Piperaquine

All participants were inoculated by IBSM with approximately 2,800 P. falciparum–infected human erythrocytes administered intravenously as previously described (36 (link)) and monitored via daily telephone calls for AEs and malaria. Parasitemia was measured daily from day 4 pi, using a previously described qPCR assay targeting DNA from the 18S ribosomal RNA gene (rDNA) and twice daily once participants were confirmed as malaria positive (Supplemental Methods) (37 (link)). Asexual parasitemia was also measured with a qRT-PCR assay that measures SBP-1 mRNA transcripts (Supplemental Methods, Supplemental Table 10, and Supplemental Figure 4) (22 (link), 26 (link)). To attenuate asexual parasite replication, participants were admitted for 48 hours confinement and given 480 mg piperaquine phosphate (Penn Pharmaceuticals) on day 7 pi (group 1) or day 8 pi (groups 2 and 3). After confinement, participants were monitored for up to 24 days and a second dose of piperaquine phosphate (960 mg) was administered to participants who experienced recrudescence. Gametocyte development was measured from day 7 pi by qRT-PCR for female-specific pfs25 mRNA and male-specific pfMGET mRNA (Pf3D7_1469900) (Supplemental Methods) (24 (link), 25 (link)). To determine infectivity of gametocytes to An. stephensi mosquitoes, feeding assays were performed on group 2 and 3 participants between day 17 and day 30 pi via either direct skin feeding (~30 mosquitoes per assay, 3 assays per participant) or membrane feeding (~50 mosquitoes per assay). Membrane-feeding assays were performed either with whole venous blood (direct membrane-feeding assay; n = 6 per participant) or after removing the participant’s plasma and replacing with control serum prior to feeding (membrane feeding with serum replacement) (Supplemental Methods). Prior to performing the mosquito feeding assays, the mosquito colony was verified as being highly susceptible to P. falciparum infection using in vitro–cultured gametocytes (Supplemental Figure 3, A and B). The mosquitoes used in all experiments were healthy and fed well on the gametocytemic blood, with an average adult mosquito mortality of 7.6% and an average mosquito blood feeding rate of 97.4% (Supplemental Tables 11 and 12). Transmission to mosquitoes was determined by detecting midgut oocysts using the 18S rDNA qPCR assay, with visual confirmation of oocysts performed by microscopy on a small random selection of midguts prior to PCR analysis (Supplemental Methods and Supplemental Figure 3C). To investigate gametocytocidal drug activity, all 6 participants in the EFITA study and 2 participants in the OZGAM study were negative controls who received no investigational drug other than piperaquine. The remaining participants received artefenomel (500 mg, n = 4) or primaquine (15 mg, n = 5) on day 22 (group 1), day 25 (group 2), or day 24 (group 3) pi. At the end of the study, all participants received a course of artemether/lumefantrine (Riamet, Novartis Pharmaceuticals Australia Pty. Ltd.; 4 tablets taken as a single dose every 12 hours for 60 hours) and, if required, a single dose of 45 mg of primaquine to clear gametocytes (Supplemental Table 1 and Supplemental Table 13). End of study visits were on day 34 (group 1) or day 36 pi (groups 2 and 3); however, participants were followed up until all outstanding abnormal laboratory test results resolved (Supplemental Table 1).

The study was conducted in Ratanakiri province located in the north-east of Cambodia. Diagnostic-based incidence rate in Ratanakiri was estimated to be 22.2 per 1,000 inhabitants in 2007, which is among the highest in the country [15 ]. Malaria transmission tends to peak during the rainy season (June – October), while it is generally acknowledged that migrants and forest workers are among the population most at risk to Plasmodium infections in the greater Mekong region [15 ].
A total of 117 villages out of 240 were selected based on malaria incidence records obtained from the Cambodian National Malaria Control Programme and collected between 2009 and 2011 [16 ]. Villages with the highest incidence were selected. The average number of inhabitants per village was 468 (range: [53-3228]). A random sample of 65 individuals per village, excluding children below two years of age, was extracted based upon the population census of 2011. Samples were collected during the dry season. Between 31 January 2012 and 24 February 2012, five teams of four people visited each village for two consecutive days. The aim was to collect at least 50 samples in each village over a two-day period. When a team indicated to be unable to reach sufficient people during the first day, an extra list of 15 randomly selected individuals was provided on the second sampling day. Blood samples were collected by finger prick and immediately stored in labeled 96-well plates and on filter paper as previously described in [14 (link)]. Each individual received a unique code number and malariometric data were recorded using a standardized questionnaire (Additional file 1). Gender, age and ethnicity were recorded as specific individual variables. Axillary temperature was measured and the participants were asked for their history of fever over the past 48 hours. Individuals with fever or other malaria related symptoms were checked by a rapid diagnostic test (CareStart™ Malaria, pLDH/HRP2 COMBO (PAN/Pf)). Malaria positive cases were treated by dihydroartemisinin plus piperaquine combination (Duo-Cotecxin®: Beijing Holley – Cotec Pharmaceutical CO., Ltd, Being, China), according to the national treatment guidelines. Behaviour related to bed net use, sleeping and waking times, overnight stays in homes at the farms located outside the village (plot huts) and overnight stays during activities in the forest were recorded in a subsequent form (Additional file 1 for detailed questions).

The adherence methodology was also applied using previously reported PK models for the antimalarial drugs piperaquine and lumefantrine, according to the overall procedures suggested in Figure 2.
A large‐scale pooled PK analysis of piperaquine, combining 11 clinical trials (8776 samples from 728 individuals) in healthy volunteers (n = 50) and patients with uncomplicated malaria (n = 678), showed that piperaquine PK properties were described accurately by a three‐compartment disposition model with transit absorption.
²⁴ (link) A total of 301 children below the age of 5 years was included in the analysis, and the age to reach 50% of full maturation of the elimination clearance (MF₅₀) was 0.575 years, and the Hill coefficient in the maturation function was 5.11. This resulted in 95% of the full elimination clearance (MF₉₅) at 1 year of age, resulting in a negligible age effect on clearance in patients greater than 1 year. The relative bioavailability was increased by 23.7% between each dosing occasion in patients with malaria infection.
The PK properties of lumefantrine has also been evaluated in a large pooled analysis based on 4122 patients from 26 different studies in adults (n = 2665), pregnant women (n = 123), and children below 10 years old (n = 1457) with uncomplicated P. falciparum malaria. This study found that lumefantrine were described accurately by a two‐compartment disposition model with first‐order absorption.
²⁵ (link) Lumefantrine exposure decreased with increasing pretreatment parasitemia, and showed dose‐dependent saturation of the absorption. Moreover, pregnancy status increased the absorption rate of lumefantrine by 35.2%.
PK parameters and variability presented in the published papers
²⁴ (link),
²⁵ (link) were used for the current simulations. All the patients were assumed to receive a daily dose of piperaquine or a twice‐daily dose of lumefantrine for a total of 3 days, following two plausible clinical dosing strategies; directly observed administration of the first dose only (DOT first‐dose) followed by non‐observed therapy for the remaining doses; and non‐observed therapy of all doses (non‐DOT). The dosage of piperaquine and lumefantrine were chosen based on bodyweight as given by the WHO malaria treatment guidelines.
¹² The simulation was based on the covariate‐parameter relationships in previous pooled PK model in patients with malaria infection. With the individual covariate data, we were able to simulate PK concentrations in all conditions for piperaquine (bodyweight and age) and lumefantrine (bodyweight, pretreatment parasitemia, dose, and pregnant status). We reported cutoff concentrations for each kg bodyweight in patients weighting more than 11 kg receiving piperaquine. For younger children (bodyweight <11 kg) receiving piperaquine, and all patients receiving lumefantrine, we used an R script (Appendix S1) to simulate and derive cutoff concentrations for different combinations of covariates.
The plasma concentrations of piperaquine and lumefantrine on day 7 post first‐dose has been reported to be a biomarker for therapeutic response,
³⁰ (link),
³¹ (link) with suggested target lumefantrine concentrations of 175 ng/mL,
³² (link) and a target piperaquine concentration of 30 ng/mL.
³³ (link),
³⁴ (link) Day 7 concentration was therefore used for adherence assessments. In addition, the overall predictive performance was evaluated when using simulated plasma concentrations on day 3, 7, 14, and 21 instead. The WHO recommends that therapeutic outcomes should be assessed on day 28, or day 42 for drugs with longer elimination half‐lives (e.g., piperaquine). This would have been the ideal sampling time for adherence assessments, but days 28 and 42 had a low predictive performance for the investigated drugs (i.e., piperaquine and lumefantrine) and was not evaluated further in this study.
Two thousand virtual individuals were simulated for full adherence and each nonadherence scenario, based on a typical non‐pregnant patient with a median bodyweight according to that reported in the modeled populations. Both the percentile and Bayesian approach were evaluated (in the same manner as stated above). However, the Bayesian method was not evaluated for lumefantrine due too many possible dose combinations (a total of 64 simulated full and nonadherence scenarios for 6 different dosing events) making this approach unpractical.

Drug concentration in plasma was measured at the Pharmacology Laboratory of the Mahidol Oxford Tropical Medicine Research Unit, using the high-performance liquid chromatography tandem mass spectrometry, as described previously (12 (link), 13 (link)). The overall method performance for each drug is summarized in Table S1. Samples were excluded from the analysis if the measured venous drug concentration was below the quantification level: the lower limit of quantification was 7.77 ng/mL for lumefantrine, 0.808 ng/mL for desbutyl-lumefantrine, 7.64 ng/mL for carboxy-mefloquine, 7.64 ng/mL for mefloquine, and 1.20 ng/mL for piperaquine.