Analysis of Breath Specimens for Biomarkers of Plasmodium falciparum Infection

Abstract

Currently, the majority of diagnoses of malaria rely on a combination of the patient's clinical presentation and the visualization of parasites on a stained blood film. Breath offers an attractive alternative to blood as the basis for simple, noninvasive diagnosis of infectious diseases. In this study, breath samples were collected from individuals during controlled malaria to determine whether specific malaria-associated volatiles could be detected in breath. We identified 9 compounds whose concentrations varied significantly over the course of malaria: carbon dioxide, isoprene, acetone, benzene, cyclohexanone, and 4 thioethers. The latter group, consisting of allyl methyl sulfide, 1-methylthio-propane, (Z)-1-methylthio-1-propene, and (E)-1-methylthio-1-propene, had not previously been associated with any disease or condition. Before the availability of antimalarial drug treatment, there was evidence of concurrent 48-hour cyclical changes in the levels of both thioethers and parasitemia. When thioether concentrations were subjected to a phase shift of 24 hours, a direct correlation between the parasitemia and volatile levels was revealed. Volatile levels declined monotonically approximately 6.5 hours after initial drug treatment, correlating with clearance of parasitemia. No thioethers were detected in in vitro cultures of Plasmodium falciparum. The metabolic origin of the thioethers is not known, but results suggest that interplay between host and parasite metabolic pathways is involved in the production of these thioethers.

Keywords: (E)-1-methylthio-1-propene; diagnostic tool; machine learning; malaria; odors; thioethers; volatile organic compound.

Figure 1.

Extracted ion gas chromatograms for a mass-to-charge ratio (m/z) of 88+90 for 1 typical subject on day 0, day 4, day 7 (after drug administration), and day 9. Peaks: 1, allyl methyl sulfide; 2, 1-methylthio-propane; 3, (E)-1-methylthio-1-propene; 4, (Z)-1-methylthio-1-propene.

Figure 2.

VOCs in breath (right y-axis) and parasite levels in blood (left y-axis) for cohort 1 during the course of experimental malaria before (during the first 7 days after infection onset) and after antimalarial treatment. Each participant in the cohort was inoculated on day 0 with approximately 1800 human erythrocytes infected with Plasmodium falciparum. Breath and blood samples were collected over the course of infection. Drug treatment (OZ439) started on day 7 for cohort 1 (n = 7), as indicated by dotted vertical lines in the plots. Multiplication and clearance of parasites are denoted by blue circles, and the abundance of compounds is denoted by squares: (Z)-1-methylthio-1-propene (m/z 88), 1-methylthio-propane (m/z 90), allyl methyl sulfide (m/z 88) and (E)-1-methylthio-1-propene (m/z 88). Error bars represent standard errors of the mean.

Figure 3.

VOCs in breath (right y-axis) and parasite levels in blood (left y-axis) for cohort 2 during the course of experimental malaria before (during the first 8 days after infection onset) and after antimalarial treatment. Each participant in the cohort was inoculated on day 0 with approximately 1800 human erythrocytes infected with Plasmodium falciparum. Breath and blood samples were collected over the course of infection. Drug treatment (piperaquine) started on day 8 for cohort 2 (n = 6), as indicated by dotted vertical lines in the plots. Multiplication and clearance of parasites are denoted by blue circles, and the abundance of compounds is denoted by squares: (Z)-1-methylthio-1-propene (m/z 88), 1-methylthio-propane (m/z 90), allyl methyl sulfide (m/z 88) and (E)-1-methylthio-1-propene (m/z 88). Error bars represent standard errors of the mean.

Figure 4.

Correlation of parasite level and abundance of malaria VOCs before (A) and after (B) antimalarial treatment of the 2 cohorts. The thioether data were subjected to a phase shift of 24 hours, which revealed a direct correlation between parasitemia and volatile levels. In cohort 1, a fast-acting synthetic ozonide drug was used on day 7, and in cohort 2, a slower-acting piperaquine drug was administered on day 8. Points denote means and standard deviations.

Figure 5.

Screening of in vitro Plasmodium falciparum blood cultures under various conditions for thioethers (m/z 88+90). Thioethers were absent in the control (A) and in the headspace of 100 mL of a red blood cell suspension (1% hematocrit; B). Thioethers were neither present following treatment with 1000 ng/mL of the antimalarial piperaquine tetraphosphate tetrahydrate (C) nor after treatment-lysing of cells (D). As a positive control, red blood cell cultures were spiked at the levels previously detected in healthy individuals (E). Peaks: 1, allyl methyl sulfide; 2, 1-methylthio-propane; 3, (E)-1-methylthio-1-propene; and 4, (Z)-1-methylthio-1-propene. Data are representative of findings obtained in 2 independent experiments with average parasitemia levels of 29.9%. Abbreviation: VOC, volatile organic compounds.