This study was conducted at Chulaimbo Health Centre in Kisumu District in western Kenya. The facility serves a predominantly rural population in an area with high perennial malaria transmission in the lowlands around Lake Victoria. Transmission peaks during the long rains (March–May) and the short rains (October–December). The annual entomological inoculation rate in the surrounding area was 31.1 infectious bites/person/year for 12 months ending June 2004 [18
]. More than 95% of infections are caused by P. falciparum
, with the remaining being mixed P. falciparum
with Plasmodium malariae
and rarely Plasmodium ovale
infections. In vivo
drug resistance to SP in children <5 years is 74.5% [19
] and 59% to amodiaquine [21
Patient screening and recruitment
A 2003 WHO protocol for the assessment and monitoring of anti-malarial drug efficacy for the treatment of uncomplicated falciparum malaria was used [22
]. Children presenting to the health facility were screened for eligibility and invited to participate in the study if they met the following inclusion criteria: aged 6–59 months; body weight ≥ 5 kg; a history of fever in the previous 24 hours or measured fever (axillary temperature ≥ 37.5°C); monoinfection with P. falciparum
, with parasitaemia in the range of 2,000–200,000 asexual parasites per microlitre of blood; no other cause for fever than suspected malaria; and, no general danger signs or signs of severe and complicated falciparum malaria as per WHO guidelines [23
Study design, randomization and treatment
A randomized, controlled, open-label trial design was used. PCR-corrected cure rate by day 28 after first dose was the primary endpoint used for computing sample size. Assuming a 94% cure rate with artemether-lumefantrine tablets and 99.9% with the artemether-lumefantrine suspension, a sample of 134 children was required (i.e. 127, including 5% adjustment for loss to follow up) in each treatment arm to detect this 6% difference in the parasitological cure rates, with 80% power using a two-sided alpha of 0.05. The randomization code was computer-generated without stratification from which treatment groups were assigned.
At enrolment, a medical history was obtained from parents/guardians including presenting symptoms, current medications, previous anti-malarial use and bed net use. A physical examination was performed, weight and axillary temperatures recorded and finger prick blood obtained for malaria smears, haemoglobin and blotted on filter paper for parasite genotyping.
Consecutively eligible children were randomly assigned into one of two treatment groups according to the randomization code. A study nurse prepared and administered the study medications, according to the treatment assignment. One group received 6 doses of artemether/lumefantrine 20/120 mg tablets (Coartem®, Novartis AG), crushed, mixed with water and administered at hours 0, 8, 24, 36, 48 and 60 over 3 days while the second group received artemether/lumefantrine powder for suspension (Co-artesiane® Dafra Pharma NV) containing 15 mg artemether and 90 mg lumefantrine per 5 ml after reconstitution. A bottle of suspension was prepared for each child and administered once daily at hour 0, 24 and 48 over 3 days. Treatment doses were calculated based on patient weight and the administration directly observed under in-patient care. All children were given a glass of milk or breast fed (for those still breastfeeding) after drug administration since treatment times did not always coincide with meals. They were then observed for 30 minutes after drug administration for vomiting. If vomiting occurred, the whole treatment dose was re-administered. If the re-treatment dose was vomited, rescue treatment with parenteral quinine was administered and the child was withdrawn from the study.
All treatment failures were treated with oral or parenteral quinine for 7 days depending on clinical presentation. Paracetamol was administered to children with temperature ≥ 38.0°C or at the clinician's discretion. Antihelminthics were given to all children >1 year who had not received any in the past 3 months while children with haemoglobin <10 g/dl were treated with ferrous sulphate for 14 days. Long lasting insecticide treated bed nets (Olyset®, Sumitomo Corp) were given to the children on completion of the study.
All children in the study were admitted to hospital for 3 days for observed drug administration and followed up for a total of 28 days. Clinical and parasitological evaluation was performed during hospitalization (days 0, 1, 2 and 3) and at the study clinic during scheduled visits on days 7, 14, and 28, after initiating study treatment, or on any other day if the child was unwell. During each scheduled visit, a brief clinical history was obtained and a physical examination was performed; blood smears for malaria parasites and filter paper spot samples were obtained. Patients who did not return to the clinic for scheduled visits by mid day were visited at home by the social worker and asked to come to the health facility. Patients were excluded from the study if they; 1) withdrew consent, 2) left the study area, or 3) reported taking anti-malarial medication during follow-up.
Blood was drawn from a finger prick to prepare thick and thin blood smears on days 0, 1, 2, 3, 7, 14, 28 and on any other unscheduled visit. The smears were air dried, stained with 3% Giemsa for 30 – 45 minutes and read independently by two technologists. Parasite density was calculated by counting asexual parasites against a 500 leucocytes assuming a leukocyte count of 8,000/μL of blood to obtain number of parasites and gametocytes per microlitre (μL). Thin smears were examined for plasmodium parasite speciation. A slide was considered negative after scanning 100 high power fields. A third microscopist independently read discrepant slides. Haemoglobin was measured using HemoCue®
(HemoCue AB Angelholm, Sweden) on Days 0, 7, 14 and 28. Filter paper blood spots were collected on Days 0, 14 and 28 or any other day of recurrent parasitaemia. Paired filter paper samples of children who had parasitaemia during follow-up were used to extract parasite DNA for PCR to distinguish recrudescent from new infections as described by Snounou et al. [24
]. Block 2 of MSP-1 and block 3 of MSP-2 were amplified by nested PCR and size polymorphisms identified by gel electrophoresis against a 100-basepair (bp) molecular weight marker (New England Biolabs, Beverly, MA). Parasites were classified as recrudescent if they shared any of the bands that were present on day 0 and as new infections if there were no common bands.
This study was approved by the Kenya National/Kenya Medical Research Institute Ethics Committee. Written informed consent was obtained from all parents or guardians of eligible children prior to enrolment.
Data from case report forms were checked, double entered and verified for errors using Epi Info 2002 (Centers for Disease Control and Prevention, Atlanta, GA USA). Data was analysed using SPSS (v.12, SPSS Inc. Chicago, IL USA) and Epi-Info 2002. The primary efficacy endpoint was day 28 PCR-corrected parasitological cure rate, defined as the proportion of patients without asexual parasitaemia within 7 days after beginning treatment, without recrudescence within 28 days after beginning treatment and who demonstrated no need for rescue treatment for signs of clinical malaria within 28 days after initiation of study treatment. Secondary end-points included PCR-corrected parasitological cure rate on day 14, gametocyte carriage, fever and parasite clearance rate. The intent to treat (ITT) population defined as all randomized patients who took at least one dose of study medication was used for safety analysis. Proportions were compared between treatment groups using the chi-square test. Normally distributed variables were compared using the Student's t-test and analysis of variance (ANOVA). For skewed data, medians were computed and comparisons made using the Kruskal-Wallis one way ANOVA. Two tailed p-values < 0.05 were considered statistically significant.