41 research outputs found

    MOESM1 of Identifying risk factors for the development of sepsis during adult severe malaria

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    Additional file 1. Sociodemographic and clinical characteristics of 86 patients with a clinical diagnosis of sepsis among patients diagnosed with severe malaria

    Additional file 2: of Performance of critical care prognostic scoring systems in low and middle-income countries: a systematic review

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    A table presenting the checklist for critical appraisal and data extraction for systematic reviews of prediction modelling studies. (XLSX 41 kb

    CNV profiling of <i>in vitro-</i>selected <i>P</i>. <i>falciparum</i>.

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    <p>Copy number variations in 6A-R and 11C-R were identified using microarray-based comparative genomic hybridization (CGH). Chromosome plots reflect the subtracted log<sub>2</sub>ratio of the artemisinin-resistant parasite lines relative to their control counterparts. Copy number variable genes in 6A-R vs. 6A, and 11C-R vs 11C are indicated in the red and green boxes, respectively. Additional data can be found in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006930#ppat.1006930.s011" target="_blank">S5 Fig</a></b> and <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006930#ppat.1006930.s012" target="_blank">S6 Table</a></b>.</p

    Effect of overexpression of <i>pftrx1</i>, <i>pf6Pgd</i> and <i>pfspp</i> on artemisinin sensitivity.

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    <p>To validate the role of stress response gene overexpression on artemisinin resistance, transfectant lines overexpressing select candidate genes were generated, and subsequently assayed for artemisinin sensitivity. <b>(A)</b>Differential mRNA expression in the copy number-amplified genes identified in chromosomes in 10, 12, and 14 was evaluated between artemisinin-resistant parasite lines and their corresponding controls. <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006930#ppat.1006930.s012" target="_blank">S6 Table</a></b> lists down corrected p- and FDR values for each gene in the Chr 10, Chr 12 and Chr 14 CNV clusters identified. Chromosome plots depict the z-score calculated for each gene based on differences in expression levels between resistant and sensitive parasites across the IDC, while the heatmaps represent the fold-difference between resistant and sensitive parasites for each gene at 6 timepoints taken at 8-hour intervals across a single IDC. Also indicated are the Normalized Enrichment Score (NES) values for transcriptional upregulation in each cluster, obtained by GSEA (p-value < 0.05, FDR < 0.25). Marked in red boxes are candidate stress response genes that were found to be significantly upregulated across the IDC (corrected p-value < 0.05, FDR < 0.25) and amplified in 6A-R. These three genes (PF3D7_1454700 (<i>pf6pgd</i>), PF3D7_1457000 (<i>pfspp)</i>, and PF3D7_1457200 (<i>pftrx1</i>)) were subsequently episomally overexpressed and investigated for their capacity to modulate artemisinin sensitivity. Prior to phenotyping, <b>(B)</b>Real-time qPCR was used to determine the relative overexpression of <i>pftrx1</i>, <i>pf6pgd</i> and <i>pfspp</i> from their respective overexpression parasite lines. Mean fold-change values are derived from three biological replicates; error bars represent the standard deviation. <b>(C)</b>Western blot analysis was also carried out on all overexpression and control parasite lines using a monoclonal mouse anti-HA antibody to validate tagged-protein production. Bands denoted by red arrows indicate the tagged proteins at their expected molecular weights. <b>(D)</b>Ring-stage artemisinin sensitivity (IC50<sub>10hpi/4hr</sub>) was then measured for all overexpression parasite lines and compared against the vector control. Drug assays were performed in biological triplicates; error bars represent the standard deviation. Pairwise comparison of IC50<sub>10hpi/4hr</sub> between each overexpression line and the vector control was performed using student’s t-test. Additional data can be found in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006930#ppat.1006930.s012" target="_blank"><b>S6</b> Fig</a> and <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006930#ppat.1006930.s013" target="_blank">S7 Table</a></b>.</p

    Chemosensitivity profiling of <i>in vitro</i>-selected <i>P</i>. <i>falciparum</i>.

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    <p>Chemosensitivity phenotypes of our artemisinin-resistant parasites were evaluated. <b>(A)</b>Apart from their ring-stage sensitivity, the artemisinin IC50<sub>4hr</sub> for the trophozoite (IC50<sub>20hpi/4hr</sub>) and schizont (IC50<sub>30hpi/4hr</sub>) stages was also measured in all parasite lines. Differential susceptibility to artemisinin between selected and control parasites, was subsequently validated using a standard <i>in vitro</i> (72-hour) drug assay (IC50) <b>(B)</b>, and the Ring stage Survival Assay (RSA) <b>(C)</b>. Additional data can be found in <b>Figs <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006930#ppat.1006930.g002" target="_blank">2A</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006930#ppat.1006930.s002" target="_blank">S2B</a></b> and <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006930#ppat.1006930.s008" target="_blank">S2 Table</a></b>. <b>(D)</b> Ring-stage susceptibility (IC50<sub>10hpi/4hr</sub>) against a pulse exposure to artemisinin derivatives Dihydroartemisinin (DHA) and Artesunate (ATS) were also monitored, while chemosensitivity against non-artemisinin antimalarials, quinine (QN), chloroquine (CQ), mefloquine (MEF) and pyrimethamine (PYR), were evaluated using a standard drug assay (IC50). Additional data can be found in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006930#ppat.1006930.s009" target="_blank">S3A and S3B Fig</a></b> and <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006930#ppat.1006930.s009" target="_blank">S3 Table</a></b>. <b>(E)</b>Apart from antimalarial drugs, the ring-stage sensitivity of all parasite lines was also measured for compounds that are related to the mechanism of action of artemisinin: hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>), dithiothreitol (DTT), and epoxomicin (EPX). Additional data can be found in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006930#ppat.1006930.s009" target="_blank">S3C Fig</a></b> and <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006930#ppat.1006930.s009" target="_blank">S3 Table</a></b>. All drug assays were performed in biological triplicates; error bars represent the standard deviation. Pairwise comparison of percent survival (RSA), IC50<sub>10hpi/4hr</sub> and IC50 values between resistant and sensitive parasites was performed using student’s t-test.</p

    <i>In vitro</i> selection of artemisinin resistance in two <i>P</i>. <i>falciparum</i> clones 6A and 11C.

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    <p>Artemisinin resistance was induced in two subclones (6A and 11C) of the 3D7 strain of <i>P</i>. <i>falciparum</i> through periodic exposure of the parasite to short pulses of a clinically relevant dose of artemisinin. <b>(A)</b>The <i>in vitro</i> artemisinin selection protocol involved repeated 4-hour pulse treatments of synchronized mid-ring stage parasites (6A and 11C) to 900 nM artemisinin. DMSO-treated parasites were grown alongside the artemisinin-treated parasites (renamed as 6A-R and 11C-R) to serve as controls. Both sets of parasites were subjected to the same number of artemisinin and DMSO treatments throughout drug selection, and at the same generations. Stage-specific artemisinin sensitivity was monitored throughout the course of selection using a 4-hour drug pulse assay at the ring (IC50<sub>10hpi/4hr</sub>), trophozoite (IC50<sub>20hpi/4hr</sub>), and schizont (IC50<sub>30hpi/4hr</sub>) stages of the IDC. <b>(B)</b>In order to monitor incremental changes in ring stage artemisinin sensitivity over time, artemisinin IC50<sub>10hpi/4hr</sub> was measured throughout increasing cycles of drug selection between artemisinin-treated parasites and their controls. Additional data can be found in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006930#ppat.1006930.s007" target="_blank">S1 Table</a></b>. <b>(C)</b>At the start of artemisinin selection, parasite viability and morphology after 4-hour treatment was monitored using microscopic evaluation of Giemsa-stained blood smears. The solid gray line depicts the proportion of surviving parasites 24 hours post treatment normalized to the starting parasitemia, while stacked bars depict proportions of ring, trophozoite and schizont stage morphologies observed among the remaining parasites that appeared to be viable. Examples of parasite morphologies after pulse artemisinin treatment are depicted in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006930#ppat.1006930.s001" target="_blank">S1 Fig</a></b>.</p

    MOESM1 of Limitations of malaria reactive case detection in an area of low and unstable transmission on the Myanmar–Thailand border

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    Additional file 1. Proportion of falciparum cases that would have been detected using random selection of the same proportion of houses selected by a given radius around index houses

    Çanakkale

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    Échelle(s) : Échelle : 1/400 000Numérisé par le partenaireAppartient à l’ensemble documentaire : BbLevt0Numérisé par le partenair
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