High prevalence of Plasmodium falciparum gametocyte infections in school-age children using molecular detection: patterns and predictors of risk from a cross-sectional study in southern Malawi

Abstract Background In endemic areas, many people experience asymptomatic Plasmodium infections, particularly older children and adults, but their transmission contribution is unknown. Though not the exclusive determinant of infectiousness, transmission from humans to mosquitoes requires blood meals containing gametocytes. Gametocytes often occur at submicroscopic densities, challenging measurement in human populations. More sensitive molecular techniques allow better characterization of gametocyte epidemiologic patterns. Methods Approximately 30 households were selected from each of eight sites in southern Malawi during two cross-sectional surveys. Blood was sampled from 623 people during the dry season and 896 the following rainy season. Among people PCR-positive for Plasmodium falciparum, mature gametocytes were detected by qRT-PCR. Regression models evaluated predictors of gametocyte carriage and density in the total population and among those with PCR-positive infections. Results The prevalence of gametocyte carriage by molecular testing was 3.5% during the dry season and 8.6% during the rainy season, and by microscopy 0.8 and 3.3%, respectively. Nearly half of PCR-positive infections carried gametocytes, regardless of recent symptom status. Among P. falciparum-infected people, only living in unfinished houses and age were significantly associated with gametocyte presence. Infected people in unfinished houses had higher odds of carrying gametocytes (OR 2.24, 95% CI 1.16–4.31), and 31% (95% CI 3–65%) higher gametocyte density than those in finished houses. School-age children (5–15 years), had higher odds than adults (≥16 years) of having gametocytes when infected (OR 2.77, 95% CI 1.47–5.19), but 31% (95% CI 11–47%) lower gametocyte density. Children <5 years did not have significantly higher odds of gametocyte carriage or density when infected than adults. Conclusions School-age children frequently carry gametocytes in communities of southern Malawi and represent an under-recognized reservoir of infection. Malaria elimination strategies should address these frequently asymptomatic reservoirs, especially in highly endemic areas. Improved household construction may also reduce the infectious reservoir.http://deepblue.lib.umich.edu/bitstream/2027.42/134670/1/12936_2016_Article_1587.pd

Integrative metabolomics and transcriptomics signatures of clinical tolerance to Plasmodium vivax reveal activation of innate cell immunity and T cell signaling

Almost invariably, humans become ill during primary infections with malaria parasites which is a pathology associated with oxidative stress and perturbations in metabolism. Importantly, repetitive exposure to Plasmodium results in asymptomatic infections, which is a condition defined as clinical tolerance. Integration of transcriptomics and metabolomics data provides a powerful way to investigate complex disease processes involving oxidative stress, energy metabolism and immune cell activation. We used metabolomics and transcriptomics to investigate the different clinical outcomes in a P. vivax controlled human malaria infection trial. At baseline, the naïve and semi-immune subjects differed in the expression of interferon related genes, neutrophil and B cell signatures that progressed with distinct kinetics after infection. Metabolomics data indicated differences in amino acid pathways and lipid metabolism between the two groups. Top pathways during the course of infection included methionine and cysteine metabolism, fatty acid metabolism and urea cycle. There is also evidence for the activation of lipoxygenase, cyclooxygenase and non-specific lipid peroxidation products in the semi-immune group. The integration of transcriptomics and metabolomics revealed concerted molecular events triggered by the infection, notably involving platelet activation, innate immunity and T cell signaling. Additional experiment confirmed that the metabolites associated with platelet activation genes were indeed enriched in the platelet metabolome. Keywords: Malaria, Plasmodium vivax, Tolerance, Metabolomics, Transcriptomics, Integration, Platelets, Immunit

Humoral immunity prevents clinical malaria during Plasmodium relapses without eliminating gametocytes.

Plasmodium relapses are attributed to the activation of dormant liver-stage parasites and are responsible for a significant number of recurring malaria blood-stage infections. While characteristic of human infections caused by P. vivax and P. ovale, their relative contribution to malaria disease burden and transmission remains poorly understood. This is largely because it is difficult to identify 'bona fide' relapse infections due to ongoing transmission in most endemic areas. Here, we use the P. cynomolgi-rhesus macaque model of relapsing malaria to demonstrate that clinical immunity can form after a single sporozoite-initiated blood-stage infection and prevent illness during relapses and homologous reinfections. By integrating data from whole blood RNA-sequencing, flow cytometry, P. cynomolgi-specific ELISAs, and opsonic phagocytosis assays, we demonstrate that this immunity is associated with a rapid recall response by memory B cells that expand and produce anti-parasite IgG1 that can mediate parasite clearance of relapsing parasites. The reduction in parasitemia during relapses was mirrored by a reduction in the total number of circulating gametocytes, but importantly, the cumulative proportion of gametocytes increased during relapses. Overall, this study reveals that P. cynomolgi relapse infections can be clinically silent in macaques due to rapid memory B cell responses that help to clear asexual-stage parasites but still carry gametocytes

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

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

Identification of metabolic features associated with CQ resistance.

<p>A) Type 1 Manhattan plot, -log₁₀ <i>p</i> vs mass-to-charge. 81 <i>m/</i>z features with a broad range of <i>m/z</i> were found significant at FDR 0.20 threshold. Green dots represent the features that were up-regulated in the CQ-Resistant group and the red dots represent the features that were higher in the CQ-Sensitive group; B) Type 2 Manhattan plot, -log₁₀ <i>p</i> vs retention time, Majority of features had retention time greater than 4 minutes, which is consistent with elution profile of lipids on a C18 column; C) Two-way hierarchical clustering analysis using discriminatory features; D) Mummichog enriched pathways.</p

Comparison of glycerophosphocholine abundance levels in NIST, pooled normal plasma (US), healthy controls (Brazil), CQ-Resistant (<i>P</i>. <i>vivax</i>), and CQ-Sensitive (<i>P</i>. <i>vivax</i>) groups along with 95% confidence intervals.

<p>The glycerophosphocholines were found to be lower in CQ-Resistant group as compared to CQ-Sensitive and other control samples (<i>p</i><0.05).</p

Demographic and laboratory characteristics of 46 patients with <i>P</i>. <i>vivax</i> infections assessed for CQ resistance in Manaus, Brazil, 2011–2012.

<p>Demographic and laboratory characteristics of 46 patients with <i>P</i>. <i>vivax</i> infections assessed for CQ resistance in Manaus, Brazil, 2011–2012.</p