This study was conducted in three countries, Cameroon in central Africa, Ivory Coast and Senegal in West Africa. In Cameroon, the study was performed in the urban health centre of Melen in Yaoundé, the capital city. In Ivory Coast, it was done in El Rapha Health Centre of Abidjan, the capital city, and in Senegal in the health district of Kaolack located 200 km from Dakar, the capital city.
Children above 6 months and under 7 years of age, who came to the local health centre, with P. falciparum parasite density between 1,000 and 100,000/μl in a low transmission area (Senegal) and between 2,000 and 200,000/μl in high transmission areas (Ivory Coast and Cameroon)
, with the presence of an axillary temperature higher than 37.5°C, ability to swallow drugs per os, were enrolled in the study. Patients presenting severe vomiting, signs of severe malaria
 or severe malnutrition (children whose weight-for-height was less than 70% of the median NCHS/WHO reference value, with a symmetrical oedema involving the feet), patients with allergy to study drugs or who had used any anti-malarial drug within 28 days prior to enrolment were excluded. Each participant’s guardian gave fully informed written consent prior to enrolment.
This was a multicentre open, comparative and randomized phase IV trial undertaken in two parallel groups to test non-inferiority between two associations: AS+AQ versus artemether/lumefantrine (AL) in children from six months to seven years. After enrolment, children were weighed and randomized into blocks of 10 to receive one of the two drugs.
The formulations of the two associations were the following: AS+AQ in a box with 1 bottle containing AS in powder dosed a 160mg/80ml to suspend and 1 bottle containing AQ in syrup dosed at 50mg/5ml. Children under two years received 10ml of AQ and 20ml of AS per day and children up to two years received 15ml of AQ plus 25 ml of AS per day, in a single administration.
AL was presented in fixed tablets containing 20mg of artemether and 120 mg of lumefantrine. Tablets were crushed and mixed with water before administration. No food was given prior the AL administration. It was given twice a day according to the patient’s bodyweight and the manufacturer’s instructions. The two drug regimens covered three days (day 0 to day 2). All the daily doses were administered at the health post, under the direct supervision of the investigators. In case of vomiting within the 30 minutes following the administration, study doses were administered again to the patient. Patients who kept vomiting were withdrawn.
Study participants were examined in the study clinic 1, 2, 3, 7, 14, 21, and 28 days after enrolment or at any time if they did not feel well. Quinine treatment was given in case of treatment failure which includes : early treatment failure (development of danger signs or severe malaria on Day 1, Day 2 or Day 3, in the presence of parasitaemia, or parasitaemia on Day 2 higher than Day 0 count irrespective of axillary temperature, or parasitaemia on Day 3 with axillary temperature ≥3 7.5°C, or parasitaemia on Day 3 ≥ 25% of count on Day 0) and late treatment failure (development of danger signs or severe malaria after Day 3 in the presence of parasitaemia, without previously meeting any of the criteria of early treatment failure or presence of parasitaemia and axillary temperature ≥ 37.5°C on any day from Day 4 to Day 28, without previously meeting any of the criteria of early treatment failure)
Biological follow up
Finger prick to obtain blood for thick and thin smears were done at Day 1, 2, 3, 7, 14, 21 and Day 28 after inclusion or at any time if the patients did not feel well. Blood smears were stained with Giemsa and 200 leucocytes were counted. Assuming a total leukocytes count of 8,000 per litre, parasite density was determined as the number of asexual parasites × 8,000/200
. The slide readers were kept blinded to the treatment allocated.
Polymerase chain reaction (PCR)
Four drops of blood were collected on filter paper for each patient at day 0 and at day of recurrent parasitaemia to distinguish recrudescence from new infection. For this, parasite genotyping by nested PCR was conducted to compare two polymorphic genetic markers from Plasmodium falciparum: Merozoite Surface Protein (msp) genes 1 and 2. The primers and schedules described by Faye et al were used.
Tolerability and safety assessment
All adverse events were closely monitored during each scheduled visit by interview with patient if possible or guardian and clinical examination by a physician. All signs noted were reported in the case report form. Adverse events were defined as occurring new events or worsening from baseline after administration of treatment. Haematology and biochemical tests were done at the enrolment and at Day 7 to evaluate haemoglobin, creatinine, aspartate amino transferase (ASAT), alanine amino transferase (ALAT) and bilirubine parameters. Biological abnormalities were noted.
Data statistical analysis
The number of patients to be included in this study was determined using Epi info software version 6.04d
, On the basis of previous studies, the cure rate of AL was estimated at 95%
[8, 9]. The maximal difference acceptable for the AS+AQ to be considered as clinically non-inferior is 10% (absolute value in percentage)
[10, 11]. For a statistical power of 80% (β = 20%) a risk α = 5% and using 95% confidence level sample size for each arm was 207. In order to prevent premature stops, this number was increased to 10% given a total of 456 patients (76 patients per arm in each country). Data were entered using Epi info software version 6.04d. All analysis was done with STATA IC 10™ software.
An intention to treat (ITT) and per protocol (PP) analysis were done. ITT included all randomized participants who took at least one full dose and had one post baseline efficacy assessment without major protocol violations as wrong dosage, wrong use of non-assigned drug by mistake, co-infection with other malaria species. Patients with major violations and patients lost during the follow-up or withdrawn (due to an adverse event or to the use of another drug with anti-malarial activity or withdrawal consent) have been considered as failure. The per protocol analysis included patients who received the three doses and who had no major protocol violation up to the day 28. Those lost to follow up, and the withdrawals of consent were excluded from the per protocol analysis.
The primary endpoint was the Adequate Clinical and Parasitological Response (ACPR) which is an absence of parasitaemia on Day 28 irrespective of axillary temperature without previously meeting any of the criteria of Early Treatment Failure or Late Clinical Failure
. The cumulative incidence of failure rate was calculated in each study arm and compared using Kaplan-Meir method. The secondary endpoints were the comparison of fever and parasites clearances, gametocytes carriage after treatment and tolerability of the two regimens.
Data were analysed by estimations of difference in proportions corresponding to 95% confidence interval. Comparison between groups was made using chi-square test or Fisher exact test for qualitative outcome and student test for quantitative outcome when applicable. Otherwise, non-parametric tests (Man Withney, Kruskall Wallis) were used. A p value (two-sided) less than 0.05 was considered statistically significant.
The protocol was reviewed and approved by the national ethical committees of each country.