Metabolic Profiling Framework for Discovery of Candidate Diagnostic Markers of Malaria

Abstract

Despite immense efforts to combat malaria in tropical and sub-tropical regions, the potency of this vector-borne disease and its status as a major driver of morbidity and mortality remain undisputed. We develop an analytical pipeline for characterizing Plasmodium infection in a mouse model and identify candidate urinary biomarkers that may present alternatives to immune-based diagnostic tools. We employ 1 H nuclear magnetic resonance (NMR) profiling followed by multivariate modeling to discover diagnostic spectral regions. Identification of chemical structures is then made on the basis of statistical spectroscopy, multinuclear NMR, and entrapment of candidates by iterative liquid chromatography (LC) and mass spectrometry (MS). We identify two urinary metabolites (i) 4-amino-1-[3-hydroxy-5-(hydroxymethyl)-2,3-dihydrofuran-2-yl]pyrimidin-2(1H)-one, (ii) 2-amino-4-({[5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4-hydroxy-4,5-dihydrofuran-2-yl]methyl}sulfanyl)butanoic acid that were detected only in Plasmodium berghei-infected mice. These metabolites have not been described in the mammalian or parasite metabolism to date. This analytical pipeline could be employed in prospecting for infection biomarkers in human populations. P lasmodium falciparum is the most deadly human parasite, inflicting the greatest mortality and global burden of all parasitic diseases 1 . In 2010, P. falciparum accounted for approximately 216 million clinical cases and more than 600,000 deaths, mostly amongst children below the age of 5 years in sub-Saharan Africa. Antigen-detecting rapid diagnostic tests (RDTs) for malaria have mushroomed in the last few years and have undergone rigorous testing. The development and large-scale deployment of RDTs as point-of-care tests has revolutionized malaria management. Clinical sensitivity, however, still leaves ground for improvement, particularly in cases of low parasitemia 2 .An additional complication is that in many tropical and sub-tropical regions, people are concurrently infected with Plasmodium and parasitic worms (helminths), particularly in remote rural areas. One quarter of all school-aged children on the African continent (,45 million) are likely to harbor concomitant infections with P. falciparum and hookworm 3 , explained by the high degree of geographic overlap of the parasites. One obvious question is to what extent a parasitic worm infection affects diagnosis, immune response, clinical manifestation, and prognosis of malaria 4,5 .Metabolic profiling has been used to gain knowledge about host-parasite interactions and to discover infectionrelated metabolite patterns. Thus far, the majority of exploratory work has been conducted in rodent models wherein each host-parasite model is represented by an infection-specific fingerprint in urine and/or plasma [6][7][8][9][10] .Despite the widely assumed universality of eukaryotic metabolism, some class-or species-specific adaptions exist in parasites, such as the trypanothione system in kinetoplasts (Leishmania spp. and Trypanosoma spp.) that replaces gl...

Metabolome-wide association study of peripheral parasitemia in Plasmodium vivax malaria

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