Rapid identification of genes controlling virulence and immunity in malaria parasites.

Identifying the genetic determinants of phenotypes that impact disease severity is of fundamental importance for the design of new interventions against malaria. Here we present a rapid genome-wide approach capable of identifying multiple genetic drivers of medically relevant phenotypes within malaria parasites via a single experiment at single gene or allele resolution. In a proof of principle study, we found that a previously undescribed single nucleotide polymorphism in the binding domain of the erythrocyte binding like protein (EBL) conferred a dramatic change in red blood cell invasion in mutant rodent malaria parasites Plasmodium yoelii. In the same experiment, we implicated merozoite surface protein 1 (MSP1) and other polymorphic proteins, as the major targets of strain-specific immunity. Using allelic replacement, we provide functional validation of the substitution in the EBL gene controlling the growth rate in the blood stages of the parasites.

STOP!: a randomised, double-blind, placebo-controlled study of the efficacy and safety of methoxyflurane for the treatment of acute pain.

OBJECTIVE: To evaluate the short-term efficacy and safety of methoxyflurane for the treatment of acute pain in patients presenting to an emergency department (ED) with minor trauma. METHODS: STOP! was a randomised, double-blind, multicentre, placebo-controlled study conducted at six sites in the UK. A total of 300 patients, 90 of whom were adolescent patients (age 12-17 years), were randomised 150:150 to receive either methoxyflurane via a Penthrox inhaler or placebo. The primary end point of the study was the change in pain intensity as measured using the visual analogue scale (VAS) from baseline to 5, 10, 15 and 20 min after the start of study drug inhalation. Patients were supplied with one inhaler containing 3 mL methoxyflurane or 5 mL placebo after enrolment and initial assessments. Age group (adolescent/adult) and baseline VAS score were controlled for in the statistical analyses. RESULTS: A total of 149 patients received methoxyflurane, and 149 patients received placebo. Demographic and baseline characteristics were comparable between the groups. Methoxyflurane reduced pain severity significantly more than placebo (p<0.0001) at all time points tested, with the greatest estimated treatment effect of -18.5 mm (adjusted change from baseline) seen at 15 min after the start of treatment. Methoxyflurane was well tolerated, with the majority of adverse reactions being mild, transient and in line with anticipated pharmacological action. CONCLUSION: The results of this study suggest that methoxyflurane administered via the Penthrox inhaler is an efficacious, safe, and rapidly acting analgesic. TRIAL REGISTRATION NUMBER: NCT01420159.

Use of human rights to meet the unmet need for family planning.

In this report, we describe how human rights can help to shape laws, policies, programmes, and projects in relation to contraceptive information and services. Applying a human rights perspective and recognising the International Conference on Population and Development and Millennium Development Goal commitments to universal access to reproductive health including family planning, we support measurement of unmet need for family planning that encompasses more groups than has been the case until recently. We outline how human rights can be used to identify, reduce, and eliminate barriers to accessing contraception; the ways in which human rights can enhance laws and policies; and governments' legal obligations in relation to contraceptive information and services. We underline the crucial importance of accountability of states and identify some of the priorities for making family planning available that are mandated by human rights.

Artemisinin resistance in rodent malaria--mutation in the AP2 adaptor µ-chain suggests involvement of endocytosis and membrane protein trafficking.

BACKGROUND: The control of malaria, caused by Plasmodium falciparum, is hampered by the relentless evolution of drug resistance. Because artemisinin derivatives are now used in the most effective anti-malarial therapy, resistance to artemisinin would be catastrophic. Indeed, studies suggest that artemisinin resistance has already appeared in natural infections. Understanding the mechanisms of resistance would help to prolong the effective lifetime of these drugs. Genetic markers of resistance are therefore required urgently. Previously, a mutation in a de-ubiquitinating enzyme was shown to confer artemisinin resistance in the rodent malaria parasite Plasmodium chabaudi. METHODS: Here, for a mutant P. chabaudi malaria parasite and its immediate progenitor, the in vivo artemisinin resistance phenotypes and the mutations arising using Illumina whole-genome re-sequencing were compared. RESULTS: An increased artemisinin resistance phenotype is accompanied by one non-synonymous substitution. The mutated gene encodes the µ-chain of the AP2 adaptor complex, a component of the endocytic machinery. Homology models indicate that the mutated residue interacts with a cargo recognition sequence. In natural infections of the human malaria parasite P. falciparum, 12 polymorphisms (nine SNPs and three indels) were identified in the orthologous gene. CONCLUSION: An increased artemisinin-resistant phenotype occurs along with a mutation in a functional element of the AP2 adaptor protein complex. This suggests that endocytosis and trafficking of membrane proteins may be involved, generating new insights into possible mechanisms of resistance. The genotypes of this adaptor protein can be evaluated for its role in artemisinin responses in human infections of P. falciparum.

Quantitative genome re-sequencing defines multiple mutations conferring chloroquine resistance in rodent malaria.

BACKGROUND: Drug resistance in the malaria parasite Plasmodium falciparum severely compromises the treatment and control of malaria. A knowledge of the critical mutations conferring resistance to particular drugs is important in understanding modes of drug action and mechanisms of resistances. They are required to design better therapies and limit drug resistance.A mutation in the gene (pfcrt) encoding a membrane transporter has been identified as a principal determinant of chloroquine resistance in P. falciparum, but we lack a full account of higher level chloroquine resistance. Furthermore, the determinants of resistance in the other major human malaria parasite, P. vivax, are not known. To address these questions, we investigated the genetic basis of chloroquine resistance in an isogenic lineage of rodent malaria parasite P. chabaudi in which high level resistance to chloroquine has been progressively selected under laboratory conditions. RESULTS: Loci containing the critical genes were mapped by Linkage Group Selection, using a genetic cross between the high-level chloroquine-resistant mutant and a genetically distinct sensitive strain. A novel high-resolution quantitative whole-genome re-sequencing approach was used to reveal three regions of selection on chr11, chr03 and chr02 that appear progressively at increasing drug doses on three chromosomes. Whole-genome sequencing of the chloroquine-resistant parent identified just four point mutations in different genes on these chromosomes. Three mutations are located at the foci of the selection valleys and are therefore predicted to confer different levels of chloroquine resistance. The critical mutation conferring the first level of chloroquine resistance is found in aat1, a putative aminoacid transporter. CONCLUSIONS: Quantitative trait loci conferring selectable phenotypes, such as drug resistance, can be mapped directly using progressive genome-wide linkage group selection. Quantitative genome-wide short-read genome resequencing can be used to reveal these signatures of drug selection at high resolution. The identities of three genes (and mutations within them) conferring different levels of chloroquine resistance generate insights regarding the genetic architecture and mechanisms of resistance to chloroquine and other drugs. Importantly, their orthologues may now be evaluated for critical or accessory roles in chloroquine resistance in human malarias P. vivax and P. falciparum.

Improving outcome in severe trauma: what's new in ABC? Imaging, bleeding and brain injury.

Appropriate imaging is critical in the initial assessment of patients with severe trauma. Plain radiographs remain integral to the primary survey. Focused ultrasonography is useful for identifying intraperitoneal fluid likely to represent haemorrhage in patients who are shocked and also has a role in identifying intrathoracic pathology. Modern scanners permit a greater role for CT, being more rapid and exposing the patient to less ionising radiation. 'Whole body' (head to pelvis) CT scanning has been shown to identify injuries missed by 'traditional' focused assessment and may be associated with an improved outcome. CT identifies more spinal injuries than plain radiographs, is the gold standard for diagnosing blunt aortic injury and facilitates non-operative management of solid organ injury and other bleeding. Coagulopathy occurs early in trauma as a direct result of injury and hypoperfusion. Damage control resuscitation with blood components is associated with an improved outcome in patients with trauma with massive haemorrhage. Packed cells and fresh frozen plasma should be used in a 1:1 to 1:2 ratio. Bedside measures of coagulopathy may prove useful. Adjuvant early treatment with tranexamic acid is of benefit in reducing blood loss and reducing mortality. Limited 'damage control surgery' with early optimisation of physiology augmented by interventional radiology to control haemorrhage is preferable to early definitive care. Limiting haemorrhage by correction of anticoagulation and minimising secondary brain injury through optimal supportive care is critical to improving outcome in neurotrauma.

Polymer-Induced Drag Reduction in Dilute Newtonian and Semi-Dilute Non-Newtonian Fluids: An Assessment of the Double-Gap Concentric Cylinder Method.

Polymer-induced drag reduction (DR) in fluids was studied using a rotational rheometer with double-gap concentric cylinder geometry. Although both polymers (polyacrylamide (PAM) and 2-acrylamido-2-methylpropane sulfonic acid (SPAM)) had molecular weights of several MDa, the contrasting polymer charge, nonionic and anionic, led to different polymer overlap concentrations (c*), PAM ≫ SPAM, and fluid rheology, with PAM fluids mostly Newtonian and SPAM fluids non-Newtonian (shear-thinning). Based on these differences, it was important to account for the infinite shear viscosity and normalize the polymer concentration by the intrinsic concentration (c int) so that the DR performance of the two polymer fluids could be accurately compared. Both polymers induced DR, and the maximum DR by SPAM (DR% = 28) was slightly higher than that by PAM (DR% = 22) when Re p ∼ 1700. For PAM, the loss of DR with time diminished at higher polymer concentrations (≥100 ppm, at Re p = 3149) but was found to be sensitive to high Re p, with polymer chain scission the likely cause of the reduced performance. For the semi-dilute SPAM fluids, the shear stability contrasted that of PAM, showing negligible dependence on the polymer concentration and Re p. The apparent rapid loss of DR was predominantly attributed to a time-dependent effect and not polymer degradation. In pipe flow, the maximum DR for SPAM was higher than that measured by rheometry and was attributed to differences in the flow conditions. However, changes in the normalized DR/c with polymer concentration were found to be consistent between the two flow geometries. Furthermore, the high fluid stresses in pipe flow (at high Re p) led to drag reduction losses consistent with PAM, as the time-dependent effect was not seen.

Genomewide scan reveals amplification of mdr1 as a common denominator of resistance to mefloquine, lumefantrine, and artemisinin in Plasmodium chabaudi malaria parasites.

Multidrug-resistant Plasmodium falciparum malaria parasites pose a threat to effective drug control, even to artemisinin-based combination therapies (ACTs). Here we used linkage group selection and Solexa whole-genome resequencing to investigate the genetic basis of resistance to component drugs of ACTs. Using the rodent malaria parasite P. chabaudi, we analyzed the uncloned progeny of a genetic backcross between the mefloquine-, lumefantrine-, and artemisinin-resistant mutant AS-15MF and a genetically distinct sensitive clone, AJ, following drug treatment. Genomewide scans of selection showed that parasites surviving each drug treatment bore a duplication of a segment of chromosome 12 (translocated to chromosome 04) present in AS-15MF. Whole-genome resequencing identified the size of the duplicated segment and its position on chromosome 4. The duplicated fragment extends for ∼393 kbp and contains over 100 genes, including mdr1, encoding the multidrug resistance P-glycoprotein homologue 1. We therefore show that resistance to chemically distinct components of ACTs is mediated by the same genetic mutation, highlighting a possible limitation of these therapies.

Experimental evolution, genetic analysis and genome re-sequencing reveal the mutation conferring artemisinin resistance in an isogenic lineage of malaria parasites.

BACKGROUND: Classical and quantitative linkage analyses of genetic crosses have traditionally been used to map genes of interest, such as those conferring chloroquine or quinine resistance in malaria parasites. Next-generation sequencing technologies now present the possibility of determining genome-wide genetic variation at single base-pair resolution. Here, we combine in vivo experimental evolution, a rapid genetic strategy and whole genome re-sequencing to identify the precise genetic basis of artemisinin resistance in a lineage of the rodent malaria parasite, Plasmodium chabaudi. Such genetic markers will further the investigation of resistance and its control in natural infections of the human malaria, P. falciparum. RESULTS: A lineage of isogenic in vivo drug-selected mutant P. chabaudi parasites was investigated. By measuring the artemisinin responses of these clones, the appearance of an in vivo artemisinin resistance phenotype within the lineage was defined. The underlying genetic locus was mapped to a region of chromosome 2 by Linkage Group Selection in two different genetic crosses. Whole-genome deep coverage short-read re-sequencing (Illumina Solexa) defined the point mutations, insertions, deletions and copy-number variations arising in the lineage. Eight point mutations arise within the mutant lineage, only one of which appears on chromosome 2. This missense mutation arises contemporaneously with artemisinin resistance and maps to a gene encoding a de-ubiquitinating enzyme. CONCLUSIONS: This integrated approach facilitates the rapid identification of mutations conferring selectable phenotypes, without prior knowledge of biological and molecular mechanisms. For malaria, this model can identify candidate genes before resistant parasites are commonly observed in natural human malaria populations.

Are drug companies living up to their human rights responsibilities? The perspective of the former United Nations Special Rapporteur (2002-2008).

BACKGROUND TO THE DEBATE: The human rights responsibilities of drug companies have been considered for years by nongovernmental organizations, but were most sharply defined in a report by the UN Special Rapporteur on the right to health, submitted to the United Nations General Assembly in August 2008. The "Human Rights Guidelines for Pharmaceutical Companies in relation to Access to Medicines" include responsibilities for transparency, management, monitoring and accountability, pricing, and ethical marketing, and against lobbying for more protection in intellectual property laws, applying for patents for trivial modifications of existing medicines, inappropriate drug promotion, and excessive pricing. Two years after the release of the Guidelines, the PLoS Medicine Debate asks whether drug companies are living up to their human rights responsibilities. Sofia Gruskin and Zyde Raad from the Harvard School of Public Health say more assessment is needed of such responsibilities; Geralyn Ritter, Vice President of Global Public Policy and Corporate Responsibility at Merck & Co. argues that multiple stakeholders could do more to help States deliver the right to health; and Paul Hunt and Rajat Khosla introduce Mr. Hunt's work as the UN Special Rapporteur on the right to the highest attainable standard of health, regarding the human rights responsibilities of pharmaceutical companies and access to medicines.

Lessons identified from the 2017 Manchester and London terrorism incidents. Part 1: introduction and the prehospital phase.

This is the first article in a three-part series detailing the lessons identified during the NHS England clinical debrief meetings which followed the response to the 2017 Manchester and London terrorist incidents. It covers the prehospital phase including the overall key learning points, timeline information, scene challenges, resource utilisation, triage, distribution and helicopter emergency medical service feedback.

Lessons identified from the 2017 Manchester and London terrorism incidents. Part 3: the postincident and recovery phase.

This is the third paper in a three-part series detailing the lessons identified during the National Health Service (NHS) England clinical debrief meetings which followed the response to the 2017 Manchester and London terrorist incidents. It covers the postincident and recovery phases including rehabilitation, bereavement support, psychological support, network and regional lessons, NHS communications and supply organisations. It also summarises the military application of these lessons and outlines the next steps for further development.

Lessons identified from the 2017 Manchester and London terrorism incidents. Part two: the reception and definitive care (hospital) phases.

The provision of medical care during the reception and definitive care phases of a terrorist incident will likely take place in designated receiving hospitals such as Major Trauma Centres. There is a need for an enhanced capability in such units to receive, initially manage and hold casualties with more serious injuries. Also, even less severely injured casualties may require significant time and clinical input such as risk management in potential bloodborne viruses.The distribution of casualties from the incident scene requires advance consideration of the injury pattern and regional network organisation of specialist services, such as maxillofacial, neurosurgery or severe burns care. Paediatric centres are also more sparsely distributed and often only in large city networks which represents a significant challenge for planners and responders in other regions. An effective response relies on a coordinated multidisciplinary approach including emergency and front-of-house teams, surgical, medical and clinical support services.

Health systems and the right to health: an assessment of 194 countries.

60 years ago, the Universal Declaration of Human Rights laid the foundations for the right to the highest attainable standard of health. This right is central to the creation of equitable health systems. We identify some of the right-to health features of health systems, such as a comprehensive national health plan, and propose 72 indicators that reflect some of these features. We collect globally processed data on these indicators for 194 countries and national data for Ecuador, Mozambique, Peru, Romania, and Sweden. Globally processed data were not available for 18 indicators for any country, suggesting that organisations that obtain such data give insufficient attention to the right-to-health features of health systems. Where they are available, the indicators show where health systems need to be improved to better realise the right to health. We provide recommendations for governments, international bodies, civil-society organisations, and other institutions and suggest that these indicators and data, although not perfect, provide a basis for the monitoring of health systems and the progressive realisation of the right to health. Right-to-health features are not just good management, justice, or humanitarianism, they are obligations under human-rights law.

Health systems and the right to the highest attainable standard of health.

The right to the highest attainable standard of health should be the cornerstone of any consideration of health and human rights. The content of this fundamental human right is now sufficiently well understood to be applied in an operational, systematic, and sustained manner. At the heart of the right to the highest attainable standard of health lies an effective and integrated health system, encompassing medical care and the underlying determinants of health, which is responsive to national and local priorities and accessible to all. Yet in many countries, health systems are failing and collapsing, giving rise to an extremely grave human rights problem. This article outlines the general approach of the right to the highest attainable standard of health toward the strengthening of health systems. It identifies some of the key right-to-health features of a health system, such as transparency, participation, equity and equality, a comprehensive national health plan, a minimum "basket" of health-related services and facilities, disaggregated data, monitoring and accountability, and so on. This general approach has to be consistently and systematically applied across the "building blocks" that together constitute a functioning health system. By way of illustration, the article applies this approach to one of these "building blocks:" leadership, governance, and stewardship. There are numerous health movements and approaches, including health equity, primary health care, social determinants, and so on. All are very important. But it is misconceived to regard human rights as yet another approach with the same status as the others. Like ethics, the right to the highest attainable standard of health is not optional--and, like ethics, it recurs throughout all other approaches. The right to the highest attainable standard of health is the only perspective that is both underpinned by universally recognized moral values and reinforced by legal obligations.