The study was conducted in the War Memorial Hospital (WMH) in Navrongo (representing the savannah zone); Hohoe Municipal Hospital (HMH) in Hohoe (representing the forest zone); and Ewim polyclinic (EWP) in Cape-Coast (representing the coastal zone) ( Figure 1 ). These sites have been described elsewhere (Abuaku et al., 2019). Briefly, malaria transmission in the savannah zone is perennial with marked seasonal variation. Malaria transmission in the forest zone is intense and perennial whilst transmission in the coastal zone is perennial but not intense (Abuaku et al., 2019).

The study was a one-arm prospective evaluation of the clinical, parasitological and hematological responses of children treated with DHAP in three of Ghana’s ten sentinel sites for monitoring therapeutic efficacy of first-line and second-line antimalarials.

Children aged between 6 months and 9 years were included in this study, if they had a history of fever during the past 24 hours or an axillary temperature ≥ 37.5°C with mono Plasmodium falciparum infection and a parasite density ranging between 1,000 and 250,000 per µL. Children with severe malaria or whose parents refused to give consent were excluded from the study. As per the WHO protocol on methods for surveillance of antimalarial drug efficacy, a prior intake of an antimalarial was not an exclusion criterion (WHO, 2009).

Following parental consent, enrolled children were treated with 20 mg/160 mg or 40 mg/320 mg of D-ARTEPP^® (products of Guilin Pharmaceutical Company Limited, Guangxi, China) based on their weight in kilograms as per the manufacturer’s instructions ( Table 1 ). All children were observed for 30 minutes after drug administration. Those who vomited within the 30 minutes after treatment received a repeated dose of D-ARTEPP^®. Children with repeated vomiting were withdrawn from the study and treated as severe malaria cases as per national case management guidelines (MOH, 2020).

Each child was followed-up for a period of 42 days. The follow-up schedule involved clinical assessment (days 0, 1, 2, 3, 7, 14, 21, 28, 35, 42, and any unscheduled visit within the 42-day follow-up period); drug administration (days 0, 1, 2); parasitological examination (days 0, 2, 3, 7, 14, 21, 28, 35, 42, and any unscheduled visit within the 42-day follow-up period); and hemoglobin level assessment (days 0, 28, and 42) using an automated hematology analyser (Mindray BC-2800™).

All malaria blood slides for parasitological examination were examined by two microscopists. Slides with discordant readings were re-examined by a third senior microscopist. Discordant readings were related to presence/absence of asexual/sexual parasites, species diagnosis, and day 0 parasite density within range of inclusion criterion (1,000 – 250,000 per µL). Parasite recrudescence was distinguished from re-infection by PCR genotyping using merozoite surface protein 2 (MSP2)-specific primers for FC 27 and 3D7 strains. List of specific sequence of primers used have been published elsewhere (Duah et al., 2016). Samples with post-treatment alleles having the same band sizes as pre-treatment alleles (base pairs) were classified as recrudescence (WHO, 2007).

A minimum sample of 55 children was estimated for each study site based on 5% PCR-corrected treatment failure rate at 95% confidence level, 6% precision, and10% loss to follow-up. Data for each child was captured using the WHO Excel^® template for therapeutic efficacy tests. The primary study outcomes were day 3 parasitemia and days 28 and 42 PCR-uncorrected and PCR-corrected efficacy outcomes. Day 3 parasitemia was evaluated as the proportion of patients seen on day 3 with parasitemia. Per protocol and Kaplan Meier survival analyses were used to describe the patterns of PCR-uncorrected and PCR-corrected treatment outcomes on days 28 and 42 post-treatment. Treatment outcomes analyzed were defined as early treatment failure (ETF), late parasitological failure (LPF), late clinical failure (LCF), and adequate clinical and parasitological response (ACPR) as per WHO protocol (WHO, 2009). Secondary study outcomes were measured fever (axillary temperature ≥ 37.5°C), parasite clearance, changes in mean hemoglobin levels, and prevalence of adverse events. Proportions were compared using Chi-square and Fisher’s exact tests whilst means were compared using student t-test/ANOVA (significant at p<0.05).

The Institutional Review Board (IRB) of the Noguchi Memorial Institute for Medical Research, University of Ghana (FWA 00001824) approved this study (NMIMR-IRB CPN 032/05-06a amend. 2020). Written informed consent was obtained from each parent/guardian prior to commencement of the study. Each parent/guardian was presented with details of the study: objectives, methods, anticipated risks and benefits. They were also informed of their right to withdraw their children from the study at anytime during the study period without penalty.

A total of 226 of the 326 children screened met the inclusion criteria and were enrolled into the study in the three sites ( Figure 2 ). The proportion of males was higher among participants in EWP (53.6%) and lower among participants in HMH (46.0%) and WMH (48.2%) but not statistically significant (p=0.671) ( Table 2 ). The mean age (years) of participants was significantly higher in EWP (5.4 ± 2.4) and WMH (5.4 ± 2.5) compared with HMH (2.2 ± 2.5) (p<0.001). As expected, the mean weight of participants was significantly lower in HMH (11.9 ± 5.8), where a higher proportion of participants (79.3%) were less than 5 years old compared with EWP and WMH ( Table 2 ). Mean axillary temperature (°C) was significantly higher in WMH (38.2 ± 0.8) and HMH (38.0 ± 0.6) compared with EWP (37.7 ± 1.1 (p<0.001). Geometric mean parasite density was significantly highest among participants in EWP whilst mean hemoglobin level was significantly higher among participants in WHM (10.9 ± 1.4) compared with HMH (9.9 ± 0.9) and EWP (9.9 ± 1.4) (p<0.001) ( Table 2 ). No participant in the three study sites was gametocytemic, and none had a history of previous intake of an antimalarial prior to visiting the clinic.

No day 3 parasitemia was detected in all three sites. Per protocol analyses on day 28 showed no ETF, LCF, and LPF yielding a day-28 DHAP cure rate of 100% in all three sites. Extending the follow-up period to the standard 42-day period showed PCR-uncorrected and PCR-corrected cure rates of 100% for HMH; PCR-uncorrected and PCR-corrected cure rates of 96.4% (95% CI: 86.4-99.4) and 100%, respectively, for EWP; PCR-uncorrected and PCR-corrected cure rates of 71.8% (95% CI: 60.3-81.1) and 90.3% (95% CI: 79.5-96.0), respectively, in WMH ( Table 3 ). The national PCR-uncorrected and PCR-corrected cure rates were therefore 89.1% (95% CI: 84.0 – 92.8) and 97.0% (95% CI: 93.4 – 98.8), respectively.

Kaplan-Meier survival analyses showed a PCR-uncorrected cumulative treatment success incidence of 1.000 for all three sites between Day 0 and Day 28 ( Figure 3A ). Cumulative success incidence in HMH remained 1.000 on Days 35 and 42 whilst incidence in EWP remained 1.000 on Day 35 and dropped to 0.960 (95% CI: 0.859-0.992) on Day 42. Cumulative PCR-uncorrected treatment success incidence in WMH dropped to 0.900 (95% CI: 0.806-0.953) on Day 35 and further dropped to 0.720 (95% CI: 0.605-0.813) on Day 42. PCR-corrected cumulative treatment success incidence in HMH was 1.000 on Day 42 whilst incidence in WMH was 0.974 (95% CI: 0.887-0.997) on Day 35 and 0.918 (95% CI: 0.813-0.969) on Day 42 ( Figure 3B ) (WHO, 2009).

The prevalence of measured fever (axillary temperature ≥ 37.5°C) significantly decreased from Day 0 to Day 1 in all three sites: 87.4% (95% CI: 78.1-93.3) to 11.5% (95% CI: 6.0-20.6) in HMH; 50.0% (95% CI: 36.5-63.5) to 7.1% (95% CI: 2.3-18.1) in EWP; and 98.8% (95% CI: 93.1-99.9) to 7.3% (95% CI: 3.0-15.8) in WMH. On Day 2, no child in HMH and WMH had axillary temperature ≥ 37.5°C ( Figure 4 ). The only child in EWP with axillary temperature ≥ 37.5°C on Day 2 was aparasitemic. On Day 3, no child in HMH and WMH had axillary temperature ≥ 37.5°C. The only child in EWP with axillary temperature ≥ 37.5°C on Day 3 was aparasitemic. On Day 7, no child in HMH and EWP had axillary temperature ≥ 37.5°C. The only child in WMH with axillary temperature ≥ 37.5°C on day 7 was aparasitemic.

Parasite prevalence on Day 2 ranged between 1.2% (95% CI: 0.1-7.5) in WMH and 2.3% (95% CI: 0.4-8.8) in HMH. No child was parasitemic on Day 3 and Day 7 ( Figure 5A ). Gametocytemia was prevalent in HMH on Day 2 only (1.1%; 95% CI: 0.6-2.1) and in EWP on Day 2 (3.6%; 95% CI: 0.6-13.6) and Day 3 (1.8%; 95% CI: 0.1-11.0). Gametocytemia was not prevalent in WMH during the follow-up period ( Figure 5B ).

Mean haemoglobin levels on days 28 and 42 were significantly higher than pre-treatment levels in all three sites ( Table 4 ). The mean changes between day 28 and day 42 were significant in HMH and EWP (p<0.001 and p=0.006, respectively) but not WMH (p=0.075).

Vomiting was the main adverse event reported. Prevalence of vomiting on day 0 was significantly highest in HMH compared with EWP and WMH (33.3%; 95% CI: 23.8-44.4 vs 1.8%; 95% CI: 0.1-10.8 vs 20.5%; 95% CI: 12.7-31.0; p<0.001) but significantly decreased to 0% on days 1 and 2 ( Figure 6 ). The changes observed in EWP (3.6%; 95% CI: 0.6-13.4 on day 1 and 0% on day 2) were not significant (p=0.421). Likewise, the changes observed in WMH (14.6%; 95% CI: 8.1-24.6 on day 1 and 12.2%; 95% CI: 6.3-21.7 on day 2) were not significant (p=0.322) ( Figure 6 ). Even though prevalence of vomiting in WMH was over 10% on days 1 and 2, there was no significant difference in treatment failure between those who vomited and those who did not (22.2% vs 33.3%; p=0.404 for PCR-uncorrected failures and 6.7% vs 12.5%; p=0.732 for PCR-corrected failures).