Abstract
BackgroundCurrent available malaria diagnostic methods each have some limitations to meet the need for real-time and large-scale screening of asymptomatic and low density malaria infection at community level. It was proposed that malaria parasite-specific low molecular-weight metabolites could be used as biomarkers for the development of a malaria diagnostic tool aimed to address this diagnostic challenge. In this study, high resolution metabolomics (HRM) was employed to identify malaria parasite-specific metabolites in Plasmodium falciparum in vitro culture samples.MethodsSupernatants were collected at 12 hours interval from 3% haematocrit in vitro 48-hour time-course asynchronized culture system of P. falciparum. Liquid chromatography coupled with high resolution mass spectrometry was applied to discover potential parasite-specific metabolites in the cell culture supernatant. A metabolome-wide association study was performed to extract metabolites using Manhattan plot with false discovery rate (FDR) and hierarchical cluster analysis. The significant metabolites based on FDR cutoff were annotated using Metlin database. Standard curves were created using corresponding chemical compounds to accurately quantify potential Plasmodium-specific metabolites in culture supernatants.ResultsThe number of significant metabolite features was 1025 in the supernatant of the Plasmodium infected culture based on Manhattan plot with FDR q=0.05. A two way hierarchical cluster analysis showed a clear segregation of the metabolic profile of parasite infected supernatant from non-infected supernatant at four time points during the 48 hour culture. Among the 1025 annotated metabolites, the intensities of four molecules were significantly increased with culture time suggesting a positive association between the quantity of these molecules and level of parasitaemia: i) 3-methylindole, a mosquito attractant, ii) succinylacetone, a haem biosynthesis inhibitor, iii) S-methyl-L-thiocitrulline, a nitric oxide synthase inhibitor, and iv) O-arachidonoyl glycidol, a fatty acid amide hydrolase inhibitor, The highest concentrations of 3-methylindole and succinylacetone were 178 ± 18.7 pmoles at 36 hours and 157±30.5 pmoles at 48 hours respectively in parasite infected supernatant.ConclusionHRM with bioinformatics identified four potential parasite-specific metabolite biomarkers using in vitro culture supernatants. Further study in malaria infected human is needed to determine presence of the molecules and its relationship with parasite densities.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0651-1) contains supplementary material, which is available to authorized users.
Highlights
Current available malaria diagnostic methods each have some limitations to meet the need for real-time and large-scale screening of asymptomatic and low density malaria infection at community level
For programmes aiming to reduce transmission by further decreasing the parasite reservoir in humans through large scale screening approaches to detect and to radically cure asymptomatic lowdensity malaria infections in real time, a field-deployable non-invasive, sensitive, low-cost, simple diagnostic tool would be very useful at the community level
This study demonstrated high resolution metabolomics (HRM) could achieve a relatively comprehensive and quantitative analysis of Plasmodium-specific metabolites in supernatant from parasite infected culture system, and explores low molecular-weight biomarkers [6,13] associated with Plasmodium
Summary
Current available malaria diagnostic methods each have some limitations to meet the need for real-time and large-scale screening of asymptomatic and low density malaria infection at community level. Current malaria diagnostic tools include: 1) parasite detection by microscopic examination of blood smears, 2) antigen-based rapid diagnostic tests (RDTs), and 3) sensitive DNA-based assays All these diagnostic methods require blood sampling by finger-prick and their implementation has been limited by either their labour/time intensive nature and requirement for specialized training and skills (microscopic method), moderate sensitivity (RDTs, microscopy), or high cost of sample preparation and supporting infrastructure needed (DNAbased methods). For programmes aiming to reduce transmission by further decreasing the parasite reservoir in humans through large scale screening approaches to detect and to radically cure asymptomatic lowdensity malaria infections in real time, a field-deployable non-invasive, sensitive, low-cost, simple diagnostic tool would be very useful at the community level. It was proposed to identify malaria parasitespecific low molecular-weight metabolites that could potentially be used for future development of such diagnostic tools
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