Using Plasmodium knowlesi as a model for screening Plasmodium vivax blood-stage malaria vaccine targets reveals new candidates.

Plasmodium vivax is responsible for the majority of malaria cases outside Africa. Unlike P. falciparum, the P. vivax life-cycle includes a dormant liver stage, the hypnozoite, which can cause infection in the absence of mosquito transmission. An effective vaccine against P. vivax blood stages would limit symptoms and pathology from such recurrent infections, and therefore could play a critical role in the control of this species. Vaccine development in P. vivax, however, lags considerably behind P. falciparum, which has many identified targets with several having transitioned to Phase II testing. By contrast only one P. vivax blood-stage vaccine candidate based on the Duffy Binding Protein (PvDBP), has reached Phase Ia, in large part because the lack of a continuous in vitro culture system for P. vivax limits systematic screening of new candidates. We used the close phylogenetic relationship between P. vivax and P. knowlesi, for which an in vitro culture system in human erythrocytes exists, to test the scalability of systematic reverse vaccinology to identify and prioritise P. vivax blood-stage targets. A panel of P. vivax proteins predicted to function in erythrocyte invasion were expressed as full-length recombinant ectodomains in a mammalian expression system. Eight of these antigens were used to generate polyclonal antibodies, which were screened for their ability to recognize orthologous proteins in P. knowlesi. These antibodies were then tested for inhibition of growth and invasion of both wild type P. knowlesi and chimeric P. knowlesi lines modified using CRISPR/Cas9 to exchange P. knowlesi genes with their P. vivax orthologues. Candidates that induced antibodies that inhibited invasion to a similar level as PvDBP were identified, confirming the utility of P. knowlesi as a model for P. vivax vaccine development and prioritizing antigens for further follow up.European Union, National Institutes of Health (US

The Malaria Cell Atlas: single parasite transcriptomes across the complete Plasmodium life cycle

Malaria parasites adopt a remarkable variety of morphological life stages as they transition through multiple mammalian host and mosquito vector environments. We profiled the single-cell transcriptomes of thousands of individual parasites, deriving the first high-resolution transcriptional atlas of the entire life cycle. We then used our atlas to precisely define developmental stages of single cells from three different human malaria parasite species, including parasites isolated directly from infected individuals. The Malaria Cell Atlas provides both a comprehensive view of gene usage in a eukaryotic parasite and an open-access reference dataset for the study of malaria parasites

Plasmodium knowlesi as a model system for characterising Plasmodium vivax drug resistance candidate genes.

Plasmodium vivax causes the majority of malaria outside Africa, but is poorly understood at a cellular level partly due to technical difficulties in maintaining it in in vitro culture conditions. In the past decades, drug resistant P. vivax parasites have emerged, mainly in Southeast Asia, but while some molecular markers of resistance have been identified, none have so far been confirmed experimentally, which limits interpretation of the markers, and hence our ability to monitor and control the spread of resistance. Some of these potential markers have been identified through P. vivax genome-wide population genetic analyses, which highlighted genes under recent evolutionary selection in Southeast Asia, where chloroquine resistance is most prevalent. These genes could be involved in drug resistance, but no experimental proof currently exists to support this hypothesis. In this study, we used Plasmodium knowlesi, the most closely related species to P. vivax that can be cultured in human erythrocytes, as a model system to express P. vivax genes and test for their role in drug resistance. We adopted a strategy of episomal expression, and were able to express fourteen P. vivax genes, including two allelic variants of several hypothetical resistance genes. Their expression level and localisation were assessed, confirming cellular locations conjectured from orthologous species, and suggesting locations for several previously unlocalised proteins, including an apical location for PVX_101445. These findings establish P. knowlesi as a suitable model for P. vivax protein expression. We performed chloroquine and mefloquine drug assays, finding no significant differences in drug sensitivity: these results could be due to technical issues, or could indicate that these genes are not actually involved in drug resistance, despite being under positive selection pressure in Southeast Asia. These data confirm that in vitro P. knowlesi is a useful tool for studying P. vivax biology. Its close evolutionary relationship to P. vivax, high transfection efficiency, and the availability of markers for colocalisation, all make it a powerful model system. Our study is the first of its kind using P. knowlesi to study unknown P. vivax proteins and investigate drug resistance mechanisms

Association Between Hospital Performance on Patient Safety and 30‐Day Mortality and Unplanned Readmission for Medicare Fee‐for‐Service Patients With Acute Myocardial Infarction

Background: Little is known regarding the relationship between hospital performance on adverse event rates and hospital performance on 30‐day mortality and unplanned readmission rates for Medicare fee‐for‐service patients hospitalized for acute myocardial infarction (AMI). Methods and Results: Using 2009–2013 medical record‐abstracted patient safety data from the Agency for Healthcare Research and Quality's Medicare Patient Safety Monitoring System and hospital mortality and readmission data from the Centers for Medicare & Medicaid Services, we fitted a mixed‐effects model, adjusting for hospital characteristics, to evaluate whether hospital performance on patient safety, as measured by the hospital‐specific risk‐standardized occurrence rate of 21 common adverse event measures for which patients were at risk, is associated with hospital‐specific 30‐day all‐cause risk‐standardized mortality and unplanned readmission rates for Medicare patients with AMI. The unit of analysis was at the hospital level. The final sample included 793 acute care hospitals that treated 30 or more Medicare patients hospitalized for AMI and had 40 or more adverse events for which patients were at risk. The occurrence rate of adverse events for which patients were at risk was 3.8%. A 1% point change in the risk‐standardized occurrence rate of adverse events was associated with average changes in the same direction of 4.86% points (95% CI, 0.79–8.94) and 3.44% points (95% CI, 0.19–6.68) for the risk‐standardized mortality and unplanned readmission rates, respectively. Conclusions: For Medicare fee‐for‐service patients discharged with AMI, hospitals with poorer patient safety performance were also more likely to have poorer performance on 30‐day all‐cause mortality and on unplanned readmissions