Expression level of chloroquine resistance genes in severe patients.

<p>Relative quantification of <i>pvcrt-o</i> (A) and <i>pvmdr1</i> (B) transcript levels in total RNA obtained from parasites from severe patients vs a pool of total RNA obtained from parasites susceptible to CQ. Severe cases (S). Non-severe cases (NS). The error bars in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105922#pone-0105922-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105922#pone-0105922-g003" target="_blank">3</a> reflect the average standard error of the Ct.</p

Expression gene levels of <i>pvcrt-o</i> and <i>pvmdr-1</i> in all the groups.

<p>Relative quantification of <i>pvcrt-o</i> (A) and <i>pvmdr1</i> (B) transcript levels in total RNA obtained from parasites from severe patients with chloroquine-resistant <i>P. vivax</i>, patients susceptible to CQ, severe patients and patients without severity symptoms. Chloroquine-resistant <i>P. vivax</i> parasites (R). Chloroquine-susceptible <i>P. vivax</i> parasites at D0 (S). Severe cases (Sev). Non-severe cases (NS). *p<0.05.</p

Expression Levels of <i>pvcrt-o</i> and <i>pvmdr-1</i> Are Associated with Chloroquine Resistance and Severe <i>Plasmodium vivax</i> Malaria in Patients of the Brazilian Amazon

<div><p>Molecular markers associated with the increase of chloroquine resistance and disease severity in <i>Plasmodium vivax</i> are needed. The objective of this study was to evaluate the expression levels of <i>pvcrt-o</i> and <i>pvmdr-1</i> genes in a group of patients presenting CQRPv and patients who developed severe complications triggered exclusively by <i>P. vivax</i> infection. Two different sets of patients were included to this comprehensive study performed in the Brazilian Amazon: 1) patients with clinically characterized chloroquine-resistant <i>P. vivax</i> compared with patients with susceptible parasites from <i>in</i><i>vivo</i> studies and 2) patients with severe vivax malaria compared with patients without severity. Quantitative real-time PCR was performed to compare the transcript levels of two main transporters genes, <i>P. vivax</i> chloroquine resistance transporter (<i>pvcrt-o</i>) and the <i>P. vivax</i> multidrug resistance transporter (<i>pvmdr-1</i>). Twelve chloroquine resistant cases and other 15 isolates from susceptible cases were included in the first set of patients. For the second set, seven patients with <i>P. vivax</i>-attributed severe and 10 mild manifestations were included. Parasites from patients with chloroquine resistance presented up to 6.1 (95% CI: 3.8–14.3) and 2.4 (95% CI: 0.53–9.1) fold increase in <i>pvcrt-o</i> and <i>pvmdr-1</i> expression levels, respectively, compared to the susceptible group. Parasites from the severe vivax group had a 2.9 (95% CI: 1.1–8.3) and 4.9 (95% CI: 2.3–18.8) fold increase in <i>pvcrt-o</i> and <i>pvmdr-1</i> expression levels as compared to the control group with mild disease. These findings suggest that chloroquine resistance and clinical severity in <i>P. vivax</i> infections are strongly associated with increased expression levels of the <i>pvcrt-o</i> and <i>pvmdr-1</i> genes likely involved in chloroquine resistance.</p></div

Expression levels of chloroquine resistance genes in patients with CQRPv parasites.

<p>Relative quantification of <i>pvcrt-o</i> (A) and <i>pvmdr1</i> (B) transcripts in total RNA obtained from parasites from patients with chloroquine-resistant <i>P. vivax</i> vs a pool of total RNA obtained from 5 patients with CQ-susceptible parasites. Chloroquine-resistant <i>P. vivax</i> parasites (R). Chloroquine-susceptible <i>P. vivax</i> parasites (S). Day of admission (D0). Day of recrudescence (DR). The error bars in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105922#pone-0105922-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105922#pone-0105922-g003" target="_blank">3</a> reflect the average standard error of the Ct.</p

Plasma metabolomics reveals membrane lipids, aspartate/asparagine and nucleotide metabolism pathway differences associated with chloroquine resistance in <i>Plasmodium vivax</i> malaria

<div><p>Background</p><p>Chloroquine (CQ) is the main anti-schizontocidal drug used in the treatment of uncomplicated malaria caused by <i>Plasmodium vivax</i>. Chloroquine resistant <i>P</i>. <i>vivax</i> (PvCR) malaria in the Western Pacific region, Asia and in the Americas indicates a need for biomarkers of resistance to improve therapy and enhance understanding of the mechanisms associated with PvCR. In this study, we compared plasma metabolic profiles of <i>P</i>. <i>vivax</i> malaria patients with PvCR and chloroquine sensitive parasites before treatment to identify potential molecular markers of chloroquine resistance.</p><p>Methods</p><p>An untargeted high-resolution metabolomics analysis was performed on plasma samples collected in a malaria clinic in Manaus, Brazil. Male and female patients with <i>Plasmodium vivax</i> were included (n = 46); samples were collected before CQ treatment and followed for 28 days to determine PvCR, defined as the recurrence of parasitemia with detectable plasma concentrations of CQ ≥100 ng/dL. Differentially expressed metabolic features between CQ-Resistant (CQ-R) and CQ-Sensitive (CQ-S) patients were identified using partial least squares discriminant analysis and linear regression after adjusting for covariates and multiple testing correction. Pathway enrichment analysis was performed using Mummichog.</p><p>Results</p><p>Linear regression and PLS-DA methods yielded 69 discriminatory features between CQ-R and CQ-S groups, with 10-fold cross-validation classification accuracy of 89.6% using a SVM classifier. Pathway enrichment analysis showed significant enrichment (<i>p</i><0.05) of glycerophospholipid metabolism, glycosphingolipid metabolism, aspartate and asparagine metabolism, purine and pyrimidine metabolism, and xenobiotics metabolism. Glycerophosphocholines levels were significantly lower in the CQ-R group as compared to CQ-S patients and also to independent control samples.</p><p>Conclusions</p><p>The results show differences in lipid, amino acids, and nucleotide metabolism pathways in the plasma of CQ-R versus CQ-S patients prior to antimalarial treatment. Metabolomics phenotyping of <i>P</i>. <i>vivax</i> samples from patients with well-defined clinical CQ-resistance is promising for the development of new tools to understand the biological process and to identify potential biomarkers of PvCR.</p></div

Correlation between cytokines levels and platelet count.

<p>Correlation between IL-6 (A) and IL-10 (B) and platelet count.</p

Description of 7 patients presenting severe vivax malaria admitted to a tertiary health center, Manaus, Amazonas, Brazil.

<p>Acute respiratory distress syndrome (ARDS).</p><p>Description of 7 patients presenting severe vivax malaria admitted to a tertiary health center, Manaus, Amazonas, Brazil.</p

Correlation between expression gene levels <i>pvcrt-o</i> and <i>pvmdr-1</i> genes and hemoglobin concentration.

<p>Correlation between expression levels of chloroquine resistance genes, <i>pvcrt-o</i> (A) and <i>pvmdr-1</i> (B) and hemoglobin in hospitalized patient.</p

Characteristics of patients with <i>Plasmodium vivax</i> (with and without thrombocytopenia).

<p>SD = standard deviation.</p><p>NT = non-thrombocytopenic; T = thrombocytopenic.</p>*<p>Non-thrombocytopenic patients×thrombocytopenic patients.</p>a<p>Mann Whitney test.</p>b<p>Chi-square or Fisher’s exact test.</p

10-fold cross-validation analysis using clinical variables and top discriminatory metabolic features.

<p>10-fold cross-validation classification accuracies varied from 65% to 89.6% using platelet count, glycerophosphocholines, top 10, top 30, and all 69 discriminatory features. The average permuted accuracies (N = 1000 permutations) varied from 55–58%.</p