Background: Exhaled breath fingerprints have the potential to change the way diseases are diagnosed in the future, offering an attractive alternative to conventional disease diagnostic tests since they are simple, painless, noninvasive and more acceptable to patients. Recent work has shown that malaria parasites produce an array of parasite-specific volatile organic compounds (VOCs) present in the exhaled breath of infected hosts, suggesting a diagnostic breath test may be feasible for malaria diagnosis. One particular class of molecules (terpenes) was identified in the exhaled breath of falciparum-positive individuals. Terpenes, if parasite derived, are likely synthesized from end products of the mevalonate-independent (MEP) pathway, of which the final enzymatic step is catalyzed by the enzyme IspH. They are further known to be strong disease-specific biomarkers as humans are not known to produce them naturally. More work is required to elucidate the metabolic origin of these terpenes as well as optimize breath collection protocols for uncooperative infants.

Objective: The goal of this study was to test the feasibility of a breath collection system adapted for uncooperative patients using healthy newborns as a model and to build an in vitro plasmid construct to investigate the breath biomarker role of the IspH locus of Plasmodium falciparum.

Methods: This was a cross-sectional study with an experimental component, from January to May 2017, in St. Louis, Missouri, USA. Breath samples were collected from 25 healthy newborns and analyzed using gas chromatography mass spectrometry (GCMS). Detection of breath VOCs was expressed by extracted-ion chromatograms compared to room air controls. To investigate the breath biomarker role of the IspH locus, an IspH knockout strategy was conceived using the CRISPR/Cas9 system to alter the erythrocytic stage of P. falciparum. In the first step, primers were designed to amplify the IspH locus and linear vector plasmids generated capable of integrating the IspH locus by In-Fusion. The recombinant plasmids were used to transform stellar competent cells and the IspH_HR1 insert verified by PCR, restriction digest and sequencing.

Results and Discussion: The neonatal extracted-ion chromatograms showed levels of VOCs, 2.3-5.7 times increased concentration compared to room air controls. The cumulative alveolar gradient was positive for acetone affirming our breath collection model captures human breath in uncooperative newborns. We also isolated the IspH locus, integrated the IspH_HR1 locus into linearized plasmids, successfully cloned the IspH_HR1 fragment and confirmed the insert by PCR, restriction digest and sequencing with a 94% identity match factor.

Conclusion: These results present a unique opportunity for better understanding the biochemical basis of diseases suggesting that VOCs of endogenous origin empirically correlate with human metabolic pathways. Because our breath collection system captures breath VOCs in uncooperative newborns, this model is amenable to mentally-ill, comatose and uncooperative infants under five, providing a useful new tool to advance research towards a malaria diagnostic breath test. Our recombinant plasmid construct containing the IspH_HR1 locus was engineered for more investigation of the IspH locus in its role as a volatile trigger and as a potential therapeutic drug target distinct from any human metabolic path.