A single centre uncontrolled trial with three dose levels was conducted during the rainy season 2004 at the district hospital of Nouna in north-western Burkina Faso, an area of intense malaria transmission . Febrile children from Nouna town were invited to the hospital for examination and treatment. Inclusion criteria were age 6–59 months, uncomplicated malaria (axillary temperature ≥ 37.5°C and ≥ 2.000 P. falciparum asexual parasites per μl blood), haemoglobin ≥ 8 g/dl, absence of severe malaria and other significant disease, and informed written consent. The study was conducted in accordance with the internationally established principles for GCP and controlled by a data safety monitoring board (DSMB). The protocol was approved by the Ethics Committee of the University of Heidelberg and the local Ethics Committee in Burkina Faso.
Children were recruited for the three dose levels sequentially. In addition to receiving a total CQ dose of 25 mg/kg (10 mg/kg on days 0 and 1, and 5 mg/kg on day 2), study children received total doses of MB of 36, 54, and 72 mg/kg respectively. At each dose level children were block-randomized by envelope to two or four MB doses per day. MB (Mayrhofer Pharmazeutika, Linz/Austria) was given as a 2.3% solution with fruit flavouring and honey supplement to mask the bitter taste. CQ (tablets or syrup) was taken from the essential drug stock of the hospital. In case of vomiting within 30 minutes after intake, the drugs were re-administered once.
The dose escalation process for a dosage regimen went into the next higher dosage level if the safety (i.e. one-sided 95% CI for the incidence of relevant adverse events below 0.1) and the efficacy criterion (i.e. one-sided 95% CI for the incidence of CF below 0.15) were fulfilled at most in one dosage level.
Study participants were hospitalized for 72 hours. Treatment failures were managed according to national guidelines. Children were systematically examined on day 0, 1, 2, 3, 4 and 14. Blood samples were processed with standard methods in the laboratory of the Centre de Recherche en Santé de Nouna (CRSN) . Methaemoglobin formation was monitored twice daily on day 0 and once on day 1, 2 and 3. Other laboratory parameters like liver enzymes, serum creatinine or the phenotypical G6PD status were available at any time if clinically indicated. Based on filter paper blood samples, the G6PD genotype was determined in Germany .
Treatment outcomes were classified according to the WHO guidelines from 2003 as adequate clinical and parasitological response (ACPR), early treatment failure (ETF), late clinical failure (LCF), late parasitological failure (LPF) and clinical failure (CF = ETF+LCF) .
Assuming a 10% drop-out rate, 72 patients per group and dose level were needed to discover relevant safety and efficacy scenarios with a power of 80% and to avoid a false positive dose effect with probability of 95%. The sample size estimation assumed the independence of the efficacy outcome and the safety outcome. The null hypothesis had to be rejected in each level if the incidence of the relevant study adverse events fell below 10%. For treatment outcomes, the null hypothesis was rejected if the CF rate was below 15%. The safety analysis was based on the children who have received at least one dose of CQ-MB (FAS = full analysis set). Efficacy data were assessed in the population of children who received the full course of treatment (per protocol population, PP). All data were double entered and SAS® 8.2 was used to analyse the data. Continuous variables in two groups were compared with the nonparametric Wilcoxon-Mann-Whitney (WMW) or Kruskal-Wallis Test (KW), categorical variables with Chi-square-test (Chi). The clinical failure rates were analysed using the two fixed factors (group and level) in a logistic regression model, likelihood ratio tests (LR) were conducted.