A randomized feasibility trial comparing four antimalarial drug regimens to induce Plasmodium falciparum gametocytemia in the controlled human malaria infection model

Abstract

Background: Malaria elimination strategies require a thorough understanding of parasite transmission from human to mosquito. A clinical model to induce gametocytes to understand their dynamics and evaluate transmission-blocking interventions (TBI) is currently unavailable. Here, we explore the use of the well-established Controlled Human Malaria Infection model (CHMI) to induce gametocyte carriage with different antimalarial drug regimens.

Methods: In a single centre, open-label randomised trial, healthy malaria-naive participants (aged 18–35 years) were infected with Plasmodium falciparum by bites of infected Anopheles mosquitoes. Participants were randomly allocated to four different treatment arms (n = 4 per arm) comprising low-dose (LD) piperaquine (PIP) or sulfadoxine-pyrimethamine (SP), followed by a curative regimen upon recrudescence. Male and female gametocyte densities were determined by molecular assays.

Results: Mature gametocytes were observed in all participants (16/16, 100%). Gametocytes appeared 8.5–12 days after the first detection of asexual parasites. Peak gametocyte densities and gametocyte burden was highest in the LD-PIP/SP arm, and associated with the preceding asexual parasite biomass (p=0.026). Male gametocytes had a mean estimated circulation time of 2.7 days (95% CI 1.5–3.9) compared to 5.1 days (95% CI 4.1–6.1) for female gametocytes. Exploratory mosquito feeding assays showed successful sporadic mosquito infections. There were no serious adverse events or significant differences in the occurrence and severity of adverse events between study arms (p=0.49 and p=0.28).

Conclusions: The early appearance of gametocytes indicates gametocyte commitment during the first wave of asexual parasites emerging from the liver. Treatment by LD-PIP followed by a curative SP regimen, results in the highest gametocyte densities and the largest number of gametocyte-positive days. This model can be used to evaluate the effect of drugs and vaccines on gametocyte dynamics, and lays the foundation for fulfilling the critical unmet need to evaluate transmission-blocking interventions against falciparum malaria for downstream selection and clinical development.

Funding: Funded by PATH Malaria Vaccine Initiative (MVI).

Clinical trial number: NCT02836002.

Keywords: p. falciparum; Malaria; Plasmodium falciparum; gametocytes; infectious disease; microbiology; transmission.

Conflict of interest statement

IR, Lv, LC, KL, LM, WG, Gv, KT, RS, Mv, Qd, Av, KI, CH, Sd, JB, KC, CO, JM, RS, TB No competing interests declared

© 2018, Reuling et al.

Figures

Figure 1.. Trial profile.

ECG = electrocardiography, BMI = body mass index, AST = aspartate aminotransferase, ALP = alkaline phosphatase

Figure 2.. Asexual parasitemia and gametocytemia.

Black line represents 18S qPCR asexual parasitemia. Black dotted-line represents 18S qPCR after treatment 1. Red line represents Pfs25 qRT-PCR gametocytemia.

Figure 2—figure supplement 1.. Asexual parasitemia and gametocytemia per study participant.

Black line represents 18S qPCR asexual parasitemia. Black dotted-line represents 18S qPCR after treatment 1. Red line represents Pfs25 qRT-PCR gametocytemia. Grey lines represent individual PCR curves of other participants of the same group.

Figure 3.. Gametocyte kinetics between study arms.

(A) Percentage gametocyte carriers between study arms (B) Estimated mean area under the curve for concentration of gametocytes per arm (Bayesian framework). The shaded area of each density curve represents the middle 95% percentiles (i.e. 2.5th to 97.5th percentiles) of the estimated mean AUC for a study arm; the density curve itself spans the middle 99% percentiles of the posterior; the posterior mean is indicated by the vertical solid line within each density plot. (C) Association of area under the curves of asexual parasitemia and gametocytemia. The different plotting shapes are the individual participants per group. (D) Thin- and thick- blood smears of concentrated gametocytes after magnetic cell sorting of blood samples from two individuals from LD-PIP/SP arm.

Figure 4.. Total female and male gametocyte density of all participants.

Dots represent individual gametocyte data. Circles and squares represent mean and error (SEM) of gametocytes per timepoint.

Figure 4—figure supplement 1.. Female and male gametocytes per study arm.

Figure 4—figure supplement 2.. Female and male gametocyte clearance dynamics per participant included in analysis.

Curves are log gametocytes/mL. Recoded days are the days of gametocyte observations from 12 days after the last detection of asexual parasites until the end of study.

Figure 5.. Standard curves of qRT-PCR and qPCR.

Standard curves (Mean, SD) obtained using 10-fold dilutions of cultured gametocytes. The highest concentration was enumerated by two independent expert microscopists. The mean and standard deviation of 54, 28, 72 replicates of the standard curve during the study was determined for the Pfs 25-, PfMGET, and 18S target genes, respectively. For PfMGET, six points starting from 106 pure male gametocytes/mL were measured. 101 was positive in 6/28 replicates (black dot).

Figure 5—figure supplement 1.. Standard curves of Pfs25 qRT-PCR – low-density trendlines.

Standard curves (Mean, SD) obtained using serial dilutions of cultured gametocytes including low-density trendlines to determine the limit of detection (LOD) and limit of quantification (LOQ) of the Pfs25 qRT-PCR.

Figure 5—figure supplement 2.. Correlation of duplo Pfs25 qRT-PCR measurements in all study samples.

All duplo- estimation data points of the study participants as measured by Pfs25 qRT PCR. All samples with ≥5 parasites/mL were duplo positive (190/190, 100%), and showed a correlation coefficient R2 of 0.94. Variation of samples < 5 parasites/mL was considerably larger and positivity could not be reliably estimated with 35/75 (47%) of samples that were positive in at least one qRT-PCR being single positives (R2 of 0.46).

Figure 6.. Adverse events.

(A) Adverse events per study arm (B) Total no. of adverse events and time course.

Figure 6—figure supplement 1.. Liver function test derangements.

ALT = alanine aminotransferase, AST = aspartate aminotransferase, γGT = gamma glutamyl transferase, ALP = alkaline phosphatase, T1 = Treatment 1, T2 = Treatment 2.

Author response image 1.. Model fit for CHMI-trans data female gametocytes.

Log female gametocyte densities at different days of follow-up (left) and residuals of non-zero female log gametocyte densities as a function of time of follow-up (right).

Author response image 2.. Model fit for CHMI-trans data male gametocytes.

Log male gametocyte densities at different days of follow-up (left) and residuals of non-zero male log gametocyte densities as a function of time of follow-up (right).

Author response image 3.. Model fit for male gametocytes in trial in Malian gametocyte carriers treated with non-gametocytocidal drugs dihydroartemisinin-piperaquine or amodiaquine plus sulfadoxine-pyrimethamine (NCT02831023).

Log male gametocyte densities at different days of follow-up (left) and residuals of non-zero male log gametocyte densities as a function of time of follow-up (right).