Artemether

The full study methodology was published by Tiono et al. [21 (link)]. A brief summary of the methodology is given here.
This was a single-center, controlled, parallel, cluster-randomized study that evaluated the effect of systematic treatment of P. falciparum asymptomatic carriers on the incidence of symptomatic malaria episodes in children (<5 years) and adults over a 12-month period after completion of three community screening and treatment campaigns, compared with no treatment of asymptomatic carriers. In total, 18 clusters (each comprising one village) were randomized and assigned in a 1:1 ratio to the intervention or control arm.
Before the study implementation phase, all inhabitants of the 18 clusters were provided with long-lasting insecticide-treated nets (LLINs; OLYSET® nets [Sumitomo Chemical Co, Ltd, Tokyo, Japan]). Compliance with mosquito net use was checked every two months during home visits to the trial participants by Demographic Surveillance System (DSS) fieldworkers.
During the implementation phase, inhabitants of the intervention and control clusters participated in the three screening campaigns that took place ~1 month apart between January and April 2011, before the start of the rainy season. A fourth campaign was conducted in January 2012 after the rainy season had ended to mark the end of the study at ~12 months (Figure 1). At each campaign, finger-prick blood samples were taken from the entire study population in the intervention arm and a randomly selected 40% in the control arm for microscopic screening for P. falciparum asexual forms and gametocytes. In the intervention arm, the population was also screened using a rapid diagnostic test (RDT; First Response® Malaria Ag, Premier Medical Corp Ltd., Nani-Daman, India) to identify asymptomatic carriers. Those individuals with a positive RDT received treatment with artemether-lumefantrine (20 mg artemether and 120 mg lumefantrine [Coartem®, Novartis Pharma AG, Basel, Switzerland]) or artemether-lumefantrine dispersible, twice a day for three consecutive days. Subjects in the control arm were not screened by RDT – microscopy alone with delayed reading was used to ensure that study personnel and screened subjects remained unaware of subject status.
Following the third screening campaign, study participants in both arms were followed up for passive detection of symptomatic malaria throughout the wet season, when malaria transmission is high. Participants were encouraged to report to their local healthcare facility or clinic as soon as they felt unwell. An RDT was performed for all participants attending the local healthcare facility with confirmed fever (axillary temperature ≥37.5°C) or history of fever within the last 24 hours. On day 1 of campaign 4, a blood sample was also collected for gametocyte assessment by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), which was conducted in 1,999 randomly selected subjects drawn from the entire intervention group and a 40% subgroup of the control population. In this study, a case of symptomatic malaria was defined as fever or history of fever within the previous 24 hours and a P. falciparum asexual parasite count >5,000/μL. Each episode of symptomatic malaria was treated and the patient followed up at day 7. Parasitological cure was assessed by microscopy for each symptomatic malaria episode on day 7.
The blood films obtained during visits and for symptomatic malaria assessment were air-dried and Giemsa-stained for examination by a light microscope fitted with a 100 X oil immersion lens at a single laboratory. At least 200 thick film fields were examined before a slide was declared negative. When P. falciparum was present, a count of the asexual forms against leukocytes was made using a tally counter. Counting was done based on at least 200 leukocytes according to WHO standards. If less than 10 parasites were identified from the 200 leukocyte screen, counting was extended to 1,000 leukocytes. If P. falciparum gametocytes were seen, a gametocyte count was performed against 1,000 leukocytes. All slides were read by two independent microscopists. If the ratio of densities from the first two readings was >1.5 or <0.67, or if less than 30 parasites were counted with an absolute difference of more than 10 in the number of parasites, the slide was evaluated by a third microscopist. The definitive result was the mean of the parasite density of the two most concordant reading results.
Microscopist competency was evaluated through two equivalent quality control (EQC) programs. The first EQC was carried out by College of American Pathology proficiency testing and included a set of 5 slides provided to each microscopist for reading thrice a year. The second EQC was performed by WHO (National Institute for Communicable Diseases) and involved the reading of a set of 20 slides every quarter by each microscopist. Only those with a score of at least 80%, graded as ‘competent’ , were involved in the reading of trial participants’ slides.
This trial is registered with ClinicalTrials.gov, number NCT01256658.

Bovine serum albumin, phosphate buffer solution (PBS), trypsin, Roswell Park Memorial Institute (RPMI) 1640 Medium, Dulbecco’s Modified Eagle Medium (DMEM), DMEM/F-12, fetal bovine serum and penicillin & streptomycin & amphotericin B solution were purchased from Gibco, USA. CLIPPKF was purchased from Bank Peptide biological technology Co., Ltd, Hefei, China with the purity above 95%. Distearoyl phosphoethanolamine (DSPE)-PEG₂₀₀₀-Mal (Maleimide) was obtained from Laysan Bio Co., Arab, AL. Cholesterol (CHO), eggplant bottle, heparin sodium and Giemsa staining solutions were purchased from Titan Scientific Co., Ltd., Shanghai, China. Egg phosphatidylcholine (EPC), H₂O₂ and Tween-80 were purchased from J&K Chemical Ltd., Shanghai, China. Nile red (NR) and DAPI dye were from Beijing Fanbo Science and Technology Co., Ltd., Beijing, China. Percoll separation solution was obtained from Yibaiju Economic and Trade Co., Ltd., Shanghai, China. Annexin V-Cy5 and 10 × Annexin V binding buffer were from BioVision Inc, USA. Artemether, insulin and SYBR Green I was provided by Dalian Meilun Biology Technology Co., Ltd., Dalian, China. Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) was purchased from Beyotime Institute of Biotechnology, Beijing, China. The Hoechst 33,342 (HO), thiazole orange (TO), 5-(dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), hematoxylin-eosin (H&E) staining kit, ethylenediaminetetraacetic acid (EDTA), bicinchoninic acid (BCA) assay kit and DAB chromogenic kit were purchased from Sigma Aldrich, USA. Zoletil™ 50 and paraformaldehyde were purchased from Youchong Biological Technology Co., Ltd., Guangzhou, China. Anti-mouse TNF and IL-6, Streptavidin HRP were purchased from Shanghai Baiye Biotechnology Center, Shanghai, China. CellROX™ Deep Red Reagent, MitoTracker™ Deep Red FM, Texas Red®-X-conjugated WGA and TUNEL cell apoptosis detection kit were from Thermo Fisher Scientific, USA. Tissue autofluorescence quencher (AutoFluo Quencher) was purchased from Beijing Pulilai Gene Technology Co., Ltd., Beijing, China. Sodium citrate, xylene, physiological saline and hydrochloric acid ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd., China. Dimethyl sulfoxide (DMSO), dichloromethane, tetrahydrofuran, anhydrous ethanol and triethylamine were purchased from Sigma-aldrich, Germany. Nuclepore hydrophilic membrane was purchased from GE Healthcare life science, Whatman, England. PVDF blotting membrane, antibody CD47, HRP-conjugated secondary antibody, and Omni-Ecltm ultrasensitive chemiluminescence detection Kit were gained from Shanghai Epizyme Biomedical Technology Co., Ltd.

The recruitment of children took place from August 2017 to August 2018 with an interruption from April to June 2018 (representing the dry season). For all consecutive children aged 0 to 15 years presenting at the paediatric emergency room at any time with a clinical presentation suggestive of severe malaria, an informed consent was obtained by study personnel from parents/legal representatives. Patients were further assessed by paediatric ward clinicians (who benefited from a refresher protocol specific training prior to the study start) and observations recorded using a standardized case report form. Data collected included the medical history, vital signs, a complete physical examination for signs of severity (affecting the airway, respiratory, circulatory, and neurological systems). Finger prick blood sample was taken for malaria smear (methods for microscopy examination of blood smears and criteria for diagnosis of severe malaria are given below), haemoglobin and glucose measurements using point of care devices (HemoCue® Hb 201 + and HemoCue® Glucose 201 + ; HaemoCue AB, Ängelholm, Sweden). If the RDT was positive, anti-malarial treatment was immediately initiated based on the NMCP guidelines.
Children received parenteral artesunate at 2.4 mg/kg body weight on admission (time zero), then after 12 and 24 h and then once a day, or artemether Injection at 3.2 mg/kg on admission, then 1.6 mg/kg body weight per day. In both cases, parenteral administration continued until the child improved and was able to take full course of oral ACT, using artemether-lumefantrine or artesunate-amodiaquine. Supportive therapy recommended for severe malaria includes the treatment of hypoglycaemia with dextrose when glucose < 2.2 mmol/l, blood transfusion for children with haemoglobin less than 5 g/dL. All treatments were free of charge. No adjusted dose of parenteral artesunate for children weighing less than 20 kg was adopted by the NMCP at the time of the study conduct.