Study design and study site
The study was conducted from November 2006 to January 2007 at the end of the rainy season, in the primary health care center of Anonkoua-kouté, 10 kms from Abidjan. This health care center is located in a crowded sub-urban area with holoendemic P. falciparum transmission.
This trial was a randomized single-blinded clinical trial.
Inclusion criteria were the following: (1) six months old or more; (2) P. falciparum monoinfection with parasitaemia level of 2,000 – 200,000 asexual parasites/μl of blood; (3) axillary temperature equal or over 37.5°C at the time of enrolment or history of fever during the preceding 24 h; (4) no history of serious side effects to study medications; (5) no evidence of concomitant febrile illness; (6) provision of written informed consent by the participant or by a parent or guardian (for children).
Exclusion criteria were as follows: (1) symptoms and/or signs of severe malaria; (2) any danger sign (persistent vomiting; inability to sit, stand, drink or breastfeed; (3) recent history of convulsions and/or lethargy or otherwise impaired consciousness; (4) haemoglobin concentration ≤ 6 mg/dl; (5) serious underlying disease or known allergy to the study drugs.
Participants were also excluded after randomization, if they repeatedly vomited their first dose of study medications.
The sample size for the study was calculated based on the assumptions that day 28 PCR-corrected cure rates for the ACTs will not be less than 95%, and that the cure rate for either AN or AL will not be less than 85%. At 95% confidence interval, 80% power, and a sample ratio of 1:1, the required sample per arm was 49. Allowing for a dropout rate of 10%, a minimum of 55 participants were to be recruited for each arm of the study.
Treatments and randomization
Participants recruited into the study were allocated to two treatment groups using a computer generated random list based on a simple random selection procedure without the use of blocking or stratification by an off-site investigator.
Sequentially numbered, sealed envelopes containing the treatment group assignments were prepared from the randomization list.
The study clinical investigators assigned treatment numbers sequentially and a third party investigator who is an appropriately qualified member of the study site, allocated treatment by opening the envelope corresponding to the treatment number. The randomization codes were secured in a locked cabinet accessible only by the third party. Participants were enrolled by the study physicians, and treatments were assigned and administered by the third party.
Only the third party was aware of treatment assignments. All other study personnel, including the study physicians and laboratory personnel involved in assessing outcomes, were blinded to the treatment assignments. Participants were not informed of their treatment regimen.
Study treatment was started on the day of randomization (day 0) and completed by day 2 according to the combination received.
The study drug Arco® (AN) was provided in blister packs by Kunming Pharmaceutical Corp. (China) The active ingredients are artemisinin and naphtoquine. Each tablet contains 125 mg of artemisinin and 50 mg of naphtoquine.
The comparator drug Coartem® (AL) was provided by Novartis SA (swiss) in blisters packs. The active ingredients are artemether 20 mg and lumefantrine 120 mg.
Administration of treatment was as described below.
The dosing for artemisinin/naphtoquine (AN) was 1/2 crushed tablet for participants weighing between 6 kg and 10 kg; one crushed tablet for participants weighing between 10 and 15 kg; two crushed tablets for participants weighing between 15 and 25 kg; three tablets for participants weighing between 25 and 35 kg, and four tablets for participants whose weight was at least equal to 35 kg. All tablets were given twice on a single day; the second dose being given eight hours after the first dose.
For artemether/lumefantrine (AL), the dosing was 1 crushed tablet for participants weighing between 5 and 15 kg; two crushed tablets for participants weighing between 15 and 25 kg; three tablets for participants weighing between 25 and 35 kg; four tablets for participants weighing between whose weight was at least equal to 35 kg. All tablets were given twice a day for three days; the second dose was given eight hours after the first one and the third dose 24 hours after the first one or 16 hours after the second one, the following doses given every 12 hours.
For AN, the morning doses were directly observed, while the evening dose was given to the participants to be taken at home. For AL, the morning doses were also directly observed over the three days of treatment, while the evening doses were given to participants to be taken at home. The empty sachets were returned to the study site as evidence of taking the drug. Paracetamol tablets (three doses per day for two days) were provided, to be taken if needed. Participants who were absent for follow-up were visited at home on the same day.
All tablets were either swallowed whole or crashed with water.
In the case a participant vomits the first dose (D0) within 30 minutes of drug administration; a repeat full dose was given. If this participant also vomits this repeat dose or any subsequent dosing, he was not re-dosed and was withdrawn from the study. A participant who was withdrawn due to vomiting received rescue medication according to local practices.
Enrolled participants were given an identity code and were assigned to receive either artemisinin/naphtoquine (125/50 mg tablet) twice on a single day (Day 0) or artemether/lumefantrine (20/120 mg tablet) twice a day for three days (Day 0, 1 and 2). At enrolment, we asked participants, children's parents or guardians about prior anti-malarial therapy, use of other medications, and presence of common symptoms. Axillary temperature and weight were recorded, and a physical examination was performed. We also collected blood by fingerprick for thick and thin blood smears, and for storage on filter paper for PCR analysis.
Participants were recommended to return to study site for follow-up visit on days 1, 2, 3, 7, 14, 21 and 28, and any other day, if they felt ill. Follow-up evaluation consisted in a standardized history and physical examination. If participants did not return for follow-up, they were visited at home.
Participants were excluded after enrolment if any of the following occurred: (1) use of antimalarial drugs outside of the study protocol; (2) parasitaemia in the presence of a concomitant febrile illness; (3) withdrawal of consent; (4) loss to follow-up, (5) protocol violation, or (6) death due to a non-malaria illness.
Treatment outcomes were classified according to 2003 World Health Organization (WHO) guidelines as early treatment failure (ETF: danger signs or complicated malaria or failure to adequately respond to therapy days 0–3); late clinical failure (LCF: danger signs or complicated malaria or fever and parasitemia on days 4–28 without previously meeting criteria for ETF); late parasitological failure (LPF: asymptomatic parasitemia on day 28 without previously meeting criteria for ETF or LCF); and adequate clinical and parasitological response (ACPR: absence of parasitemia on day 28 without previously meeting criteria for ETF, LCF, or LPF) [WHO assessment].
The overall rate of treatment failure (Total Treatment Failure TTF) was computed as if the participant had an ETF, LCF or a LPF. Only parasitaemia confirmed by PCR as recrudescence was considered a treatment failure. Participants were also considered treatment failures if they received rescue treatment on or before day 28.
An adverse event is defined as any unfavourable and unintended sign, symptom, syndrome, or illness that develops or worsens during the period of observation in the study.
Clinically relevant abnormal results of diagnostic procedures including abnormal laboratory findings which was considered by the investigator to be detrimental was recorded as adverse events whether or not they have a causal relationship with the study drug.
All observed adverse events were monitored actively and passively from the time the participant has taken one dose of study treatment through last visit, and were recorded on the Case Report Form (CRF) according to Good Clinical Practice (GCP) and ICH guidelines.
Thick and thin blood smears were prepared and stained in 10% Giemsa solution for 30 minutes. The smears were read to 100 fields with quantification of P. falciparum asexual parasitaemia on the thick smear. Parasites were enumerated using thick film, as described by Shute . The parasite density (per μl of blood) was calculated, assuming a normal leucocyte level of 8,000/μl. The thin film was used to speciate the parasites.
A smear was considered negative if no parasite were seen after review of 100 high-power fields. We also assessed gametocytemia from thick blood smears. Thin blood smears were reviewed for non- falciparum infections. A second microscopist, who was unaware of the results of the first reading, re-read all slides. A third microscopist unaware of the first two readings resolved discrepant slides.
A complete blood count plus serum alanine aminotransferase (ALT), serum asparate aminotransferase, serum total bilirubin and serum creatinine assessments were performed on the total participants at baseline and seven days after initiation of treatment. Tests were also performed if clinically indicated or if a significant abnormality was detected on day 7. Molecular genotyping techniques were used to distinguish recrudescence from new infection for all participants failing therapy after day 7. Briefly, filter paper blood samples collected on the day of enrolment and on the day of failure were analyzed for polymorphisms in the genes for merozoite surface protein-1 (msp -1) and merozoite surface protein-2 (msp -2) using nested-PCR as previously described .
Management of recrudescent infections
Participants with uncomplicated recrudescent infections were re-treated with quinine, 24 mg/kg/day for 5 days. However, their response to repeat therapy was not assessed.
Data generated were recorded in a log book and individual participants case record files. Data were entered and analysed with EPI-Info version 6.4. Analysis of treatment outcome was per protocol, which only included participants with treatment outcomes. Frequencies were compared by chi-squared tests and Fisher exact tests, and continuous variables by Student's t-tests, Mann-Whitney U-tests, analysis of variance or Kruskal-Wallis tests as applicable.
All the clinical and laboratory data collected were subjected to quality control. The study was carried out according to standard operating procedures following the guidelines of good clinical practice.
The study was approved by our Ethical Committee. This work was conducted in compliance with ICH-GCP guidelines. Patient's informed consent was obtained according to the ethical principles stated in the declaration of Helsinki 2000 version (amended in Tokyo 2004), the applicable guidelines for ICH-GCP. The applicable laws and regulations of Ivory Coast Informed consent document was used to explain the risks and benefits of study participation to the participant if adults or parent or guardian of children participants in simple terms before the participant was entered into the study. The informed consent document contains a statement that the consent is freely given, parent or guardian of the participant was aware of the risks and benefits of entering the study, and the participant is free to withdraw from the study at any time. Written consent was provided by participant or parent or guardian. If the participant or parent or guardian was unable to write, witnessed consent was permitted.