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Radical cure of Plasmodium vivax malaria must include elimination of quiescent "hypnozoite" forms in the liver; however, the only FDA-approved treatments are contraindicated in many vulnerable populations. To identify new drugs and drug targets, we screened the Repurposing, Focused Rescue, and Accelerated Medchem library against P. vivax liver stages and identified the DNA methyltransferase inhibitors hydralazine and cadralazine as active against hypnozoites. We then used bisulfite sequencing and immunostaining to identify cytosine modifications in the infectious stage (sporozoites) and liver stages, respectively. A subsequent screen of epigenetic inhibitors revealed hypnozoites are broadly sensitive to histone acetyltransferase and methyltransferase inhibitors, indicating that several epigenetic mechanisms are likely modulating hypnozoite persistence. Our data present an avenue for the discovery and development of improved radical cure antimalarials.

Introduction: Emergency departments (ED) are rapidly replacing conventional troponin assays with high-sensitivity troponin tests. We sought to evaluate emergency physician utilization of troponin tests before and after high-sensitivity troponin introduction in our ED. Methods: We retrospectively examined 9,477 ED encounters, identifying the percentage in which physicians ordered a serum troponin both before and after our institution adopted a high-sensitivity troponin test. Results: After introduction of high-sensitivity troponin testing, the percentage of ED encountersin which physicians ordered troponin studies decreased (28.3% before vs 22% after; P

To discover leads for next-generation chemoprotective antimalarial drugs, we tested more than 500,000 compounds for their ability to inhibit liver-stage development of luciferase-expressing Plasmodium spp. parasites (681 compounds showed a half-maximal inhibitory concentration of less than 1 micromolar). Cluster analysis identified potent and previously unreported scaffold families as well as other series previously associated with chemoprophylaxis. Further testing through multiple phenotypic assays that predict stage-specific and multispecies antimalarial activity distinguished compound classes that are likely to provide symptomatic relief by reducing asexual blood-stage parasitemia from those which are likely to only prevent malaria. Target identification by using functional assays, in vitro evolution, or metabolic profiling revealed 58 mitochondrial inhibitors but also many chemotypes possibly with previously unidentified mechanisms of action.

Background: Malaria, a disease caused by parasites of the genus Plasmodium, continues to impact many regions globally. The rise in resistance to artemisinin-based anti-malarial drugs highlights the need for new treatments. Ideally, new anti-malarials will kill the asymptomatic liver stages as well as the symptomatic blood stages. While blood stage screening assays are routine and efficient, liver stage screening assays are more complex and costly. To decrease the cost of liver stage screening, a previously reported luciferase detection protocol requiring only common laboratory reagents was adapted for testing against luciferase-expressing Plasmodium berghei liver stage parasites. Methods: After optimizing cell lysis conditions, the concentration of reagents, and the density of host hepatocytes (HepG2), the protocol was validated with 28 legacy anti-malarials to show this simple protocol produces a stable signal useful for obtaining quality small molecule potency data similar to that obtained from a high content imaging endpoint. The protocol was then used to screen the Global Health Priority Box (GHPB) and confirm the potency of hits in dose–response assays. Selectivity was determined using a galactose-based, 72 h HepG2 assay to avoid missing mitochondrial-toxic compounds due to the Crabtree effect. Receiver-operator characteristic plots were used to retroactively characterize the screens' predictive value. Results: Optimal luciferase signal was achieved using a lower HepG2 seed density (5 × 103 cells/well of a 384-well microtitre plate) compared to many previously reported luciferase-based screens. While producing lower signal compared to a commercial alternative, this luciferase detection method was found much more stable, with a > 3 h half-life, and robust enough for producing dose–response plots with as few as 500 sporozoites/well. A screen of the GHPB resulted in 9 hits with selective activity against P. berghei liver schizonts, including MMV674132 which exhibited 30.2 nM potency. Retrospective analyses show excellent predictive value for both anti-malarial activity and cytotoxicity. Conclusions: This method is suitable for high-throughput screening at a cost nearly 20-fold less than using commercial luciferase detection kits, thereby enabling larger liver stage anti-malarial screens and hit optimization make-test cycles. Further optimization of the hits detected using this protocol is ongoing. [ABSTRACT FROM AUTHOR]

Introduction: Patients with malignancy represent a particular challenge for the emergency department (ED) given their higher acuity, longer ED length of stay, and higher admission rate. It is unknown if patients with malignancies and hyperlactatemia are at increased risk of mortality. If serum lactic acid could improve detection of at-risk patients with cancer, it would be useful in risk stratification. There is also little evidence that “alarm” values of serum lactate (such as >/=4 mmol/L) are appropriate for the population of patients with cancer. Methods: This was a continuous retrospective cohort study of approximately two years (2012-2014) at a single, tertiary hospital ED; 5,440 patients had serum lactic acid measurements performed in the ED. Of the 5,440 patients in whom lactate was drawn, 1,837 were cancer patients, and 3,603 were non-cancer patients.Cumulative unadjusted mortality (determined by hospital records and an external death tracking system) was recorded at one day, three days, seven days, and 30 days. We used logistic regression to examine the risk of mortality 30 days after the ED visit after adjusting for confounders.  Results: In an unadjusted analysis, we found no statistically significant difference in the mortality of cancer vs. non-cancer patients at one day and three days. Significant differences in mortality were found at seven days (at lactate levels of

Abstract Background Much of the Plasmodium falciparum genome encodes hypothetical proteins with limited homology to other organisms. A lack of robust tools for genetic manipulation of the parasite limits functional analysis of these hypothetical proteins and other aspects of the Plasmodium genome. Transposon mutagenesis has been used widely to identify gene functions in many organisms and would be extremely valuable for functional analysis of the Plasmodium genome. Results In this study, we investigated the lepidopteran transposon, piggyBac, as a molecular genetic tool for functional characterization of the Plasmodium falciparum genome. Through multiple transfections, we generated 177 unique P. falciparum mutant clones with mostly single piggyBac insertions in their genomes. Analysis of piggyBac insertion sites revealed random insertions into the P. falciparum genome, in regards to gene expression in parasite life cycle stages and functional categories. We further explored the possibility of forward genetic studies in P. falciparum with a phenotypic screen for attenuated growth, which identified several parasite genes and pathways critical for intra-erythrocytic development. Conclusion Our results clearly demonstrate that piggyBac is a novel, indispensable tool for forward functional genomics in P. falciparum that will help better understand parasite biology and accelerate drug and vaccine development.

Abstract Background Cloning of parasites by limiting dilution is an essential and rate-limiting step in many aspects of malaria research including genomic and genetic manipulation studies. The standard Giemsa-stained blood smears to detect parasites is time-consuming, whereas the more sensitive parasite lactate dehydrogenase assay involves multiple steps and requires fresh reagents. A simple PCR-based method was therefore tested for parasite detection that can be adapted to high throughput studies. Methods Approximately 1 μL of packed erythrocytes from each well of a microtiter cloning plate was directly used as template DNA for a PCR reaction with primers for the parasite 18s rRNA gene. Positive wells containing parasites were identified after rapid separation of PCR products by gel electrophoresis. Results The PCR-based method can consistently detect a parasitaemia as low as 0.0005%, which is equivalent to 30 parasite genomes in a single well of a 96-well plate. Parasite clones were easily detected from cloning plates using this method and a comparison of PCR results with Giemsa-stained blood smears showed that PCR not only detected all the positive wells identified in smears, but also detected wells not identified otherwise, thereby confirming its sensitivity. Conclusion The PCR-based method reported here is a simple, sensitive and efficient method for detecting parasite clones in culture. This method requires very little manual labor and can be completely automated for high throughput studies. The method is sensitive enough to detect parasites a week before they can be seen in Giemsa smears and is highly effective in identifying slow growing parasite clones.

Objective: To describe the relationship between emergency department resource utilization and admission rate at the level of the individual physician.Methods: Retrospective observational study of physician resource utilization and admitting data at two emergency departments. We calculated observed to expected (O/E) ratios for four measures of resource utilization (intravenous medications and fluids, laboratory testing, plain radiographs, and advanced imaging studies) as well as for admission rate. Expected values reflect adjustment for patient- and time-based variables. We compared O/E ratios for each type of resource utilization to the O/E ratio for admission for each provider. We report degree of correlation (slope of the trendline) and strength of correlation (adjusted R2 value) for each association, as well as categorical results after clustering physicians based on the relationship of resource utilization to admission rate.Results: There were statistically significant positive correlations between resource utilization and physician admission rate. Physicians with lower resource utilization rates were more likely to have lower admission rates, and those with higher resource utilization rates were more likely to have higher admission rates.Conclusions: In a two-facility study, emergency physician resource utilization and admission rate were positively correlated: those who used more ED resources also tended to admit more patients. These results add to a growing understanding of emergency physician variability. [ABSTRACT FROM AUTHOR]

Malaria liver stages represent an ideal therapeutic target with a bottleneck in parasite load and reduced clinical symptoms; however, current in vitro pre-erythrocytic (PE) models for Plasmodium vivax and P. falciparum lack the efficiency necessary for rapid identification and effective evaluation of new vaccines and drugs, especially targeting late liver-stage development and hypnozoites. Herein we report the development of a 384-well plate culture system using commercially available materials, including cryopreserved primary human hepatocytes. Hepatocyte physiology is maintained for at least 30 days and supports development of P. vivax hypnozoites and complete maturation of P. vivax and P. falciparum schizonts. Our multimodal analysis in antimalarial therapeutic research identifies important PE inhibition mechanisms: immune antibodies against sporozoite surface proteins functionally inhibit liver stage development and ion homeostasis is essential for schizont and hypnozoite viability. This model can be implemented in laboratories in disease-endemic areas to accelerate vaccine and drug discovery research. [ABSTRACT FROM AUTHOR]

Background Plasmodium vivax preferentially infects Duffy-positive reticulocytes and infections typically have few parasite-infected cells in the peripheral circulation. These features complicate detection and quantification by flow cytometry (FC) using standard nucleic acid-based staining methods. A simple antibody-based FC method was developed for rapid parasite detection along with simultaneous detection of other parasite and erythrocyte markers. Methods Clinical samples were collected from patients diagnosed with P. vivax at a district Malaria Clinic in Kanchanaburi, Thailand. One µL of infected blood was washed, fixed, stained with a Plasmodium pan-specific anti-PfBiP antibody conjugated with Alexa Fluor 660, and analysed by FC. Additional primary conjugated antibodies for stage-specific markers of P. vivax for late trophozoite-early schizonts (MSP1-Alexa Fluor 660), late-stage schizonts (DBP-Alexa Fluor 555), and sexual stages (Pvs16) were used to differentiate intraerythrocytic developmental stages. Results The percentages of P. vivax-infected cells determined by the FC method and manually by microscopic examination of Giemsa-stained thick blood smears were positively correlated by Spearman's rank correlation coefficient (R2 = 0.93843) from 0.001 to 1.00% P. vivax-infected reticulocytes. Conclusions The FC-based method is a simple, robust, and efficient method for detecting P. vivax-infected reticulocytes. [ABSTRACT FROM AUTHOR]

The article presents a discussion concerning risk management and inherently safer design (ISD) phases in plants in the chemical process industries (CPI). The authors examine the front-end engineering and design (FEED) phase, hazard identification studies (HAZID), and conceptual design. The authors suggest that CPI companies actively seek ways to simplify complex designs during the detailed design phase.