High resolution sequencing of hepatitis C virus reveals limited intra-hepatic compartmentalization in end-stage liver disease

Background & Aims The high replication and mutation rate of hepatitis C virus (HCV) results in a heterogeneous population of viral sequences in vivo. HCV replicates in the liver and infected hepatocytes occur as foci surrounded by uninfected cells that may promote compartmentalization of viral variants. Given recent reports showing interferon stimulated gene (ISG) expression in chronic hepatitis C, we hypothesized that local interferon responses may limit HCV replication and evolution. Methods To investigate the spatial influence of liver architecture on viral replication we measured HCV RNA and ISG mRNA from each of the 8 Couinaud segments of the liver from 21 patients undergoing liver transplant. Results HCV RNA and ISG mRNA levels were comparable across all sites from an individual liver but showed up to 500-fold difference between patients. Importantly, there was no association between ISG and HCV RNA expression across all sites in the liver or plasma. Deep sequencing of HCV RNA isolated from the 8 hepatic sites from two subjects showed a similar distribution of viral quasispecies across the liver and uniform sequence diversity. Single genome amplification of HCV E1E2-envelope clones from 6 selected patients at 2 hepatic sites supported these data and showed no evidence for HCV compartmentalization. Conclusions We found no differences between the hepatic and plasma viral quasispecies in all patients sampled. We conclude that in end-stage liver disease HCV RNA levels and the genetic pool of HCV envelope sequences are indistinguishable between distant sites in the liver and plasma, arguing against viral compartmentalization

Effect of scavenger receptor BI antagonist ITX5061 in patients with hepatitis C virus infection undergoing liver transplantation

Hepatitis C virus (HCV) entry inhibitors have been hypothesized to prevent infection of the liver after transplantation. ITX5061 is a Scavenger Receptor B-I (SR-BI) antagonist that blocks HCV entry and infection in vitro. We assessed the safety and efficacy of ITX5061 to limit HCV infection of the graft. The study included 23 HCV infected patients undergoing liver transplantation. The first 13 "control" patients did not receive drug. The subsequent 10 patients received ITX5061 150 mg immediately pre- and post-transplant, and daily for 1 week thereafter. ITX5061 pharmacokinetics and plasma HCV RNA were quantified. Viral genetic diversity was measured by ultradeep pyrosequencing. ITX5061 was well tolerated with measurable plasma concentrations during therapy. Whilst the median HCV RNA reduction was greater in ITX treated patients at all time points in the first week after transplantation there was no difference in the overall change in the area over the HCV RNA curve in the 7-day treatment period. However, in genotype 1 infected patients treatment was associated with a sustained reduction in HCV RNA levels compared to the control group (area over the HCV RNA curve analysis, p=0.004). Ultradeep pyrosequencing revealed a complex and evolving pattern of HCV variants infecting the graft during the first week. ITX5061 significantly limited viral evolution where the median divergence between day 0 and day 7 was 3.5% in the control group compared to 0.1% in the treated group.CONCLUSIONS: ITX5061 reduces plasma HCV RNA post transplant notably in genotype 1 infected patients and slows viral evolution. Following liver transplantation the likely contribution of extrahepatic reservoirs of HCV necessitates combining entry inhibitors such as ITX5061 with inhibitors of replication in future studies. Clinicaltrials.gov NCT01292824. This article is protected by copyright. All rights reserved.</p

Setting up a Paediatric Rapid Access Outpatient Unit: Views of general practice teams

<p>Abstract</p> <p>Background</p> <p>Rapid Access Outpatient Units (RAOUs) have been suggested as an alternative to hospital inpatient units for the management of some acutely unwell children. These units can provide ambulatory care, delivered close to home, and may prevent unnecessary hospital admission. There are no qualitative data on the views of primary care practitioners regarding these types of facilities. The aim of the study was to explore the opinions of primary care practitioners regarding a newly established RAOU.</p> <p>Methods</p> <p>The RAOU was established locally at a district general hospital when inpatient beds were closed and moved to an inpatient centre, based six miles away at the tertiary teaching hospital.</p> <p>Qualitative, practice based group interviews with primary care practitioners (general practitioners (GPs), nurse practitioners and practice nurses) on their experiences of the RAOU. The data collection consisted of three practice based interviews with 14 participants. The interviews were recorded and transcribed verbatim. Thematic content analysis was used to evaluate the data.</p> <p>Results</p> <p>There was positive feedback regarding ease of telephone access for referral, location, and the value of a service staffed by senior doctors where children could be observed, investigated and discharged quickly. There was confusion regarding the referral criteria for the assessment unit and where to send certain children. A majority of the practitioners felt the utility of the RAOU was restricted by its opening hours. Most participants felt they lacked sufficient information regarding the remit and facilities of the unit and this led to some uneasiness regarding safety and long term sustainability.</p> <p>Conclusion</p> <p>Practitioners considered that the RAOU offered a rapid senior opinion, flexible short term observation, quick access to investigations and was more convenient for patients. There were concerns regarding opening hours, safety of patients and lack of information about the unit's facilities. There was confusion about which children should be sent to the unit. This study raises questions regarding policy in regard to the organisation of paediatric services. It highlights that when establishing alternative services to local inpatient units, continual communication and engagement of primary care is essential if the units are to function effectively.</p

Multifragmentation in Xe(50A MeV)+Sn Confrontation of theory and data

We compare in detail central collisions Xe(50A MeV) + Sn, recently measured by the INDRA collaboration, with the Quantum Molecular Dynamics (QMD) model in order to identify the reaction mechanism which leads to multifragmentation. We find that QMD describes the data quite well, in the projectile/target region as well as in the midrapidity zone where also statistical models can be and have been employed. The agreement between QMD and data allows to use this dynamical model to investigate the reaction in detail. We arrive at the following observations: a) the in medium nucleon nucleon cross section is not significantly different from the free cross section, b) even the most central collisions have a binary character, c) most of the fragments are produced in the central collisions and d) the simulations as well as the data show a strong attractive in-plane flow resembling deep inelastic collisions e) at midrapidity the results from QMD and those from statistical model calculations agree for almost all observables with the exception of ${d^2 \sigma \over dZdE}$. This renders it difficult to extract the reaction mechanism from midrapidity fragments only. According to the simulations the reaction shows a very early formation of fragments, even in central collisions, which pass through the reaction zone without being destroyed. The final transverse momentum of the fragments is very close to the initial one and due to the Fermi motion. A heating up of the systems is not observed and hence a thermal origin of the spectra cannot be confirmed.Comment: figures 1 and 2 changed (no more ps -errors

Epidemiology, prehospital care and outcomes of patients arriving by ambulance with dyspnoea: An observational study

Background: This study aimed to determine epidemiology and outcome for patients presenting to emergency departments (ED) with shortness of breath who were transported by ambulance. Methods: This was a planned sub-study of a prospective, interrupted time series cohort study conducted at three time points in 2014 and which included consecutive adult patients presenting to the ED with dyspnoea as a main symptom. For this sub-study, additional inclusion criteria were presentation to an ED in Australia or New Zealand and transport by ambulance. The primary outcomes of interest are the epidemiology and outcome of these patients. Analysis was by descriptive statistics and comparisons of proportions. Results: One thousand seven patients met inclusion criteria. Median age was 74 years (IQR 61-68) and 46.1 % were male. There was a high rate of co-morbidity and chronic medication use. The most common ED diagnoses were lower respiratory tract infection (including pneumonia, 22.7 %), cardiac failure (20.5%) and exacerbation of chronic obstructive pulmonary disease (19.7 %). ED disposition was hospital admission (including ICU) for 76.4 %, ICU admission for 5.6 % and death in ED in 0.9 %. Overall in-hospital mortality among admitted patients was 6.5 %. Discussion: Patients transported by ambulance with shortness of breath make up a significant proportion of ambulance caseload and have high comorbidity and high hospital admission rate. In this study, >60 % were accounted for by patients with heart failure, lower respiratory tract infection or COPD, but there were a wide range of diagnoses. This has implications for service planning, models of care and paramedic training. Conclusion: This study shows that patients transported to hospital by ambulance with shortness of breath are a complex and seriously ill group with a broad range of diagnoses. Understanding the characteristics of these patients, the range of diagnoses and their outcome can help inform training and planning of services

Measurements of sideward flow around the balance energy

Sideward flow values have been determined with the INDRA multidetector for Ar+Ni, Ni+Ni and Xe+Sn systems studied at GANIL in the 30 to 100 A.MeV incident energy range. The balance energies found for Ar+Ni and Ni+Ni systems are in agreement with previous experimental results and theoretical calculations. Negative sideward flow values have been measured. The possible origins of such negative values are discussed. They could result from a more important contribution of evaporated particles with respect to the contribution of promptly emitted particles at mid-rapidity. But effects induced by the methods used to reconstruct the reaction plane cannot be totally excluded. Complete tests of these methods are presented and the origins of the ``auto-correlation'' effect have been traced back. For heavy fragments, the observed negative flow values seem to be mainly due to the reaction plane reconstruction methods. For light charged particles, these negative values could result from the dynamics of the collisions and from the reaction plane reconstruction methods as well. These effects have to be taken into account when comparisons with theoretical calculations are done.Comment: 27 pages, 15 figure

Use of Dried Blood Spots to Elucidate Full-Length Transmitted/Founder HIV-1 Genomes.

BACKGROUND: Identification of HIV-1 genomes responsible for establishing clinical infection in newly infected individuals is fundamental to prevention and pathogenesis research. Processing, storage, and transportation of the clinical samples required to perform these virologic assays in resource-limited settings requires challenging venipuncture and cold chain logistics. Here, we validate the use of dried-blood spots (DBS) as a simple and convenient alternative to collecting and storing frozen plasma. METHODS: We performed parallel nucleic acid extraction, single genome amplification (SGA), next generation sequencing (NGS), and phylogenetic analyses on plasma and DBS. RESULTS: We demonstrated the capacity to extract viral RNA from DBS and perform SGA to infer the complete nucleotide sequence of the transmitted/founder (TF) HIV-1 envelope gene and full-length genome in two acutely infected individuals. Using both SGA and NGS methodologies, we showed that sequences generated from DBS and plasma display comparable phylogenetic patterns in both acute and chronic infection. SGA was successful on samples with a range of plasma viremia, including samples as low as 1,700 copies/ml and an estimated ∼50 viral copies per blood spot. Further, we demonstrated reproducible efficiency in gp160 env sequencing in DBS stored at ambient temperature for up to three weeks or at -20°C for up to five months. CONCLUSIONS: These findings support the use of DBS as a practical and cost-effective alternative to frozen plasma for clinical trials and translational research conducted in resource-limited settings

Study of intermediate velocity products in the Ar+Ni collisions between 52 and 95 A.MeV

Intermediate velocity products in Ar+Ni collisions from 52 to 95 A.MeV are studied in an experiment performed at the GANIL facility with the 4$\pi$ multidetector INDRA. It is shown that these emissions cannot be explained by statistical decays of the quasi-projectile and the quasi-target in complete equilibrium. Three methods are used to isolate and characterize intermediate velocity products. The total mass of these products increases with the violence of the collision and reaches a large fraction of the system mass in mid-central collisions. This mass is found independent of the incident energy, but strongly dependent on the geometry of the collision. Finally it is shown that the kinematical characteristics of intermediate velocity products are weakly dependent on the experimental impact parameter, but strongly dependent on the incident energy. The observed trends are consistent with a participant-spectator like scenario or with neck emissions and/or break-up.Comment: 37 pages, 13 figure

"Sleepiness" is serious in adolescence: Two surveys of 3235 Canadian students

BACKGROUND: Evidence is growing that sleep problems in adolescents are significant impediments to learning and negatively affect behaviour, attainment of social competence and quality of life. The objectives of the study were to determine the level of sleepiness among students in high school, to identify factors to explain it, and to determine the association between sleepiness and performance in both academic and extracurricular activities METHODS: A cross-sectional survey of 2201 high school students in the Hamilton Wentworth District School Board and the Near North District School Board in Ontario was conducted in 1998/9. A similar survey was done three years later involving 1034 students in the Grand Erie District School Board in the same Province. The Epworth Sleepiness Scale (ESS) was used to measure sleepiness and we also assessed the reliability of this tool for this population. Descriptive analysis of the cohort and information on various measures of performance and demographic data were included. Regression analysis, using the generalised estimating equation (GEE), was utilized to investigate factors associated with risk of sleepiness (ESS>10). RESULTS: Seventy per cent of the students had less than 8.5 hours weeknight sleep. Bedtime habits such as a consistent bedtime routine, staying up late or drinking caffeinated beverages before bed were statistically significantly associated with ESS, as were weeknight sleep quantity and gender. As ESS increased there was an increase in the proportion of students who felt their grades had dropped because of sleepiness, were late for school, were often extremely sleepy at school, and were involved in fewer extracurricular activities. These performance measures were statistically significantly associated with ESS. Twenty-three percent of the students felt their grades had dropped because of sleepiness. Most students (58–68%) reported that they were "really sleepy" between 8 and 10 A.M. CONCLUSION: Sleep deprivation and excessive daytime sleepiness were common in two samples of Ontario high school students and were associated with a decrease in academic achievement and extracurricular activity. There is a need to increase awareness of this problem in the education and health communities and to translate knowledge already available to strategies to address it