Study sites
The AL study was conducted in four of the 10 sentinel sites representing the three main ecological zones of Ghana. These were Navrongo War Memorial Hospital in the savannah zone; Bekwai Municipal Hospital and Begoro Government Hospital in the forest zone; and Ewim Health Centre in the coastal zone.
The Navrongo War Memorial Hospital is located in the Kassena Nakana East district in the Upper East region of Ghana. The Kassena Nankana East district has an estimated population of 109,944 with an annual average rainfall of 950 mm. Malaria in the district is perennial with marked seasonal variation. The peak transmission season coincides with the major rains between June and October. The Bekwai Municipal Hospital is located in the Bekwai Municipality in the Ashanti region of Ghana. The Bekwai Municipality has an estimated population of 118,024. Annual rainfall in the municipality is 1,600 – 1,800 mm with double maxima rainfall in May and October each year. Malaria transmission in the Municipality is intense and perennial. The Begoro Government Hospital is located in the Fanteakwa district in the Eastern region of Ghana. The Fanteakwa district has an estimated population of 108,614. Annual rainfall in the district is 150 – 2,000 mm with double maxima rainfall in June and October each year. Malaria transmission in the district is intense and perennial. The Ewim Health Centre is located within the Cape-Coast Metropolis in the Central region of Ghana. The Cape-Coast metropolis has an estimated population of 169,894. Annual rainfall in the metropolis is 750 – 1,000 mm with double maxima rainfall in June and December each year. Malaria transmission in the metropolis is perennial [
9‐
13].
Study population
The study population involved all children aged between six and 59 months presenting at the Out-Patient Department (OPD) of a study site clinic with symptoms suggestive of malaria. Once a clinical diagnosis of malaria was made by the study Nurse, samples of blood were obtained from a finger prick to prepare thick and thin smears for malaria microscopy and haemoglobin level determination. Children meeting the inclusion criteria were recruited into the study and followed up for a minimum of 14 days and a maximum of 28 days. Briefly, the inclusion criteria included axillary temperature ≥ 37.5°C or history of fever during the past 24 hrs; mono-infection with Plasmodium falciparum; parasite count ranging between 1,000 and 250,000 per μl; haemoglobin level > 5 g/dl; absence of signs/symptoms of severe malaria; and parent’s willingness to give their consent.
Children recruited into the study received weight-based 20/120 mg AL (Coartem® – batch number x1435 supplied by WHO, Geneva) at 0, 8, 24, 36, 48, and 60 hrs. All treatments were given under direct observation by a study nurse and children were observed for 30 minutes to ascertain retention of medicine. Children who vomited during the observation period were re-treated with the same dose of medicine and observed for an additional 30 minutes. Children with repeated vomiting were given parenteral therapy with quinine as per national standard treatment guidelines and excluded from the study. All children were allowed use of antipyretics. Children who showed signs/symptoms of severe malaria, had serious adverse events or required blood transfusion were withdrawn from the study.
Outpatient follow-up visits were scheduled for days 1, 2, 3, 7, 14, 21, and 28 after treatment (day of treatment was counted as day-0). At each visit to the clinic, children were examined physically and information on symptoms, axillary temperature, and respiratory rate recorded on a Case Record Form (CRF). Parasitaemia levels (asexual and sexual) were assessed on days 2, 3, 7, 14, 21, 28, and any day within the 28-day follow-up period that a child is brought to the clinic with fever. Thick and thin smears were stained with 3% Giemsa for 30–45 minutes for quantification of asexual parasites against 200 white blood cells using a hand tally counter. Sexual parasite counts were done per 1,000 white blood cells. Parasitaemia levels were expressed per μl blood assuming white blood cell count of 8,000 per μl blood. A smear was declared negative when examination of 100 thick-film fields did not show presence of asexual parasites. For quality control purposes, all blood slides were read by two qualified independent microscopists, and discordant readings were re-examined by a third qualified independent microscopist. Discordance was defined as differences between the first and second microscopists regarding presence/absence of asexual or sexual parasites; species diagnosis; and day-0 counts meeting the inclusion criterion of 1,000 – 250,000 per μl blood. The first or second reading was taken as final depending on whichever agrees with the third reading. Filter paper blots were obtained on day-0 and at recurrence of parasitaemia for PCR genotyping, and merozoite surface proteins 1 and 2 (msp1, msp2), and glutamate-rich protein (glurp) used to distinguish between re-infection and recrudescence. Haemoglobin levels were assessed for all study children on days 0, 14, and 28.
Data analysis
Per protocol analysis was applied in this study. Primary outcomes were treatment outcomes on day-14 and day-28 for the different ecological zones based on the WHO 2009 criteria: Early treatment failure (ETF), Late Parasitological Failure (LPF), Late Clinical Failure (LCF), and Adequate Clinical and Parasitological Response (ACPR) [
8]. Secondary outcomes were patterns of fever and parasite clearance assessed for the different ecological zones using proportions (with 95% CI) of children febrile/with temperature ≥ 37.5°C or parasitaemic within the follow-up period. Haematological responses were also assessed using mean haemoglobin levels on day-14 and day-28. Proportions were compared using chi-square and Fisher’s exact tests. Normally distributed variables were compared using Student’s t-test while skewed distributions such as parasite counts were log transformed before using the normal approximation. Differences were considered significant at
p < 0.05.
Ethics
The Institutional Review Board of the Noguchi Memorial Institute for Medical Research, University of Ghana, reviewed and approved the study. Written informed consent was obtained from each parent/guardian at the start of the study. Each parent/guardian was informed of the objectives, methods, anticipated benefits and potential hazards of the study. They were also informed that they were at liberty to withdraw their children from the study at any time without penalty.