Therapeutic efficacy of artesunate–amodiaquine and artemether–lumefantrine combinations for uncomplicated malaria in 10 sentinel sites across Ghana: 2015–2017

The study was a one-arm, prospective, evaluation of the clinical, parasitological and haematological responses to directly observed therapy for uncomplicated malaria among children aged 6 months to 9 years in 10 sentinel sites across Ghana. Primary objectives were to assess prevalence of day 3 parasitaemia as well as the clinical and parasitological cure rates on day 28 following treatment with AS–AQ and AL. Secondary objectives were to assess the patterns of fever and parasite clearance, changes in haemoglobin concentration, gametocyte carriage rates, and prevalence of adverse events. Five of the 10 sentinel sites were scheduled to first study AS–AQ followed by AL whilst the other five were scheduled to first study AL followed by AS–AQ.

The study was conducted in all 10 sentinel sites for anti-malarial drug efficacy monitoring in Ghana (Fig. 1). The sites were Navrongo War Memorial Hospital (NWMH), Yendi Municipal Hospital (YMH) and Wa regional Hospital (WRH), located within the northern savannah zone of the country; Sunyani Municipal Hospital (SMH), Bekwai Municipal Hospital (BMH), Begoro Government Hospital (BGH), Hohoe Municipal Hospital (HMH), and Tarkwa Apinto Government Hospital (TAGH), located within the forest zone of the country; and, Ledzokuku Krowor Municipal Hospital (LEKMH) and Ewim Polyclinic (EWP) located, within the coastal zone of the country. Malaria transmission in the northern savannah zone is perennial with marked seasonal variation and estimated annual entomological inoculation rate (EIR) of up to 1132 infective bites per person per year [16‐18]. Malaria transmission in the forest zone is intense and perennial with estimated annual EIR of up to 866 infective bites per person per year [17, 19, 20]. Malaria transmission in the coastal zone is perennial but not intense with estimated annual EIR of fewer than 50 infective bites per person per year [21].

Seven sites (NWMH, SMH, HMH, BMH, BGH, TAGH, EWP) were able to study both AS–AQ and AL during the study period whilst WRH and LEKMH studied only AS–AQ, and YMH studied only AL.

All anti-malarials used in the study were fixed-dose combinations, and were supplied by WHO, Geneva. The AS–AQ used were from Sanofi Aventis whilst the AL used were from Ipca Laboratories Ltd, India. Treatment doses for AS–AQ were as follows: children weighing: (i) 4.5 to < 9 kg received a tablet of the 25/67.5 mg product daily for 3 days; (ii) 9 to < 18 kg received a tablet of the 50/135 mg product daily for 3 days; and, (iii) 18 to < 36 kg received a tablet of the 100/270 mg product daily for 3 days. The administration of AL (20/120 mg), which was also by weight, was at 0, 8, 24, 36, 48, and 60 h. Treatment doses were as follows: children weighing: (i) 5 to < 15 kg received one tablet per treatment hour; (ii) 15 to < 25 kg received two tablets per treatment hour; and, (iii) 25 to < 35 kg received three tablets per treatment hour. All treatments were given under direct observation of a study nurse after satisfying himself/herself that the child had eaten. The child was then observed for 30 min to ascertain retention of anti-malarial. Children who vomited during the observation period were re-treated with the same dose of anti-malarial and observed for an additional 30 min. Children with repeated vomiting were withdrawn from the study and treated as severe malaria according to national standard treatment guidelines using the following options: (i) intravenous artesunate (2.4 mg and 3.0 mg/kg body weight for children weighing ≥ 20 kg and < 20 kg, respectively, on admission, then at 12, 24 h, and daily until patient was able to swallow); or, (ii) intramuscular artemether (3.2 mg/kg body weight as loading dose and 1.6 mg/kg body weight once daily until patient was able to swallow). Parenteral treatment of severe malaria cases was given for a minimum of 24 h. A full 3-day course of oral ACT was given when child was able to swallow oral medication [7]. All children in the study were allowed use of antipyretics (acetaminophen).

All children enrolled were scheduled to visit the outpatient clinic on days 1, 2, 3, 7, 14, 21, and 28 (day 0 being the day treatment was started). Enrolled children were clinically examined by the study physician any time they visited the clinic. Asexual parasite densities and presence of gametocytes were assessed on days 2, 3, 7, 14, 21, 28, and any unscheduled visit within the 28 days of follow-up.

Thick and thin blood smears prepared on the days of parasitological assessment were stained with 3% Giemsa for 30–45 min. Asexual parasites were counted against 200 white blood cells using a hand tally counter whilst the presence of gametocytes was noted. Parasite densities were finally expressed per µl blood assuming white blood cell count of 8000/µl blood. A blood smear was declared negative after examination of 100 thick-film fields. All blood slides were read by two qualified independent microscopists, and discordant readings (in terms of presence or absence of parasites: asexual or sexual, species identification, and day 0 count meeting the inclusion criterion of 1000–250,000/µl blood) were re-examined by a third qualified independent microscopist, whose reading was considered final. Filter paper blots were obtained on day 0 and at recurrence of parasitaemia for PCR genotyping using merozoite surface protein 2 (MSP2)-specific primers: FC 27 and 3D7 to distinguish between recrudescence and re-infection [22, 23]. Samples were classified as recrudescence when pre-treatment and post-treatment alleles had the same band sizes (base pairs). Haemoglobin levels were assessed for all study children on days 0 and 28 using an automated haematology analyzer (Sysmex KX-21N™).

A minimum sample size of 50 was computed for each sentinel site based on an estimated treatment failure rate of 5% at 95% confidence level, 10% precision, and 20% loss to follow-up. Data were captured using WHO Microsoft Excel^® template for therapeutic efficacy tests [15]. Primary treatment outcomes analysed for each ACT were prevalence of day 3 parasitaemia and day 28 PCR-uncorrected and PCR-corrected outcomes as per WHO criteria: early treatment failure (ETF), late parasitological failure (LPF), late clinical failure (LCF), and adequate clinical and parasitological response (ACPR) [15]. Day 28 treatment outcomes were analysed per protocol and Kaplan–Meier. Briefly, PCR-uncorrected per protocol analysis excluded children lost to follow-up and withdrawn whilst Kaplan–Meier analysis censored last day of follow-up for such children. PCR-corrected per protocol analysis excluded children lost to follow-up, withdrawn, with falciparum re-infection, and undetermined PCR whilst Kaplan–Meier analysis censored last day of follow-up for those lost to follow-up as well as those withdrawn or with falciparum re-infection. Children with undetermined PCR were also excluded in the Kaplan–Meier analysis [15]. Secondary treatment outcomes analysed were patterns of fever (i.e. temperature ≥ 37.5 °C) and parasite clearance during the first week of follow-up, mean differences between pre- (day 0) and post-treatment (day 28) haemoglobin concentration, and prevalence of adverse events. Chi-square and Fisher’s exact tests were used to compare proportions whilst Student’s t-test was used to compare means (significant at p < 0.05).