Artemisinins

The blood samples and clearance data analyzed came from efficacy trials of artemisinin-based therapy in Pailin, Western Cambodia, in 2007-2008 (5 (link)). The trials are registered under ClinicalTrials.gov number NCT00493363 and Current Controlled Trials number ISRCTN15351875. Ethical approval was obtained from the Ministry of Health in Cambodia, the Oxford Tropical Medicine Ethical Committee, the WHO Research Ethics Review Committee, and the Technical Review Group of the WHO Western Pacific Regional Office. Parasite density was measured on admission and every 6 hours until clearance from the peripheral blood after treatment with artemisinin mono- or combination-therapy. The patients enrolled in the drug efficacy trials were given either artesunate [Guilin Pharmaceuticals] for 7 days in a dose of 2 mg/kg or 6 mg/kg (as a single or split dose), or artesunate for 3 days in a dose of 4 mg/kg or 8 mg/kg (as a single or split dose) followed by mefloquine [Medochemie] at 72 h (15 mg/kg) and 96 h (10 mg/kg) after admission (5 (link)). We plotted the natural log of parasite density against time for each patient and measured the slope to evaluate first-order clearance rate (CR).
We genotyped 18 microsatellite loci (12 (link)). Oligos, genomic location and polymorphism are detailed in the Supplementary online material. Parasites that were identical at all loci are referred as being CI. We compared variance of CR within and among CI parasites recovered from two or more patients and estimated H² from the mean squares terms in the ANOVA, following methods using for heritability estimation using identical twins or clonal plants (10 ). In brief, we determined the within and among clone mean squares (MS_e and MS_b). The total genetic variance in CR ( ${\sigma }_G^2$ ) was estimated as (MS_b − MS_e/n, where n is the weighted mean number of patients infected with each CI genotype. n is calculated as follows: $n=[T-(\sum n_i^2∕T\left)\right]∕(N-1)$ , where T is the total number of patients, N is the number of different CI genotypes, and n_i is the number of patients infected with the ith CI genotype. The environmental variance ( ${\sigma }_E^2$ ) is estimated from the within clone variation as MS_e and so $H^2={\sigma }_G^2∕({\sigma }_G^2+{\sigma }_E^2)$ . This analysis is directly equivalent to methods used in identical twin studies in humans or studies of clonal plants. To account for other covariates that could influence CR we included patient age as a continuous variable and gender and treatment regime as categorical variables in a regression analysis, and used the residuals in the ANOVA.