluorescence analysis shows that transcription sites form an exclusive nuclear compartment, different from the compartments defined by the silenced or active chromatin markers. In conclusion, these data suggest that transcription is spatially contained in discrete foci that are developmentally regulated during the asexual cycle of malaria parasites and located in areas of low chromatin density.

An image-based drug susceptibility assay targeting the placental sequestration of Plasmodium falciparum-infected erythrocytes.

Placental malaria is a significant cause of all malaria-related deaths globally for which no drugs have been developed to specifically disrupt its pathogenesis. To facilitate the discovery of antimalarial drugs targeting the cytoadherence process of Plasmodium-infected erythrocytes in the placenta microvasculature, we have developed an automated image-based assay for high-throughput screening for potent cytoadherence inhibitors in vitro. Parasitized erythrocytes were drug-treated for 24 h and then allowed to adhere on a monolayer of placental BeWo cells prior to red blood cell staining with glycophorin A antibodies. Upon image-acquisition, drug effects were quantified as the proportion of treated parasitized erythrocytes to BeWo cells compared to the binding of untreated iRBCs. We confirmed the reliability of this new assay by comparing the binding ratios of CSA- and CD36-panned parasites on the placental BeWo cells, and by quantifying the effects of chondroitin sulfate A, brefeldin A, and artemisinin on the binding. By simultaneously examining the drug effects on parasite viability, we could discriminate between cytoadherence-specific inhibitors and other schizonticidal compounds. Taken together, our data establish that the developed assay is highly suitable for drug studies targeting placental malaria, and will facilitate the discovery and rapid development of new therapies against malaria.