Ethical approval for the study was granted by the Ethics Committee of the Faculty of Medicine, Pharmacy, and Dentistry of the University of Science, Techniques, and Technologies of Bamako (Bamako, Mali), the Committee on Human Research at the University of California San Francisco (UCSF; San Francisco, CA, USA), and the Research Ethics Committee of the London School of Hygiene & Tropical Medicine (London, UK).
Study cohort and sample collection
This study used samples obtained from participants of a trial in Ouélessébougou, Mali who were randomized 1:1:1:1 to receive either sulfadoxine–pyrimethamine (SP–AQ), SP–AQ with single low-dose primaquine (SP–AQ + PQ), dihydroartemisinin-piperaquine (DP), or dihydroartemisinin–piperaquine with methylene blue (DP + MB) . Eligible participants were males with asymptomatic P. falciparum mono-infection, between 5 and 50 years of age, who were glucose-6-phosphate dehydrogenase (G6PD)-normal by CareStart G6PD rapid diagnostic test (Access Bio, Somerset, NJ, USA), had a hemoglobin concentration of ≥ 10 g/dL, and had a P. falciparum gametocyte density of ≥ 2 gametocytes/500 white blood cells by thick film microscopy. Participants were excluded if they had a serious or chronic illness (including signs of severe malaria), weighed 80 kg or more, reported anti-malarial use within 7 days of screening (artemether–lumefantrine and artesunate–amodiaquine being first-line treatments), or reported allergies to study drugs.
Details on the study procedures are described in the original paper . In brief, participants were followed for 42 days and blood samples were obtained on days 0, 1, 2, 7, 14, 28 and 42 after initiation of treatment. Blood smear microscopy was conducted on blood samples taken by finger prick on day 0 and all days of follow-up to assess for asexual parasite and gametocyte density. For the measurement of ring-stage and gametocyte density by qRT-PCR, 100 μL of blood was collected in EDTA tubes and immediately transferred to RNAprotect (Qiagen) and stored at − 80 °C until extraction by MagNAPure LC automated extractor (Total Nucleic Acid Isolation Kit-High Performance; Roche Applied Science, Indianapolis, IN, USA).
Ring-stage parasites were quantified by qRT-PCR targeting the sbp1 mRNA transcript using previously described methods . The limit of quantification of this assay is in the range of 1 parasite/µL, the limit of detection is 0.01 parasite/µL Male and female gametocyte densities were quantified by qRT-PCR, targeting male PfMGET and female Pfs25 mRNA transcripts as described elsewhere . For samples that were microscopy-positive for asexual P. falciparum parasites on days 7, 14, 28, and 42, PCR genotyping of glurp (glurp2), msp2 (Fc27), msp1 (K1, MAD20 and RO33) and lc1 alleles were performed on samples obtained at enrollment and day of post-treatment failure. Pre- and post-treatment pairs were analysed and classified as either recrudescent, re-infection, or indeterminate infections according to World Health Organization guidelines [8, 17].
Sample size considerations
The sample size for this study was dictated by the original clinical trial, with 20 individuals per study arm to allow a within-person change in infectivity after treatment . It is acknowledged that this sample size is insufficient to quantify with precision the proportion of individuals with persisting ring-stage parasites post treatment. Despite modest in sample size, the current study samples do allow, a unique comparison of parasite persistence following ACT and non-ACT treatment. The latter group (i.e. individuals with confirmed parasite carriage receiving non-ACT treatment) is rare among clinical trials and allows exploration of the hypothesis that ring-stage parasite persistence is specific for ACT treatment [12,13,14].
All analyses were performed using Stata 14.0 (StataCorp, College Station, TX, USA) and R (version 3.5.0; R Project for Statistical Computing; http://www.r-project.org/). Comparisons between proportions were conducted using Chi-squared or Fisher’s exact test and Mann–Whitney tests were used to compare differences in parasite densities, unless otherwise specified. Correlations between ring-stage and gametocyte parasite densities were assessed by Spearman’s rank correlation coefficient using the log10 transformed versions of these variables. Generalized estimating equations were used to compare SBP-1 parasite prevalence and density between SP–AQ and DP arms, accounting for repeated observations for individuals. Log-binomial regression was used to model relative risk ratios and linear regression was used to model mean differences in log10-transformed SBP-1 parasite density. Models adjusted for baseline log10-transformed SBP-1 parasite density. An interaction term between treatment and follow-up visit was included to assess whether participants of the DP group cleared parasitaemia at a more rapid rate than SP–AQ. An overall F-test was used compute the p-value testing joint effect of the interaction terms. Log-binomial regression models were used to assess whether age, weight, treatment arm, or baseline SBP-1 parasite density were associated with persistent parasitaemia on day 7 post-treatment. All tests were two-sided with alpha = 0.05. p-values < 0.05 were considered statistically significant.