Study design and participants
Fourteen adults with severe malaria, admitted to Mulago National Referral Hospital, Kampala, Uganda were enrolled. Participants were enrolled consecutively if they were 18 years of age and above, with a positive blood smear for Plasmodium falciparum mono-infection, no other obvious cause of the fever or symptoms, with at least one laboratory or clinical feature of severe malaria and requiring parenteral therapy in accordance with the 2010 World Health Organization Guidelines . Pregnant women, patients with history of anti-malarial intake within the last 72 hours and those receiving any herbal medication, known inhibitors or inducers of cytochrome P450 were excluded.
The study was approved by the Makerere University Faculty of Medicine Research and Ethics Committee (Ref2010-015) and Uganda National Council of Science and Technology (HS605) and registered with ClinicalTrials.gov (NCT01122134). Study procedures were explained to participants or their guardians in the local languages and information leaflets were provided. All participants provided written informed consent prior to enrollment.
Participants were admitted to Mulago Hospital for treatment and monitoring. On admission, all participants received baseline evaluation including; thorough history, physical examination and laboratory investigations. All participants were weighed and blood samples were collected by finger-prick for malaria smears and venepuncture for haematocrit, plasma lactate, glucose, renal and liver function tests.
All participants received intravenous artesunate at a dose of 2.4 mg/kg at time 0, 2.4 mg/kg at 12 hours and 2.4 mg/kg/daily until they could tolerate oral therapy. Artesunate was dispensed in a 60-mg ampoule which was dissolved in 1 mL of 5% sodium bicarbonate to form sodium artesunate and diluted with 5 mL of 5% dextrose. The dose was injected as a slow bolus into an indwelling intravenous cannula over 3–4 minutes. Supportive therapy was given according to national malaria treatment guidelines. When participants could tolerate oral therapy, anti-malarial therapy was completed with a full three-day course of oral artemether-lumefantrine (Coartem®, Novartis Pharma AG, Basel, Switzerland). Participants administered oral therapy as unsupervised therapy at home but were given instructions to administer it with food or milk.
Serial thick blood films and measurement of parasite densities were performed until parasites were cleared following the schedule; 0, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 16, 18, 20, 24 hours and every six hours till six hours post-parasite clearance. Blood smears were stained with 2% Giemsa for thirty minutes and parasite densities calculated by counting the number of asexual parasites per 200 white blood cells (WBC) using the patient’s actual WBC count per uL of blood.
Blood for artesunate and dihydroartemisinin assays was drawn after the initial dose of artesunate in chilled fluoride-oxalate tubes from the arm opposite that used for drug administration at 0 (pre-dosing), 5, 10, 15, 30, 45 minutes, 1, 1.5, 2, 3, 4, 5, 6, 8 and 12 hours post-dosing. The twelfth hour sample was drawn pre-the twelve hour IV artesunate dose. Four mL of venous blood were collected at each sampling time. Blood samples were chilled immediately and transported to the laboratory on ice to avoid artesunate and dihydroartemisinin degradation. Samples were centrifuged within 30 minutes and the plasma was stored below −80°C until shipment on dry ice to the Clinical Pharmacology Laboratory at the Mahidol-Oxford Tropical Medicine Research Unit in Thailand for artesunate and dihydroartemisinin quantification. The maximum duration of sample storage was approximately three months at −80°C.
Complete physical examination was performed daily for each patient. Vital signs were monitored and recorded every four hours until parasite clearance. Participants were discharged home when they were afebrile, aparasitaemic and able to take oral therapy. They returned one week after discharge for review of symptoms, clinical examination and a repeat blood smear. Assessment of adverse events was performed on admission and 1 week post discharge using history and physical examination.
Pharmacokinetic and statistical analysis
Pharmacokinetic analysis was performed with WinNonlin software, version 5.2 (Pharsight Corp., Mountain View, CA, USA) using an infusion non-compartmental analysis model. Complete bioconversion of artesunate to dihydroartemisinin was assumed. Calculated parameters included maximal observed concentration (Cmax), terminal elimination half-life (T1/2), total exposure measured as area under the plasma concentration-time curve (AUClast) from the start of drug infusion until the last quantifiable observation, elimination clearance (CL) and apparent volume of distribution (V). The AUC was calculated by application of the trapezoidal rule (linear-up/log-down). All parameters were calculated using time in hours after the time of first drug administration (T = 0). Drug concentrations below the LLOQ of the bioanalytical assays were treated as missing data.
Data were analysed using STATA® version 10.0 (StataCorp, College Station, TX). Baseline characteristics were summarized into medians with interquartile range (IQR). Pharmacokinetic parameters were summarized into medians with range. Parasite clearance time was defined as the time taken to clear all parasites from circulation ie time until the first of two sequential negative thick blood smears.