Engineering a Model Cell for Rational Tuning of GPCR Signaling.

G protein-coupled receptor (GPCR) signaling is the primary method eukaryotes use to respond to specific cues in their environment. However, the relationship between stimulus and response for each GPCR is difficult to predict due to diversity in natural signal transduction architecture and expression. Using genome engineering in yeast, we constructed an insulated, modular GPCR signal transduction system to study how the response to stimuli can be predictably tuned using synthetic tools. We delineated the contributions of a minimal set of key components via computational and experimental refactoring, identifying simple design principles for rationally tuning the dose response. Using five different GPCRs, we demonstrate how this enables cells and consortia to be engineered to respond to desired concentrations of peptides, metabolites, and hormones relevant to human health. This work enables rational tuning of cell sensing while providing a framework to guide reprogramming of GPCR-based signaling in other systems.

Global estimates and determinants of antituberculosis drug pharmacokinetics in children and adolescents: a systematic review and individual patient data meta-analysis.

BACKGROUND: Suboptimal exposure to antituberculosis (anti-TB) drugs has been associated with unfavourable treatment outcomes. We aimed to investigate estimates and determinants of first-line anti-TB drug pharmacokinetics in children and adolescents at a global level. METHODS: We systematically searched MEDLINE, Embase and Web of Science (1990-2021) for pharmacokinetic studies of first-line anti-TB drugs in children and adolescents. Individual patient data were obtained from authors of eligible studies. Summary estimates of total/extrapolated area under the plasma concentration-time curve from 0 to 24 h post-dose (AUC0-24) and peak plasma concentration (C max) were assessed with random-effects models, normalised with current World Health Organization-recommended paediatric doses. Determinants of AUC0-24 and C max were assessed with linear mixed-effects models. RESULTS: Of 55 eligible studies, individual patient data were available for 39 (71%), including 1628 participants from 12 countries. Geometric means of steady-state AUC0-24 were summarised for isoniazid (18.7 (95% CI 15.5-22.6) h·mg·L-1), rifampicin (34.4 (95% CI 29.4-40.3) h·mg·L-1), pyrazinamide (375.0 (95% CI 339.9-413.7) h·mg·L-1) and ethambutol (8.0 (95% CI 6.4-10.0) h·mg·L-1). Our multivariate models indicated that younger age (especially <2 years) and HIV-positive status were associated with lower AUC0-24 for all first-line anti-TB drugs, while severe malnutrition was associated with lower AUC0-24 for isoniazid and pyrazinamide. N-acetyltransferase 2 rapid acetylators had lower isoniazid AUC0-24 and slow acetylators had higher isoniazid AUC0-24 than intermediate acetylators. Determinants of C max were generally similar to those for AUC0-24. CONCLUSIONS: This study provides the most comprehensive estimates of plasma exposures to first-line anti-TB drugs in children and adolescents. Key determinants of drug exposures were identified. These may be relevant for population-specific dose adjustment or individualised therapeutic drug monitoring.

Rapid acquisition and model-based analysis of cell-free transcription-translation reactions from nonmodel bacteria.

Native cell-free transcription-translation systems offer a rapid route to characterize the regulatory elements (promoters, transcription factors) for gene expression from nonmodel microbial hosts, which can be difficult to assess through traditional in vivo approaches. One such host, Bacillus megaterium, is a giant Gram-positive bacterium with potential biotechnology applications, although many of its regulatory elements remain uncharacterized. Here, we have developed a rapid automated platform for measuring and modeling in vitro cell-free reactions and have applied this to B. megaterium to quantify a range of ribosome binding site variants and previously uncharacterized endogenous constitutive and inducible promoters. To provide quantitative models for cell-free systems, we have also applied a Bayesian approach to infer ordinary differential equation model parameters by simultaneously using time-course data from multiple experimental conditions. Using this modeling framework, we were able to infer previously unknown transcription factor binding affinities and quantify the sharing of cell-free transcription-translation resources (energy, ribosomes, RNA polymerases, nucleotides, and amino acids) using a promoter competition experiment. This allows insights into resource limiting-factors in batch cell-free synthesis mode. Our combined automated and modeling platform allows for the rapid acquisition and model-based analysis of cell-free transcription-translation data from uncharacterized microbial cell hosts, as well as resource competition within cell-free systems, which potentially can be applied to a range of cell-free synthetic biology and biotechnology applications.

Population Pharmacokinetic Properties of Sulfadoxine and Pyrimethamine: a Pooled Analysis To Inform Optimal Dosing in African Children with Uncomplicated Malaria.

Sulfadoxine-pyrimethamine with amodiaquine is recommended by the World Health Organization as seasonal malaria chemoprevention for children aged 3 to 59 months in the sub-Sahel regions of Africa. Suboptimal dosing in children may lead to treatment failure and increased resistance. Pooled individual patient data from four previously published trials on the pharmacokinetics of sulfadoxine and pyrimethamine in 415 pediatric and 386 adult patients were analyzed using nonlinear mixed-effects modeling to evaluate the current dosing regimen and, if needed, to propose an optimized dosing regimen for children under 5 years of age. The population pharmacokinetics of sulfadoxine and pyrimethamine were both best described by a one-compartment disposition model with first-order absorption and elimination. Body weight, age, and nutritional status (measured as the weight-for-age Z-score) were found to be significant covariates. Allometric scaling with total body weight and the maturation of clearance in children by postgestational age improved the model fit. Underweight-for-age children were found to have 15.3% and 26.7% lower bioavailabilities of sulfadoxine and pyrimethamine, respectively, for each Z-score unit below -2. Under current dosing recommendations, simulation predicted that the median day 7 concentration was below the 25th percentile for a typical adult patient (50 kg) for sulfadoxine for patients in the weight bands of 8 to 9, 19 to 24, 46 to 49, and 74 to 79 kg and for pyrimethamine for patients in the weight bands of 8 to 9, 14 to 24, and 42 to 49 kg. An evidence-based dosing regimen was constructed that would achieve sulfadoxine and pyrimethamine exposures in young children and underweight-for-age young children that were similar to those currently seen in a typical adult.

Late Ebola virus relapse causing meningoencephalitis: a case report.

BACKGROUND: There are thousands of survivors of the 2014 Ebola outbreak in west Africa. Ebola virus can persist in survivors for months in immune-privileged sites; however, viral relapse causing life-threatening and potentially transmissible disease has not been described. We report a case of late relapse in a patient who had been treated for severe Ebola virus disease with high viral load (peak cycle threshold value 13.2). METHODS: A 39-year-old female nurse from Scotland, who had assisted the humanitarian effort in Sierra Leone, had received intensive supportive treatment and experimental antiviral therapies, and had been discharged with undetectable Ebola virus RNA in peripheral blood. The patient was readmitted to hospital 9 months after discharge with symptoms of acute meningitis, and was found to have Ebola virus in cerebrospinal fluid (CSF). She was treated with supportive therapy and experimental antiviral drug GS-5734 (Gilead Sciences, San Francisco, Foster City, CA, USA). We monitored Ebola virus RNA in CSF and plasma, and sequenced the viral genome using an unbiased metagenomic approach. FINDINGS: On admission, reverse transcriptase PCR identified Ebola virus RNA at a higher level in CSF (cycle threshold value 23.7) than plasma (31.3); infectious virus was only recovered from CSF. The patient developed progressive meningoencephalitis with cranial neuropathies and radiculopathy. Clinical recovery was associated with addition of high-dose corticosteroids during GS-5734 treatment. CSF Ebola virus RNA slowly declined and was undetectable following 14 days of treatment with GS-5734. Sequencing of plasma and CSF viral genome revealed only two non-coding changes compared with the original infecting virus. INTERPRETATION: Our report shows that previously unanticipated, late, severe relapses of Ebola virus can occur, in this case in the CNS. This finding fundamentally redefines what is known about the natural history of Ebola virus infection. Vigilance should be maintained in the thousands of Ebola survivors for cases of relapsed infection. The potential for these cases to initiate new transmission chains is a serious public health concern. FUNDING: Royal Free London NHS Foundation Trust.

Physiotherapists clinical reasoning to prescribe exercise for patients with chronic pain: A qualitative study research protocol.

BACKGROUND: Physiotherapists' play a key role in the management of chronic pain, and as part of the National Institute for Health and Care Excellence (NICE) guidelines, prescribe exercise to support patients with chronic pain. However, there is very limited evidence supporting physiotherapists on what type of exercise or dose of exercise should be prescribed. Physiotherapists' therefore have more onus on their ability to clinically reason how to prescribe exercise. At present, there is no research investigating how physiotherapists' working with patients that have chronic pain, clinically reason when prescribing exercise. This study proposes to investigate how physiotherapists experienced in pain management prescribe exercise, to understand what the key influences are on their reasoning, and how these impact on clinical practice. METHODS: This will be a qualitative study, utilising semi-structured individual interviews. Participants will be Health and Care Professions Council registered physiotherapists, working predominantly with patients that have chronic pain. Recruitment will focus on physiotherapists working within the United Kingdom (UK). Up to twenty participants will be recruited. The study, including the interview guide, will be supported by a steering group consisting of academics and physiotherapists experienced in chronic pain. The data will be analysed using framework analysis. RESULTS: The study will be reported using the COnsolidated criteria for REporting Qualitative research (COREQ) guidelines. The findings of the study will be disseminated through publication in a peer reviewed journal. CONCLUSION: This study will provide novel insight into how physiotherapists experienced working with and managing chronic pain patients, prescribe exercise, and will gain new insight into clinical practice to help inform future research and education.

Intrinsic calf factors associated with the behavior of healthy pre-weaned group-housed dairy-bred calves.

Technology-derived behaviors are researched for disease detection in artificially-reared calves. Whilst existing studies demonstrate differences in behaviors between healthy and diseased calves, intrinsic calf factors (e.g., sex and birthweight) that may affect these behaviors have received little systematic study. This study aimed to understand the impact of a range of calf factors on milk feeding and activity variables of dairy-bred calves. Calves were group-housed from ~7 days to 39 days of age. Seven liters of milk replacer was available daily from an automatic milk feeder, which recorded feeding behaviors and live-weight. Calves were health scored daily and a tri-axial accelerometer used to record activity variables. Healthy calves were selected by excluding data collected 3 days either side of a poor health score or a treatment event. Thirty-one calves with 10 days each were analyzed. Mixed models were used to identify which of live-weight, age, sex, season of birth, age of inclusion into the group, dam parity, birthweight, and sire breed type (beef or dairy), had a significant influence on milk feeding and activity variables. Heavier calves visited the milk machine more frequently for shorter visits, drank faster and were more likely to drink their daily milk allowance than lighter calves. Older calves had a shorter mean standing bout length and were less active than younger calves. Calves born in summer had a longer daily lying time, performed more lying and standing bouts/day and had shorter mean standing bouts than those born in autumn or winter. Male calves had a longer mean lying bout length, drank more slowly and were less likely to consume their daily milk allowance than their female counterparts. Calves that were born heavier had fewer lying and standing bouts each day, a longer mean standing bout length and drank less milk per visit. Beef-sired calves had a longer mean lying bout length and drank more slowly than their dairy sired counterparts. Intrinsic calf factors influence different healthy calf behaviors in different ways. These factors must be considered in the design of research studies and the field application of behavior-based disease detection tools in artificially reared calves.

Modular Metabolic Engineering and Synthetic Coculture Strategies for the Production of Aromatic Compounds in Yeast.

Microbial-derived aromatics provide a sustainable and renewable alternative to petroleum-derived chemicals. In this study, we used the model yeast Saccharomyces cerevisiae to produce aromatic molecules by exploiting the concept of modularity in synthetic biology. Three different modular approaches were investigated for the production of the valuable fragrance raspberry ketone (RK), found in raspberry fruits and mostly produced from petrochemicals. The first strategy used was modular cloning, which enabled the generation of combinatorial libraries of promoters to optimize the expression level of the genes involved in the synthesis pathway of RK. The second strategy was modular pathway engineering and involved the creation of four modules, one for product formation: RK synthesis module (Mod. RK); and three for precursor synthesis: aromatic amino acid synthesis module (Mod. Aro), p-coumaric acid synthesis module (Mod. p-CA), and malonyl-CoA synthesis module (Mod. M-CoA). The production of RK by combinations of the expression of these modules was studied, and the best engineered strain produced 63.5 mg/L RK from glucose, which is the highest production described in yeast, and 2.1 mg RK/g glucose, which is the highest yield reported in any organism without p-coumaric acid supplementation. The third strategy was the use of modular cocultures to explore the effects of division of labor on RK production. Two two-member communities and one three-member community were created, and their production capacity was highly dependent on the structure of the synthetic community, the inoculation ratio, and the culture media. In certain conditions, the cocultures outperformed their monoculture controls for RK production, although this was not the norm. Interestingly, the cocultures showed up to 7.5-fold increase and 308.4 mg/L of 4-hydroxy benzalacetone, the direct precursor of RK, which can be used for the semi-synthesis of RK. This study illustrates the utility of modularity in synthetic biology tools and their applications to the synthesis of products of industrial interest.

Reconstituting the complete biosynthesis of D-lysergic acid in yeast.

The ergot alkaloids are a class of natural products known for their pharmacologically privileged molecular structure that are used in the treatment of neurological ailments, such as Parkinsonism and dementia. Their synthesis via chemical and biological routes are therefore of industrial relevance, but suffer from several challenges. Current chemical synthesis methods involve long, multi-step reactions with harsh conditions and are not enantioselective; biological methods utilizing ergot fungi, produce an assortment of products that complicate product recovery, and are susceptible to strain degradation. Reconstituting the ergot alkaloid pathway in a strain strongly amenable for liquid fermentation, could potentially resolve these issues. In this work, we report the production of the main ergoline therapeutic precursor, D-lysergic acid, to a titre of 1.7 mg L-1 in a 1 L bioreactor. Our work demonstrates the proof-of-concept for the biological production of ergoline-derived compounds from sugar in an engineered yeast chassis.

The Effect of the Climatic Housing Environment on the Growth of Dairy-Bred Calves in the First Month of Life on a Scottish Farm.

Calf housing is naturally thermodynamic, with interactions between various elements such as wind speed, air temperature, and humidity. This study investigated the effect of the proportion of time for which calves were exposed to effective environmental temperatures below their lower critical temperature (LCT) on their daily liveweight gain (DLWG) within their first month of life. This study used the naturally occurring climatic environment, whereas other such studies have been conducted under climatically controlled conditions. Air temperature (°C), relative humidity (%), and wind speed (m/s) were recorded within the calf housing from birth until approximately 28 days of age, with calves being health-scored and weighed at regular intervals. Calves were housed from birth until 6-14 days old in individual hutches, and then moved into group housing igloo pens. Whilst individually housed, calves that spent less than 32% of their time below their LCT had a DLWG of 0.06 ± 0.34 kg/d (mean ± SE) compared to calves that spent more than 97% of their time below their LCT, which had a DLWG of -0.19 ± 0.045 kg/d. When group housed, calves that spent less than 1% of their time below their LCT had a DLWG of 0.59 ± 0.18 kg/d, whereas calves that spent more than 28% of their time below their LCT had a DLWG of 0.53 ± 0.23 kg/d. The proportion of time for which calves were exposed to effective environmental temperatures below their LCT had a significant effect on DLWG when calves were individually housed. Therefore, exposure to effective environmental temperatures below the LCT can be detrimental to the growth of the calf in the early stages of its life.

Measurement of adherence, drug concentrations and the effectiveness of artemether-lumefantrine, chlorproguanil-dapsone or sulphadoxine-pyrimethamine in the treatment of uncomplicated malaria in Malawi.

BACKGROUND: Sulphadoxine-pyrimethamine (SP) is the only single dose therapy for uncomplicated malaria, but there is widespread resistance. At the time of this study, artemether-lumefantrine (AL) and chlorproguanil-dapsone (CPD), both multi-dose regimes, were considered possible alternatives to SP in Malawi. The aim of this study was to investigate the impact of poor adherence on the effectiveness of AL and CPD. METHODS: Children > or =12 months and adults with uncomplicated malaria were randomized to receive AL, CPD or SP. Adherence was measured using a questionnaire and electronic monitoring devices, MEMS, pill bottles that recorded the date and time of opening. Day-7 plasma dapsone or lumefantrine concentrations were measured to examine their relationship with adherence and clinical response. RESULTS: 841 patients were recruited. The day-28 adequate clinical and parasitological response (ACPR) rates, using intention to treat analysis (missing data treated as failure), were AL 85.2%, CPD 63.7% and SP 50%. ACPR rates for AL were higher than CPD or SP on days 28 and 42 (p < or = 0.002 for all comparisons). CPD was more effective than SP on day-28 (p = 0.01), but not day-42.Very high adherence was reported using the questionnaire, 100% for AL treated patients and 99.2% for the CPD group. Only three CPD participants admitted missing any doses. 164/181 (90.6%) of CPD treated patients took all their doses out of the MEMS container and they were more likely to have a day-28 ACPR than those who did not take all their medication out of the container, p = 0.024. Only 7/87 (8%) AL treated patients did not take all of their doses out of their MEMS container and none had treatment failure.Median day-7 dapsone concentrations were higher in CPD treated patients with ACPR than in treatment failures, p = 0.012. There were no differences in day-7 dapsone or lumefantrine concentrations between those who took all their doses from the MEMS container and those who did not. A day-7 lumefantrine concentration reported to be predictive of AL treatment failure in Thailand was not useful in this population; only one of 16 participants with a concentration below this threshold (175 ng/ml) had treatment failure. CONCLUSION: This study provides reassurance of the effectiveness of AL, even with unsupervised dosing, as it is rolled out across sub-Saharan Africa. Self-reported adherence appears to be an unreliable measure of adherence in this population.

Management of suspected herpes simplex virus encephalitis in adults in a U.K. teaching hospital.

The outcome of herpes simplex virus (HSV) encephalitis is improved with prompt initiation of aciclovir treatment. Delays are common, but there is little understanding of why they occur. The case notes of 21 adults admitted with suspected HSV encephalitis over one year were reviewed. The median (range) duration of illness was 2.5 (1-99) days. Seventeen (81%) patients had a lumbar puncture (LP) performed, at a median (range) time of 24 (2-114) hours after encephalitis was suspected. Lumbar puncture was delayed for a computed tomography (CT) scan in 15 patients, but only one of these had contraindications to an immediate LP. The median (range) time from presentation to starting aciclovir was 48 (2-432) hours. HSV-PCR (polymerase chain reaction) was requested on cerebrospinal fluid from 12 patients, one of whom was positive. Five (24%) patients were given the wrong dose of aciclovir. Overall the management of suspected HSV encephalitis was often sub-optimal, with delays in LP occurring due to unnecessary CT scans, and the wrong aciclovir dose administered. Guidelines for the management of suspected encephalitis are needed.

Yellow fever vaccine-associated neurological disease: it is not just the silver generation at risk.

A 35-year-old man presented to his optician with sudden onset diplopia and a 1-week history of headaches. He was noted to have sixth nerve palsy. The following day he was admitted to hospital with confusion and expressive dysphasia. He had been due to travel to Ghana on business and had received yellow fever (YF) vaccination 18 days prior to onset of headaches. His initial cerebrospinal fluid (CSF) revealed elevated protein, increased white cell count but was PCR negative for standard viral pathogens. Herpes simplex virus (HSV)-1 was detected by PCR in CSF at a very low level from a second lumbar puncture performed 6 days later, and the patient was treated for HSV meningoencephalitis. However, retrospective investigation for yellow fever vaccine-associated neurological disease revealed increasing titres of YF IgG in three serial CSF samples, and no evidence of HSV antibodies in CSF or plasma, ruling out HSV encephalitis.

Author Correction: Building a global alliance of biofoundries.

The original version of this Comment contained errors in the legend of Figure 2, in which the locations of the fifteenth and sixteenth GBA members were incorrectly given as '(15) Australian Genome Foundry, Macquarie University; (16) Australian Foundry for Advanced Biomanufacturing, University of Queensland.'. The correct version replaces this with '(15) Australian Foundry for Advanced Biomanufacturing (AusFAB), University of Queensland and (16) Australian Genome Foundry, Macquarie University'. This has been corrected in both the PDF and HTML versions of the Comment.

Plasmodium falciparum strains harboring dihydrofolate reductase with the I164L mutation are absent in Malawi and Zambia even under antifolate drug pressure.

The Plasmodium falciparum dihydrofolate reductase (PfDHFR) enzyme is the target of pyrimethamine, a component of the antimalarial pyrimethamine-sulfadoxine. Resistance to this drug is associated primarily with mutations in the Pfdhfr gene. The I164L mutant allele is of particular interest, because strains possessing this mutation are highly resistant to pyrimethamine and to chlorproguanil, a component of chlorproguanil-dapsone. A recent study from Malawi reported this mutation at a prevalence of 4.7% in parasites from human immunodeficiency virus-positive pregnant women by using a real-time PCR method. These observations have huge implications for the use of pyrimethamine-sulfadoxine, chlorproguanil-dapsone, and future antifolate-artemisinin combinations in Africa. It was imperative that this finding be rigorously tested. We identified a number of critical limitations in the original genotyping strategy. Using a refined and validated real-time PCR strategy, we report here that this mutation was absent in 158 isolates from Malawi and 42 isolates from Zambia collected between 2003 and 2005.

A Cell-Free Biosensor for Detecting Quorum Sensing Molecules in P. aeruginosa-Infected Respiratory Samples.

Synthetic biology designed cell-free biosensors are a promising new tool for the detection of clinically relevant biomarkers in infectious diseases. Here, we report that a modular DNA-encoded biosensor in cell-free protein expression systems can be used to measure a bacterial biomarker of Pseudomonas aeruginosa infection from human sputum samples. By optimizing the cell-free system and sample extraction, we demonstrate that the quorum sensing molecule 3-oxo-C12-HSL in sputum samples from cystic fibrosis lungs can be quantitatively measured at nanomolar levels using our cell-free biosensor system, and is comparable to LC-MS measurements of the same samples. This study further illustrates the potential of modular cell-free biosensors as rapid, low-cost detection assays that can inform clinical practice.

Biosynthesis of the antibiotic nonribosomal peptide penicillin in baker's yeast.

Fungi are a valuable source of enzymatic diversity and therapeutic natural products including antibiotics. Here we engineer the baker's yeast Saccharomyces cerevisiae to produce and secrete the antibiotic penicillin, a beta-lactam nonribosomal peptide, by taking genes from a filamentous fungus and directing their efficient expression and subcellular localization. Using synthetic biology tools combined with long-read DNA sequencing, we optimize productivity by 50-fold to produce bioactive yields that allow spent S. cerevisiae growth media to have antibacterial action against Streptococcus bacteria. This work demonstrates that S. cerevisiae can be engineered to perform the complex biosynthesis of multicellular fungi, opening up the possibility of using yeast to accelerate rational engineering of nonribosomal peptide antibiotics.

Prospective Assessment of Virtual Screening Heuristics Derived Using a Novel Fusion Score.

High-throughput screening (HTS) is a widespread method in early drug discovery for identifying promising chemical matter that modulates a target or phenotype of interest. Because HTS campaigns involve screening millions of compounds, it is often desirable to initiate screening with a subset of the full collection. Subsequently, virtual screening methods prioritize likely active compounds in the remaining collection in an iterative process. With this approach, orthogonal virtual screening methods are often applied, necessitating the prioritization of hits from different approaches. Here, we introduce a novel method of fusing these prioritizations and benchmark it prospectively on 17 screening campaigns using virtual screening methods in three descriptor spaces. We found that the fusion approach retrieves 15% to 65% more active chemical series than any single machine-learning method and that appropriately weighting contributions of similarity and machine-learning scoring techniques can increase enrichment by 1% to 19%. We also use fusion scoring to evaluate the tradeoff between screening more chemical matter initially in lieu of replicate samples to prevent false-positives and find that the former option leads to the retrieval of more active chemical series. These results represent guidelines that can increase the rate of identification of promising active compounds in future iterative screens.

UK malaria treatment guidelines 2016.

1.Malaria is the tropical disease most commonly imported into the UK, with 1300-1800 cases reported each year, and 2-11 deaths. 2. Approximately three quarters of reported malaria cases in the UK are caused by Plasmodium falciparum, which is capable of invading a high proportion of red blood cells and rapidly leading to severe or life-threatening multi-organ disease. 3. Most non-falciparum malaria cases are caused by Plasmodium vivax; a few cases are caused by the other species of plasmodium: Plasmodium ovale, Plasmodium malariae or Plasmodium knowlesi. 4. Mixed infections with more than one species of parasite can occur; they commonly involve P. falciparum with the attendant risks of severe malaria. 5. There are no typical clinical features of malaria; even fever is not invariably present. Malaria in children (and sometimes in adults) may present with misleading symptoms such as gastrointestinal features, sore throat or lower respiratory complaints. 6. A diagnosis of malaria must always be sought in a feverish or sick child or adult who has visited malaria-endemic areas. Specific country information on malaria can be found at http://travelhealthpro.org.uk/. P. falciparum infection rarely presents more than six months after exposure but presentation of other species can occur more than a year after exposure. 7. Management of malaria depends on awareness of the diagnosis and on performing the correct diagnostic tests: the diagnosis cannot be excluded until more than one blood specimen has been examined. Other travel related infections, especially viral haemorrhagic fevers, should also be considered. 8. The optimum diagnostic procedure is examination of thick and thin blood films by an expert to detect and speciate the malarial parasites. P. falciparum and P. vivax (depending upon the product) malaria can be diagnosed almost as accurately using rapid diagnostic tests (RDTs) which detect plasmodial antigens. RDTs for other Plasmodium species are not as reliable. 9. Most patients treated for P. falciparum malaria should be admitted to hospital for at least 24 h as patients can deteriorate suddenly, especially early in the course of treatment. In specialised units seeing large numbers of patients, outpatient treatment may be considered if specific protocols for patient selection and follow up are in place. 10. Uncomplicated P. falciparum malaria should be treated with an artemisinin combination therapy (Grade 1A). Artemether-lumefantrine (Riamet(®)) is the drug of choice (Grade 2C) and dihydroartemisinin-piperaquine (Eurartesim(®)) is an alternative. Quinine or atovaquone-proguanil (Malarone(®)) can be used if an ACT is not available. Quinine is highly effective but poorly-tolerated in prolonged treatment and should be used in combination with an additional drug, usually oral doxycycline. 11. Severe falciparum malaria, or infections complicated by a relatively high parasite count (more than 2% of red blood cells parasitized) should be treated with intravenous therapy until the patient is well enough to continue with oral treatment. Severe malaria is a rare complication of P. vivax or P. knowlesi infection and also requires parenteral therapy. 12. The treatment of choice for severe or complicated malaria in adults and children is intravenous artesunate (Grade 1A). Intravenous artesunate is unlicensed in the EU but is available in many centres. The alternative is intravenous quinine, which should be started immediately if artesunate is not available (Grade 1A). Patients treated with intravenous quinine require careful monitoring for hypoglycemia. 13. Patients with severe or complicated malaria should be managed in a high-dependency or intensive care environment. They may require haemodynamic support and management of: acute respiratory distress syndrome, disseminated intravascular coagulation, acute kidney injury, seizures, and severe intercurrent infections including Gram-negative bacteraemia/septicaemia. 14. Children with severe malaria should also be treated with empirical broad spectrum antibiotics until bacterial infection can be excluded (Grade 1B). 15. Haemolysis occurs in approximately 10-15% patients following intravenous artesunate treatment. Haemoglobin concentrations should be checked approximately 14 days following treatment in those treated with IV artemisinins (Grade 2C). 16. Falciparum malaria in pregnancy is more likely to be complicated: the placenta contains high levels of parasites, stillbirth or early delivery may occur and diagnosis can be difficult if parasites are concentrated in the placenta and scanty in the blood. 17. Uncomplicated falciparum malaria in the second and third trimester of pregnancy should be treated with artemether-lumefantrine (Grade 2B). Uncomplicated falciparum malaria in the first trimester of pregnancy should usually be treated with quinine and clindamycin but specialist advice should be sought. Severe malaria in any trimester of pregnancy should be treated as for any other patient with artesunate preferred over quinine (Grade 1C). 18. Children with uncomplicated malaria should be treated with an ACT (artemether-lumefantrine or dihydroartemisinin-piperaquine) as first line treatment (Grade 1A). Quinine with doxycycline or clindamycin, or atovaquone-proguanil at appropriate doses for weight can also be used. Doxycycline should not be given to children under 12 years. 19. Either an oral ACT or chloroquine can be used for the treatment of non-falciparum malaria. An oral ACT is preferred for a mixed infection, if there is uncertainty about the infecting species, or for P. vivax infection from areas where chloroquine resistance is common (Grade 1B). 20. Dormant parasites (hypnozoites) persist in the liver after treatment of P. vivax or P. ovale infection: the only currently effective drug for eradication of hypnozoites is primaquine (1A). Primaquine is more effective at preventing relapse if taken at the same time as chloroquine (Grade 1C). 21. Primaquine should be avoided or given with caution under expert supervision in patients with Glucose-6-phosphate dehydrogenase deficiency (G6PD), in whom it may cause severe haemolysis. 22. Primaquine (for eradication of P. vivax or P. ovale hypnozoites) is contraindicated in pregnancy and when breastfeeding (until the G6PD status of child is known); after initial treatment for these infections a pregnant woman should take weekly chloroquine prophylaxis until after delivery or cessation of breastfeeding when hypnozoite eradication can be considered. 23. An acute attack of malaria does not confer protection from future attacks: individuals who have had malaria should take effective anti-mosquito precautions and chemoprophylaxis during future visits to endemic areas.