Diurnal variation in expired breath volatiles in malaria-infected and healthy volunteers

Abstract

We previously showed that thioether levels in the exhaled breath volatiles of volunteers undergoing controlled human malaria infection (CHMI) with P. falciparum increase as infection progresses. In this study, we show that thioethers have diurnal cyclical increasing patterns and their levels are significantly higher in P. falciparum CHMI volunteers compared to those of healthy volunteers. The synchronized cycle and elevation of thioethers were not present in P. vivax-infection, therefore it is likely that the thioethers are associated with unique factors in the pathology of P. falciparum. Moreover, we found that time-of-day of breath collection is important to accurately predict (98%) P. falciparum-infection. Critically, this was achieved when the disease was asymptomatic and parasitemia was below the level detectable by microscopy. Although these findings are encouraging, they show limitations because of the limited and logistically difficult diagnostic window and its utility to P. falciparum malaria only. We looked for new biomarkers in the breath of P. vivax CHMI volunteers and found that a set of terpenes increase significantly over the course of the malaria infection. The accuracy of predicting P. vivax using breath terpenes was up to 91%. Moreover, some of the terpenes were also found in the breath of P. falciparum CHMI volunteers (accuracy up to 93.5%). The results suggest that terpenes might represent better biomarkers than thioethers to predict malaria as they were not subject to malaria pathogens diurnal changes.

Figure 1

Time course of mean levels of MTPNZ for P. falciparum IBSM trials (N = 7), healthy controls (N = 8) and parasitaemia. P. falciparum infection was initiated immediately after the time zero breath sample. The red vertical dotted line represents the time when antimalarial treatment was administered in the P. falciparum trial. The healthy control data shows the mean levels of the compound found at that time of day, derived from three consecutive days of measurements. The diurnal cycle is repeated over the duration of the IBSM trial, and the collection time matched to collections from the P. falciparum-infected subjects. Figure shows mean (SEM). SEM is used as a measure of precision for the estimated mean. Blue asterisks denote sampling times between 19:00 and 21:00.

Figure 2

(A) Extract of time course of mean levels of (Z)-1-methylthio-1-propene for P. falciparum CHMI trials (N = 7) (∎) and for healthy controls (N = 8) (Δ). The healthy control data shows the mean levels of the compound at the specified time of day and were derived from three consecutive days of measurement. The diurnal cycle is repeated over the duration of the CHMI trial and the collection time matched to CHMI collections. Figure shows mean(sem). Arrows indicate the peak of the compound which corresponds to 12 h after the first sample of the day is collected (i.e. between 19:00 and 21:00). Green asterisks indicate sampling times between 7:00 and 8:00. (B) Comparisons of mean (Z)-1-methylthio-1-propene levels for P. falciparum (N = 7) CHMI trial (∎) and healthy controls (N = 8) (Δ) for samples collected between 19:00 and 21:00. For the CHMI trial, breath samples were collected at 131 h (Day 5), 155 h (Day 6) and 179 h (Day 7) after infection. Values above red symbols represent the average parasitaemia (parasites μl-1) at each breath collection time point.

Figure 3

(A) Time course of levels of MTPNZ in breath of participants undergoing CHMI with P. vivax (N = 8), healthy controls (N = 8) and parasitaemia levels. Blue vertical dotted line represents the time when experimental antimalarial was administered. The healthy control data shows the mean levels of the compound found at the specified times of day, derived from three consecutive days of measurements. The diurnal cycle is repeated over the duration of the IBSM trial, and the collection time was matched to collections from the P. vivax-infected subjects. Figure shows mean(sem). (B) Comparisons of mean of MTPNZ thioether levels for P. falciparum (N = 7) and P. vivax (N = 8) CHMI trials. Both infections were initiated immediately after the baseline breath sample, taken at time zero. Red (P. falciparum) and blue (P. vivax) vertical dotted lines represent the times when the treatment was administered. Figure shows mean(sem).

Figure 4

(A) Time course of α-terpinene abundance in the breath of CHMI P. vivax volunteers (N = 8), healthy controls (N = 8) and parasitaemia. (B) Time course of α-terpinene levels for P. falciparum CHMI trials (N = 7), healthy controls (N = 8) and parasitaemia. P. vivax and P. falciparum infections were started immediately after the breath sample at time zero. The vertical dotted lines represent the time when treatments were administered. The healthy control data shows the mean levels of the compound at that the nominated time of day, derived from three consecutive days of measurement. The diurnal cycle is repeated over the duration of the IBMS trial. The collection time matched to CHMI breath collections. Figure shows mean(sem).