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Abstract Background The application of rehabilitation robots has grown during the last decade. While meta-analyses have shown beneficial effects of robotic interventions for some patient groups, the evidence is less in others. We established the Advanced Robotic Therapy Integrated Centers (ARTIC) network with the goal of advancing the science and clinical practice of rehabilitation robotics. The investigators hope to exploit variations in practice to learn about current clinical application and outcomes. The aim of this paper is to introduce the ARTIC network to the clinical and research community, present the initial data set and its characteristics and compare the outcome data collected so far with data from prior studies. Methods ARTIC is a pragmatic observational study of clinical care. The database includes patients with various neurological and gait deficits who used the driven gait orthosis Lokomat® as part of their treatment. Patient characteristics, diagnosis-specific information, and indicators of impairment severity are collected. Core clinical assessments include the 10-Meter Walk Test and the Goal Attainment Scaling. Data from each Lokomat® training session are automatically collected. Results At time of analysis, the database contained data collected from 595 patients (cerebral palsy: n = 208; stroke: n = 129; spinal cord injury: n = 93; traumatic brain injury: n = 39; and various other diagnoses: n = 126). At onset, average walking speeds were slow. The training intensity increased from the first to the final therapy session and most patients achieved their goals. Conclusions The characteristics of the patients matched epidemiological data for the target populations. When patient characteristics differed from epidemiological data, this was mainly due to the selection criteria used to assess eligibility for Lokomat® training. While patients included in randomized controlled interventional trials have to fulfill many inclusion and exclusion criteria, the only selection criteria applying to patients in the ARTIC database are those required for use of the Lokomat®. We suggest that the ARTIC network offers an opportunity to investigate the clinical application and effectiveness of rehabilitation technologies for various diagnoses. Due to the standardization of assessments and the use of a common technology, this network could serve as a basis for researchers interested in specific interventional studies expanding beyond the Lokomat®.

Nonmotor symptoms (NMS) of Parkinson’s disease (PD) have devastating impacts on both patients and their caregivers. Jiawei-Liujunzi Tang (JLT) has been used to treat some NMS of PD based on the Chinese medicine theory since Qing dynasty. Here we report a double-blind, randomized, placebo-controlled, add-on clinical trial aiming at evaluating the efficacy and safety of the JLT in treating NMS in PD patients. We randomly assigned 111 patients with idiopathic PD to receive either JLT or placebo for 32 weeks. Outcome measures were baseline to week 32 changes in Movement Disorder Society-Sponsored Revision of Unified PD Rating Scale (MDS-UPDRS) Parts I–IV and in NMS assessment scale for PD (NMSS). We observed improvements in the NMSS total score (p=0.019), mood/cognition (p=0.005), and reduction in hallucinations (p=0.024). In addition, post hoc analysis showed a significant reduction in constipation (p

Objectives To explore the experiences and perceptions of children with bronchiectasis and their parents regarding an 8-week play-based therapeutic exercise programme.Design Qualitative study with inductive content analysis.Setting Individual semistructured interviews were conducted. Interview recordings were transcribed verbatim, and coding was guided by the content. Content categories were established via consensus moderation.Participants 10 parents and 10 children with bronchiectasis aged 5–12 years.Results From the perspective of children, the most important components of the programme were fun with friends and being active at home as a family. Parents valued the community-based sessions, perceived the programme to be engaging and motivating. Parents perceived improvements in their child’s endurance, coordination and physical activity level. They described the home programme as fun but noted that finding time was difficult. Both parents and children thought that in-person exercise sessions would be better than exercise sessions delivered online.Conclusions Children who participated in the play-based exercise programme, found it fun, motivating and accessible. Parents perceived positive impacts on fitness, coordination and physical activity.Trial registration number The trial was registered with, Australian and New Zealand Clinical Trials Register (ACTRN12619001008112).

Abstract The incidence of type 2 diabetes mellitus has risen globally, from 108 million cases in 1980 to 422 million cases in 2014. Although controlling glycemic levels in patients with diabetes is crucial, insulin and sulfonylureas can cause hypoglycemic episodes and even potentially fatal events such as comas, seizures, life-threatening arrhythmias, and myocardial infarctions. Several antibiotics have been documented to cause hypoglycemic episodes; the use of antibiotics along with insulin or sulfonylureas might further increase the risk of hypoglycemia. Therefore, researchers must determine which antibiotics carry a risk of inducing severe hypoglycemic events. The prevalence of H. pylori infection remains high in most countries, and the infection is often treated with triple therapy involving amoxicillin, clarithromycin, and a proton pump inhibitor (PPI). Several case reports have reported that hypoglycemia can occur when used with patients who also take diabetes medication. Therefore, we hypothesized that patients with diabetes have an increased risk of hypoglycemic episodes when being treated with triple therapy for H. pylori infection. By analyzing medical records from Taiwan’s National Health Insurance Research Database, we found a significant association between hypoglycemia and triple therapy treatment for diabetic patients with peptic ulcer disease. Prescribing triple therapy to patients with diabetes and peptic ulcers significantly increased the risk of a hypoglycemic episode (adjusted odds ratio [aOR] = 1.75, 95% confidence interval [CI]: 1.64 to 1.88, P

Background: Intracranial atherosclerotic disease (ICAD) is globally a major ischaemic stroke subtype with high recurrence. Understanding the morphology of symptomatic ICAD plaques, largely unknown by far, may help identify vulnerable lesions prone to relapse.Methods: We prospectively recruited patients with acute ischaemic stroke or transient ischaemic attack attributed to high-grade ICAD (60%-99% stenosis). Plaque morphological parameters were assessed in three-dimensional rotational angiography, including surface contour, luminal stenosis, plaque length/thickness, upstream shoulder angulation, axial/longitudinal plaque distribution and presence of adjoining branch atheromatous disease (BAD). We compared morphological features of smooth, irregular and ulcerative plaques and correlated them with cerebral ischaemic lesion load downstream in MRI.Results: Among 180 recruited patients (median age=60 years; 63.3% male; median stenosis=75%), plaque contour was smooth (51 (28.3%)), irregular (101 (56.1%)) or ulcerative (28 (15.6%)). Surface ulcers were mostly at proximal (46.4%) and middle one-third (35.7%) of the lesions. Most (84.4%) plaques were eccentric, and half had their maximum thickness over the distal end. Ulcerative lesions were thicker (medians 1.6 vs 1.3 mm; p=0.003), had steeper upstream shoulder angulation (56.2° vs 31.0°; p<0.001) and more adjoining BAD (83.3% vs 57.0%; p=0.033) than non-ulcerative plaques. Ulcerative plaques were significantly associated with coexisting acute and chronic infarcts downstream (35.7% vs 12.5%; adjusted OR 4.29, 95% CI 1.65 to 11.14, p=0.003). Sensitivity analyses in patients with anterior-circulation ICAD lesions showed similar results in the associations between the plaque types and infarct load.Conclusions: Ulcerative intracranial atherosclerotic plaques were associated with vulnerable morphological features and had a higher cumulative infarct load downstream. [ABSTRACT FROM AUTHOR]

Abstract The genus Phytophthora comprises many economically and ecologically important plant pathogens. Hybrid species have previously been identified in at least six of the 12 phylogenetic clades. These hybrids can potentially infect a wider host range and display enhanced vigour compared to their progenitors. Phytophthora hybrids therefore pose a serious threat to agriculture as well as to natural ecosystems. Early and correct identification of hybrids is therefore essential for adequate plant protection but this is hampered by the limitations of morphological and traditional molecular methods. Identification of hybrids is also important in evolutionary studies as the positioning of hybrids in a phylogenetic tree can lead to suboptimal topologies. To improve the identification of hybrids we have combined genotyping-by-sequencing (GBS) and genome size estimation on a genus-wide collection of 614 Phytophthora isolates. Analyses based on locus- and allele counts and especially on the combination of species-specific loci and genome size estimations allowed us to confirm and characterize 27 previously described hybrid species and discover 16 new hybrid species. Our method was also valuable for species identification at an unprecedented resolution and further allowed correct naming of misidentified isolates. We used both a concatenation- and a coalescent-based phylogenomic method to construct a reliable phylogeny using the GBS data of 140 non-hybrid Phytophthora isolates. Hybrid species were subsequently connected to their progenitors in this phylogenetic tree. In this study we demonstrate the application of two validated techniques (GBS and flow cytometry) for relatively low cost but high resolution identification of hybrids and their phylogenetic relations.

Bronchiectasis in children can progress to a severe lung condition if not diagnosed and treated early. The radiological diagnostic criteria for the diagnosis of bronchiectasis is an increased broncho-arterial (BA) ratio. From high-resolution computed tomography (HRCT) scans, the BA pairs must be detected first to derive the BA ratio. This study aims to identify potential BA pairs from HRCT scans of children undertaken to evaluate suppurative lung disease through an automated approach. After segmenting the lung regions, the HRCT scans are cleaned using a histogram analysis-based approach followed by a potential arteries identification process comprising four conditions based on imaging features. Potential arteries and their connected components are extracted, and potential bronchi are identified. Finally, the coordinates of potential arteries and potential bronchi are matched as the last step of BA pairs extraction. A total of 8–50 BA pairs are detected for each patient. Additionally, the area and several diameters of the bronchi and arteries are measured, and BA ratios based on these are calculated. Through this approach, the BA pairs of a CT scan datasets are detected and utilizing a deep learning model, a high classification test accuracy of 98.53% is achieved, validating the robustness of the proposed BA detection approach. The results show that visible BA pairs can be identified and segmented automatically, and the BA ratio calculated may help diagnose bronchiectasis with less effort and time.

Coastal regions in Southwest Europe have experienced major interventions and transformations of the territory with unprecedented urban development, primarily related to growing tourism activity. The coast is the place where marine and terrestrial processes converge, making it highly vulnerable to the effects of climate change. However, the lack of information on the frequency of these extreme weather events and their impacts on the coast hampers an accurate analysis of the consequences of global change. This paper provides a detailed analysis of the extreme weather events (EWE) that have affected the Atlantic and Mediterranean coasts of Southwest Europe during the period from 1 January 2009 to 28 February 2020, as well as a quantification of their impacts: fatalities, injuries and economic damage. Official sources from France, Portugal and Spain were consulted, along with technical reports, scientific articles, etc., to generate a unified database. A total of 95 significant extreme events have caused 168 fatalities, 137 injuries and almost €4000 M in direct economic losses. Cyclone Xynthia (February 2010) on the French Atlantic coast stands out, having caused 47 fatalities, 79 injuries and substantial economic losses valued at €3000 M. The study shows a slight upward trend in the number of events recorded, especially during the last three years of the analysis, as well as in human losses and damages. The results reveal a higher exposure of the Mediterranean coast of Southwest Europe when compared to the Atlantic, especially the Spanish Mediterranean coast, with 61% of the fatalities recorded there during the study period. This is primarily due to a model of exponential tourism growth on the Mediterranean coast, with an enormous urban and infrastructure development during the last decades. Traditionally, the Mediterranean coast is less prepared to reduce the effects of marine storms, extreme events that are becoming more frequent and virulent in the context of climate and global change. This work highlights the need to create a continuous monitoring system–at the European level–of the impacts of extreme weather events on the coast, where 40% of the European population is concentrated. This observatory should serve as a source of information for risk mitigation policies (predictive, preventive and corrective), as well as for emergency management during disasters.

Abstract Background Metastatic Merkel cell carcinoma (mMCC) has traditionally been managed with palliative chemotherapy regimens or best supportive care (BSC). Avelumab, a novel anti‐programmed death‐ligand 1 (PD‐L1) human monoclonal antibody for mMCC treatment, is being studied in the pivotal JAVELIN Merkel 200 trial. Aim Incorporating trial results, this analysis aimed to evaluate the cost‐utility of avelumab in Taiwan. Methods and results A de novo partitioned‐survival model with three key health states related to survival (progression‐free disease, progressed disease, and death) was applied in this study. The data of clinical efficacy, safety, and patient utilities were obtained from the JAVELIN Merkel 200 trial, literature review, and Taiwanese clinical expert opinion. Cost‐utility analysis was performed, and results were presented as cost per quality‐adjusted life year (QALY) gained. For treatment‐naïve patients, the incremental cost‐effectiveness ratios (ICERs) for avelumab vs BSC and avelumab vs chemotherapy were US$44885.06 and US$42993.06 per QALY gained, respectively. As to treatment‐experienced mMCC patients, avelumab was associated with ICERs of US$27243.06 (vs BSC)/US$26557.43 (vs chemotherapy) per QALY gained. All ICERs remained consistently within the willingness‐to‐pay (WTP) threshold of US$53,333.33 per QALY gained. Conclusion This study demonstrated avelumab to be a cost‐effective treatment option for both treatment‐experienced and treatment‐naïve mMCC patients with very poor prognosis in Taiwan.

Abstract Background Undergraduate students across health professions are required to be capable users of evidence in their clinical practice after graduation. Gaining the essential knowledge and clinical behaviors for evidence-based practice can be enhanced by theory-based strategies. Limited evidence exists on the effect of underpinning undergraduate EBP curricula with a theoretical framework to support EBP competence. A systematic review was conducted to determine the effectiveness of EBP teaching strategies for undergraduate students, with specific focus on efficacy of theory-based strategies. Methods This review critically appraised and synthesized evidence on the effectiveness of EBP theory-based teaching strategies specifically for undergraduate health students on long or short-term change in multiple outcomes, including but not limited to, EBP knowledge and attitudes. PubMed, CINAHL, Scopus, ProQuest Health, ERIC, The Campbell Collaboration, PsycINFO were searched for published studies and The New York Academy of Medicine, ProQuest Dissertations and Mednar were searched for unpublished studies. Two independent reviewers assessed studies using the Joanna Briggs Institute Meta-Analysis of Statistics Assessment and Review Instrument. Results Twenty-eight studies reporting EBP teaching strategies were initially selected for review with methodological quality ranging from low to high. Studies varied in course duration, timing of delivery, population and course content. Only five included papers reported alignment with, and detail of, one or more theoretical frameworks. Theories reported included Social Cognitive Theory (one study), Roger’s Diffusion of Innovation Theory (two studies) and Cognitive Apprenticeship Theory (one study). Cognitive Flexibility Theory and Cognitive Load Theory were discussed in two separate papers by the same authors. All but one study measured EBP knowledge. Mixed results were reported on EBP knowledge, attitudes and skills across the five studies. Conclusions EBP programs for undergraduate health students require consideration of multiple domains, including clinical behaviors, attitudes and cognitive learning processes; Interventions grounded in theory were found to have a small but positive effect on EBP attitudes. The most effective theory for developing and supporting EBP capability is not able to be determined by this review therefore additional rigorous research is required.

Ceratocystis platani (CP), an ascomycetous fungus, is the agent of canker stain, a lethal vascular disease of Platanus species. Ceratocystis platani has been listed as a quarantine pest (EPPO A2 list) due to extensive damage caused in Southern Europe and the Mediterranean region. As traditional diagnostic assays are ineffective, a Real-Time PCR detection method based on EvaGreen, SYBR Green, and Taqman assays was previously developed, validated in-house, and included in the official EPPO standard PM7/14 (2). Here, we describe the results of a test performance study performed by nine European laboratories for the purpose of an interlaboratory validation. Verification of the DNA extracted from biological samples guaranteed the high quality of preparations, and the stability and the homogeneity of the aliquots intended for the laboratories. All of the laboratories reproduced nearly identical standard curves with efficiencies close to 100%. Testing of blind-coded DNA extracted from wood samples revealed that all performance parameters—diagnostic sensitivity, diagnostic specificity, accuracy and reproducibility—were best fit in most cases both at the laboratory and at the assay level. The previously established limit of detection, 3 fg per PCR reaction, was also validated with similar excellent results. The high interlaboratory performance of this Real-Time PCR method confirms its value as a primary tool to safeguard C. platani-free countries by way of an accurate monitoring, and to investigate the resistance level of potentially canker stain-resistant Platanus genotypes.

Introduction: Chronic respiratory disorders are highly prevalent among Australian Aboriginal people living in the Top End Health Service region in the Northern Territory, Australia. Bronchiectasis is a heterogenous disease that features among these chronic respiratory conditions in this population. However, there are sparse comparative data between Aboriginal and non-Aboriginal patients with bronchiectasis from this region. Methods: In this retrospective study, demographics, clinical characteristics and relevant laboratory parameters were compared among adult Aboriginal and non-Aboriginal patients diagnosed with bronchiectasis between 2012 and 2017. Results: A total of 388 adults had radiology-confirmed bronchiectasis and 258 (66%) were Aboriginal. Compared to non-Aboriginal patients, Aboriginal patients were significantly younger (mean age 54 v 67 years), the majority lived in rural and remote communities (80% v 9 %), had higher rates of self-reported smoking (52% v 19%), alcohol consumption (29% v 12%) and co-occurrence of chronic obstructive pulmonary disease (65% v 38%) and other chronic co-morbidities. Sputum microbiology was also different between the groups with Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis being more common in Aboriginal patients, while Pseudomonas aeruginosa, Aspergillus species and non-tuberculous mycobacteria were higher in non-Aboriginal patients. Further, Aboriginal patients had poorer lung function compared to non-Aboriginal patients (forced expiratory volume after 1 second predicted 33% v 53%, forced vital capacity predicted 49% v 60% respectively), higher exacerbation rates (29% v 18%) and poorer overall outcomes (age at death 60 v 76 years). Conclusion: Within a single health service, Aboriginal patients with bronchiectasis have significantly poorer outcomes with differing manifestations and higher comorbidities than non-Aboriginal patients. This warrants further studies to identify feasible interventions to reduce this inequity.

Introduction Streptococcus pneumoniae and non-typeable Haemophilus influenzae (NTHi) are major otitis media pathogens that densely co-colonise the nasopharynx and infect the middle ear of Australian Aboriginal infants from very early in life. Our co-primary hypotheses are that at 18 months of age infants receiving 10-valent pneumococcal Haemophilus influenzae protein D conjugate vaccine (PHiD-CV10) compared with those receiving 13-valent pneumococcal conjugate vaccine (PCV13) as a booster at 12 months of age will have higher antibody levels to Haemophilus influenzae protein D and that infants receiving PCV13 will have higher antibody levels to PCV13-only serotypes 3, 6A and 19A.Methods and analyses Our randomised controlled trial will enrol 270 Aboriginal children at 12 months of age to a booster dose of either PHiD-CV10 or PCV13. Children who completed the three-dose primary course schedules of PHiD-CV10 at 2, 4, 6 months of age; PCV13 at 2, 4, 6 months of age; or a combination schedule of PHiD-CV10 at 1, 2, 4 months of age plus PCV13 at 6 months of age are eligible. The co-primary assessor-blinded outcomes when the infants are 18 months of age are as follows: (a) IgG geometric mean concentration (GMC) and proportion with IgG ≥100 EU/mL for protein D, and (b) IgG GMC and the proportion with IgG ≥0.35 µg/mL for pneumococcal serotypes 3, 6A and 19A. Secondary immunogenicity comparisons of six primary and booster dose schedules of 10 shared serotypes at 18 months of age, nasopharyngeal carriage, all forms of otitis media, hearing loss and developmental milestones at 18, 24, 30 and 36 months of age will be reported.Ethics and dissemination Ethics committees of NT Department of Health, Menzies, WA Department of Health and WA Aboriginal Health approved the study. Results will be presented to communities, at conferences and published in peer-reviewed journals.Trial registration number NCT01735084.

BackgroundThe safety of methods of labor induction in women with previous cesarean deliveries is still debated. We investigated perinatal outcomes associated with labor induction among women with a trial of labor after one cesarean delivery.MethodsThis retrospective study included 339 women with a trial of labor after one prior cesarean and a singleton term fetus in cephalic presentation in 2013-2016 in a French maternity unit. Labor induction was performed with oxytocin, artificial rupture of membranes and/or prostaglandin E2, according to the Bishop score. The primary outcome was a composite of uterine rupture, low Apgar score, neonatal resuscitation or admission to a neonatal unit. The secondary outcomes included cesarean delivery after onset of labor, postpartum hemorrhage and maternal hospital stay after delivery. We used logistic regression to estimate odds ratios adjusted (aOR) for potential confounders.ResultsIn our sample, 67.3% of women had spontaneous labor and 32.7% were induced. More than half of the women received oxytocin during labor regardless of the mode of labor. The proportions of the composite outcome and of cesarean after onset of labor were higher in the induced group compared to the spontaneous group (26.1% vs 15.8%, p = 0.02 and 45.0% vs 27.6%, pConclusionLabor induction after one prior cesarean was associated with a higher risk of adverse perinatal outcome. Nevertheless, the higher proportion of uterine rupture did not differ significantly from that in the spontaneous labor group.

Plasmodium falciparum is the major cause of malaria globally and is transmitted by mosquitoes. During parasitic development, P. falciparum-infected erythrocytes (P. falciparum-IEs) express multiple polymorphic proteins known as variant surface antigens (VSAs), including the P. falciparum erythrocyte membrane protein 1 (PfEMP1). VSA-specific antibodies are associated with protection from symptomatic and severe malaria. However, the importance of the different VSA targets of immunity to malaria remains unclear, which has impeded an understanding of malaria immunity and vaccine development. In this study, we developed assays using transgenic P. falciparum with modified PfEMP1 expression to quantify serum antibodies to VSAs among individuals exposed to malaria. We found that the majority of the human antibody response to the IE targets PfEMP1. Furthermore, our longitudinal studies showed that individuals with PfEMP1-specific antibodies had a significantly reduced risk of developing symptomatic malaria, whereas antibodies to other surface antigens were not associated with protective immunity. Using assays that measure antibody-mediated phagocytosis of IEs, an important mechanism in parasite clearance, we identified PfEMP1 as the major target of these functional antibodies. Taken together, these data demonstrate that PfEMP1 is a key target of humoral immunity. These findings advance our understanding of the targets and mediators of human immunity to malaria and have major implications for malaria vaccine development. [ABSTRACT FROM AUTHOR]

A letter to the editor related to a study revealing that the rapid eye movement sleep behavior disorder (RBD) is an early precursor of the Parkinson's disease, is presented.

Summary: Background: Human immune responses to infection and vaccination are heterogenous, driven by multiple factors including genetics, environmental exposures and personal infection histories. For malaria caused by Plasmodium falciparum parasites, host factors that impact on humoral immunity are poorly understood. Methods: We investigated the role of latent cytomegalovirus (CMV) on the host immune response to malaria using samples obtained from individuals in previously conducted Phase 1 trials of blood stage P. falciparum Controlled Human Malaria Infection (CHMI) and in a MSP1 vaccine clinical trial. Induced antibody and functions of antibodies, as well as CD4 T cell responses were quantified. Findings: CMV seropositivity was associated with reduced induction of parasite specific antibodies following malaria infection and vaccination. During infection, reduced antibody induction was associated with modifications to the T -follicular helper (Tfh) cell compartment. CMV seropositivity was associated with a skew towards Tfh1 cell subsets before and after malaria infection, and reduced activation of Tfh2 cells. Protective Tfh2 cell activation was only associated with antibody development in individuals who were CMV seronegative, and a higher proportion of Tfh1 cells was associated with lower antibody development in individuals who were CMV seropositive. During MSP1 vaccination, reduced antibody induction in individuals who were CMV seropositive was associated with CD4 T cell expression of terminal differentiation marker CD57. Interpretation: These findings suggest that CMV seropositivity may be negatively associated with malaria antibody development. Further studies in larger cohorts, particularly in malaria endemic regions are required to investigate whether CMV infection may modify immunity to malaria gained during infection or vaccination in children. Funding: Work was funded by National Health and Medical Research Council of Australia, CSL Australia and Snow Medical Foundation. Funders had no role in data generation, writing of manuscript of decision to submit for publication.

BACKGROUND: The objective is to estimate the cost-effectiveness of an intervention that reduces hospital re-admission among older people at high risk. A cost-effectiveness model to estimate the costs and health benefits of the intervention was implemented. METHODOLOGY/PRINCIPAL FINDINGS: The model used data from a randomised controlled trial conducted in an Australian tertiary metropolitan hospital. Participants were acute medical admissions aged >65 years with at least one risk factor for re-admission: multiple comorbidities, impaired functionality, aged >75 years, recent multiple admissions, poor social support, history of depression. The intervention was a comprehensive nursing and physiotherapy assessment and an individually tailored program of exercise strategies and nurse home visits with telephone follow-up; commencing in hospital and continuing following discharge for 24 weeks. The change to cost outcomes, including the costs of implementing the intervention and all subsequent use of health care services, and, the change to health benefits, represented by quality adjusted life years, were estimated for the intervention as compared to existing practice. The mean change to total costs and quality adjusted life years for an average individual over 24 weeks participating in the intervention were: cost savings of $333 (95% Bayesian credible interval $ -1,932:1,282) and 0.118 extra quality adjusted life years (95% Bayesian credible interval 0.1:0.136). The mean net-monetary-benefit per individual for the intervention group compared to the usual care condition was $7,907 (95% Bayesian credible interval $5,959:$9,995) for the 24 week period. CONCLUSIONS/SIGNIFICANCE: The estimation model that describes this intervention predicts cost savings and improved health outcomes. A decision to remain with existing practices causes unnecessary costs and reduced health. Decision makers should consider adopting this program for elderly hospitalised patients.

Despite being key drivers of protective antibodies against malaria, little is known regarding the host and parasite factors that influence CD4 T-follicular helper cell and antibody development. Authors utilise samples from a study of children living in an area of high malaria transmission in Uganda, to characterize Tfh cells and functional antibodies to multiple parasites stages.

Abstract Background There is a clear need for novel approaches to malaria vaccine development. We aimed to develop a genetically attenuated blood-stage vaccine and test its safety, infectivity, and immunogenicity in healthy volunteers. Our approach was to target the gene encoding the knob-associated histidine-rich protein (KAHRP), which is responsible for the assembly of knob structures at the infected erythrocyte surface. Knobs are required for correct display of the polymorphic adhesion ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1), a key virulence determinant encoded by a repertoire of var genes. Methods The gene encoding KAHRP was deleted from P. falciparum 3D7 and a master cell bank was produced in accordance with Good Manufacturing Practice. Eight malaria naïve males were intravenously inoculated (day 0) with 1800 (2 subjects), 1.8 × 105 (2 subjects), or 3 × 106 viable parasites (4 subjects). Parasitemia was measured using qPCR; immunogenicity was determined using standard assays. Parasites were rescued into culture for in vitro analyses (genome sequencing, cytoadhesion assays, scanning electron microscopy, var gene expression). Results None of the subjects who were administered with 1800 or 1.8 × 105 parasites developed parasitemia; 3/4 subjects administered 3× 106 parasites developed significant parasitemia, first detected on days 13, 18, and 22. One of these three subjects developed symptoms of malaria simultaneously with influenza B (day 17; 14,022 parasites/mL); one subject developed mild symptoms on day 28 (19,956 parasites/mL); and one subject remained asymptomatic up to day 35 (5046 parasites/mL). Parasitemia rapidly cleared with artemether/lumefantrine. Parasitemia induced a parasite-specific antibody and cell-mediated immune response. Parasites cultured ex vivo exhibited genotypic and phenotypic properties similar to inoculated parasites, although the var gene expression profile changed during growth in vivo. Conclusions This study represents the first clinical investigation of a genetically attenuated blood-stage human malaria vaccine. A P. falciparum 3D7 kahrp– strain was tested in vivo and found to be immunogenic but can lead to patent parasitemia at high doses. Trial registration Australian New Zealand Clinical Trials Registry (number: ACTRN12617000824369 ; date: 06 June 2017).

Abstract Background The pathogenesis of malaria in pregnancy (MiP) involves accumulation of P. falciparum-infected red blood cells (pRBCs) in the placenta, contributing to poor pregnancy outcomes. Parasite accumulation is primarily mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1). Magnitude of IgG to pRBCs has been associated with reduced risk of MiP in some studies, but associations have been inconsistent. Further, antibody effector mechanisms are poorly understood, and the role of antibody complement interactions is unknown. Methods Studying a longitudinal cohort of pregnant women (n=302) from a malaria-endemic province in Papua New Guinea (PNG), we measured the ability of antibodies to fix and activate complement using placental binding pRBCs and PfEMP1 recombinant domains. We determined antibody-mediated complement inhibition of pRBC binding to the placental receptor, chondroitin sulfate A (CSA), and associations with protection against placental parasitemia. Results Some women acquired antibodies that effectively promoted complement fixation on placental-binding pRBCs. Complement fixation correlated with IgG1 and IgG3 antibodies, which dominated the response. There was, however, limited evidence for membrane attack complex activity or pRBC lysis or killing. Importantly, a higher magnitude of complement fixing antibodies was prospectively associated with reduced odds of placental infection at delivery. Using genetically modified P. falciparum and recombinant PfEMP1 domains, we found that complement-fixing antibodies primarily targeted a specific variant of PfEMP1 (known as VAR2CSA). Furthermore, complement enhanced the ability of antibodies to inhibit pRBC binding to CSA, which was primarily mediated by complement C1q protein. Conclusions These findings provide new insights into mechanisms mediating immunity to MiP and reveal potential new strategies for developing malaria vaccines that harness antibody-complement interactions.

Antibodies plays critical roles in the adaptive immune response to infectious agents including malaria. Here the authors defined antibody interactions with -Fcγ-receptors expressed on immune cells with sporozoites of Plasmodium falciparum, and identified specific target epitopes of antibodies.

IntroductionUnderstanding the human immune response to Plasmodium falciparum gametocytes and its association with gametocytemia is essential for understanding the transmission of malaria as well as progressing transmission blocking vaccine candidates.MethodsIn a multi-national clinical efficacy trial of artemisinin therapies (13 sites of varying transmission over South-East Asia and Africa), we measured Immunoglobulin G (IgG) responses to recombinant P. falciparum gametocyte antigens expressed on the gametocyte plasma membrane and leading transmission blocking vaccine candidates Pfs230 (Pfs230c and Pfs230D1M) and Pfs48/45 at enrolment in 1,114 participants with clinical falciparum malaria. Mixed effects linear and logistic regression were used to determine the association between gametocyte measures (gametocytemia and gametocyte density) and antibody outcomes at enrolment.ResultsMicroscopy detectable gametocytemia was observed in 11% (127/1,114) of participants at enrolment, and an additional 9% (95/1,114) over the follow-up period (up to day 42) (total 20% of participants [222/1,114]). IgG levels in response to Pfs230c, Pfs48/45 and Pfs230D1M varied across study sites at enrolment (p < 0.001), as did IgG seroprevalence for anti-Pfs230c and D1M IgG (p < 0.001), but not for anti-Pfs48/45 IgG (p = 0.159). In adjusted analyses, microscopy detectable gametocytemia at enrolment was associated with an increase in the odds of IgG seropositivity to the three gametocyte antigens (Pfs230c OR [95% CI], p: 1.70 [1.10, 2.62], 0.017; Pfs48/45: 1.45 [0.85, 2.46], 0.174; Pfs230D1M: 1.70 [1.03, 2.80], 0.037), as was higher gametocyte density at enrolment (per two-fold change in gametocyte density Pfs230c OR [95% CI], p: 1.09 [1.02, 1.17], 0.008; Pfs48/45: 1.05 [0.98, 1.13], 0.185; Pfs230D1M: 1.07 [0.99, 1.14], 0.071).ConclusionPfs230 and Pfs48/45 antibodies are naturally immunogenic targets associated with patent gametocytemia and increasing gametocyte density across multiple malaria endemic settings, including regions with emerging artemisinin-resistant P. falciparum.

Abstract Background Chimeric virus-like particles (VLP) allow the display of foreign antigens on their surface and have proved valuable in the development of safe subunit vaccines or drug delivery. However, finding an inexpensive production system and a VLP scaffold that allows stable incorporation of diverse, large foreign antigens are major challenges in this field. Results In this study, a versatile and cost-effective platform for chimeric VLP development was established. The membrane integral small surface protein (dS) of the duck hepatitis B virus was chosen as VLP scaffold and the industrially applied and safe yeast Hansenula polymorpha (syn. Pichia angusta, Ogataea polymorpha) as the heterologous expression host. Eight different, large molecular weight antigens of up to 412 amino acids derived from four animal-infecting viruses were genetically fused to the dS and recombinant production strains were isolated. In all cases, the fusion protein was well expressed and upon co-production with dS, chimeric VLP containing both proteins could be generated. Purification was accomplished by a downstream process adapted from the production of a recombinant hepatitis B VLP vaccine. Chimeric VLP were up to 95% pure on protein level and contained up to 33% fusion protein. Immunological data supported surface exposure of the foreign antigens on the native VLP. Approximately 40 mg of chimeric VLP per 100 g dry cell weight could be isolated. This is highly comparable to values reported for the optimized production of human hepatitis B VLP. Purified chimeric VLP were shown to be essentially stable for 6 months at 4 °C. Conclusions The dS-based VLP scaffold tolerates the incorporation of a variety of large molecular weight foreign protein sequences. It is applicable for the display of highly immunogenic antigens originating from a variety of pathogens. The yeast-based production system allows cost-effective production that is not limited to small-scale fundamental research. Thus, the dS-based VLP platform is highly efficient for antigen presentation and should be considered in the development of future vaccines.

Abstract Background Malaria in pregnancy is a major cause of poor maternal and infant health, and is associated with the sequestration of P. falciparum-infected erythrocytes (IE) in the placenta. The leading vaccine candidate for pregnancy malaria, VAR2CSA, has been shown to induce antibodies that inhibit IE adhesion to the placental receptor chondroitin sulfate A (CSA), potentially preventing placental infection. However, the ability of vaccination-induced antibodies to promote opsonic phagocytosis is not well defined, but likely to be an important component of protective immunity. Methods We investigated the use of an opsonic phagocytosis assay to evaluate antibodies induced by pregnancy malaria vaccine candidate antigens based on VAR2CSA. Opsonic phagocytosis was measured by flow cytometry and visualized by electron microscopy. We measured vaccine-induced antibody reactivity to placental type IEs from different geographical origins, and the functional ability of antibodies raised in immunized rabbits to induce phagocytosis by a human monocyte cell line. Results Immunization-induced antibodies showed a mixture of strain-specific and cross-reactive antibody recognition of different placental-binding parasite lines. Antibodies generated against the DBL5 and DBL3 domains of VAR2CSA effectively promoted the opsonic phagocytosis of IEs by human monocytes; however, these functional antibodies were largely allele-specific and not cross-reactive. This has significant implications for the development of vaccines aiming to achieve a broad coverage against diverse parasite strains. Using competition ELISAs, we found that acquired human antibodies among pregnant women targeted both cross-reactive and allele-specific epitopes, consistent with what we observed with vaccine-induced antibodies. Conclusions Vaccines based on domains of VAR2CSA induced opsonic phagocytosis of IEs in a strain-specific manner. Assays measuring this phagocytic activity have the potential to aid the development and evaluation of vaccines against malaria in pregnancy.

The development of effective malaria vaccines remains a global health priority. Currently, the most advanced vaccine, known as RTS,S, has only shown modest efficacy in clinical trials. Thus, the development of more efficacious vaccines by improving the formulation of RTS,S for increased efficacy or to interrupt malaria transmission are urgently needed. The RTS,S vaccine is based on the presentation of a fragment of the sporozoite antigen on the surface of virus-like particles (VLPs) based on human hepatitis B virus (HBV). In this study, we have developed and evaluated a novel VLP platform based on duck HBV (known as Metavax) for malaria vaccine development. This platform can incorporate large and complex proteins into VLPs and is produced in a Hansenula cell line compatible with cGMP vaccine production. Here, we have established the expression of leading P. falciparum malaria vaccine candidates as VLPs. This includes Pfs230 and Pfs25, which are candidate transmission-blocking vaccine antigens. We demonstrated that the VLPs effectively induce antibodies to malaria vaccine candidates with minimal induction of antibodies to the duck-HBV scaffold antigen. Antibodies to Pfs230 also recognised native protein on the surface of gametocytes, and antibodies to both Pfs230 and Pfs25 demonstrated transmission-reducing activity in standard membrane feeding assays. These results establish the potential utility of this VLP platform for malaria vaccines, which may be suitable for the development of multi-component vaccines that achieve high vaccine efficacy and transmission-blocking immunity.

BACKGROUND:Malaria caused by Plasmodium falciparum is one of the major threats to human health globally. Despite huge efforts in malaria control and eradication, highly effective vaccines are urgently needed, including vaccines that can block malaria transmission. Chimeric virus-like particles (VLP) have emerged as a promising strategy to develop new malaria vaccine candidates. METHODS:We developed yeast cell lines and processes for the expression of malaria transmission-blocking vaccine candidates Pfs25 and Pfs230 as VLP and VLP were analyzed for purity, size, protein incorporation rate and expression of malaria antigens. RESULTS:In this study, a novel platform for the display of Plasmodium falciparum antigens on chimeric VLP is presented. Leading transmission-blocking vaccine candidates Pfs25 and Pfs230 were genetically fused to the small surface protein (dS) of the duck hepatitis B virus (DHBV). The resulting fusion proteins were co-expressed in recombinant Hansenula polymorpha (syn. Pichia angusta, Ogataea polymorpha) strains along with the wild-type dS as the VLP scaffold protein. Through this strategy, chimeric VLP containing Pfs25 or the Pfs230-derived fragments Pfs230c or Pfs230D1M were purified. Up to 100 mg chimeric VLP were isolated from 100 g dry cell weight with a maximum protein purity of 90% on the protein level. Expression of the Pfs230D1M construct was more efficient than Pfs230c and enabled VLP with higher purity. VLP showed reactivity with transmission-blocking antibodies and supported the surface display of the malaria antigens on the native VLP. CONCLUSION:The incorporation of leading Plasmodium falciparum transmission-blocking antigens into the dS-based VLP scaffold is a promising novel strategy for their display on nano-scaled particles. Competitive processes for efficient production and purification were established in this study.

Vaccines that target Plasmodium falciparum gametocytes have the potential to reduce malaria transmission and are thus attractive targets for malaria control. However, very little is known about human immune responses to gametocytes present in human hosts. We evaluated naturally-acquired antibodies to gametocyte-infected erythrocytes (gametocyte-IEs) of different developmental stages compared to other asexual parasite stages among naturally-exposed Kenyan residents. We found that acquired antibodies strongly recognized the surface of mature asexual-IEs, but there was limited reactivity to the surface of gametocyte-IEs of different stages. We used genetically-modified P. falciparum with suppressed expression of PfEMP1, the major surface antigen of asexual-stage IEs, to demonstrate that PfEMP1 is a dominant target of antibodies to asexual-IEs, in contrast to gametocyte-IEs. Antibody reactivity to gametocyte-IEs was similar to asexual-IEs lacking PfEMP1. Significant antibody reactivity to the surface of gametocytes was observed when outside of the host erythrocyte, including recognition of the major gametocyte antigen, Pfs230. This indicates that there is a deficiency of acquired antibodies to gametocyte-IEs despite the acquisition of antibodies to gametocyte antigens and asexual IEs. Our findings suggest that the acquisition of substantial immunity to the surface of gametocyte-IEs is limited, which may facilitate immune evasion to enable malaria transmission even in the face of substantial host immunity to malaria. Further studies are needed to understand the basis for the limited acquisition of antibodies to gametocytes and whether vaccine strategies can generate substantial immunity.

Malaria is caused by five different Plasmodium spp. in humans each of which modifies the host erythrocyte to survive and replicate. The two main causes of malaria, P. falciparum and P. vivax, differ in their ability to cause severe disease, mainly due to differences in the cytoadhesion of infected erythrocytes (IE) in the microvasculature. Cytoadhesion of P. falciparum in the brain leads to a large number of deaths each year and is a consequence of exported parasite proteins, some of which modify the erythrocyte cytoskeleton while others such as PfEMP1 project onto the erythrocyte surface where they bind to endothelial cells. Here we investigate the effects of knocking out an exported Hsp70-type chaperone termed Hsp70-x that is present in P. falciparum but not P. vivax. Although the growth of Δhsp70-x parasites was unaffected, the export of PfEMP1 cytoadherence proteins was delayed and Δhsp70-x IE had reduced adhesion. The Δhsp70-x IE were also more rigid than wild-type controls indicating changes in the way the parasites modified their host erythrocyte. To investigate the cause of this, transcriptional and translational changes in exported and chaperone proteins were monitored and some changes were observed. We propose that PfHsp70-x is not essential for survival in vitro, but may be required for the efficient export and functioning of some P. falciparum exported proteins.