Efficacy of Artemether-Lumefantrine and Dihydroartemisinin-Piperaquine for the Treatment of Uncomplicated Plasmodium falciparum Malaria among Children in Western Kenya, 2016 to 2017

ABSTRACT

Antimalarial resistance threatens global malaria control efforts. The World Health Organization (WHO) recommends routine antimalarial efficacy monitoring through a standardized therapeutic efficacy study (TES) protocol. From June 2016 to March 2017, children with uncomplicated P. falciparum mono-infection in Siaya County, Kenya were enrolled into a standardized TES and randomized (1:1 ratio) to a 3-day course of artemether-lumefantrine (AL) or dihydroartemisinin-piperaquine (DP). Efficacy outcomes were measured at 28 and 42 days. A total of 340 children were enrolled. All but one child cleared parasites by day 3. PCR-corrected adequate clinical and parasitological response (ACPR) was 88.5% (95% CI: 80.9 to 93.3%) at day 28 for AL and 93.0% (95% CI: 86.9 to 96.4%) at day 42 for DP. There were 9.6 times (95% CI: 3.4 to 27.2) more reinfections in the AL arm compared to the DP arm at day 28, and 3.1 times (95% CI: 1.9 to 4.9) more reinfections at day 42. Both AL and DP were efficacious (per WHO 90% cutoff in the confidence interval) and well tolerated for the treatment of uncomplicated malaria in western Kenya, but AL efficacy appears to be waning. Further efficacy monitoring for AL, including pharmacokinetic studies, is recommended.

INTRODUCTION

In the past 2 decades, the malaria burden has declined considerably in sub-Saharan Africa, largely due to investments in the scale-up of long-lasting insecticidal nets, indoor residual spraying, and case management using rapid diagnostic tests (RDTs) and artemisinin-based combination therapy (ACT) (1). Despite this, malaria remains a major source of morbidity and mortality in the region (2, 3), and new global targets have been established to reduce malaria incidence and mortality by 90% by 2030 (4). Antimalarial resistance is a major threat to these global efforts (5–7).

Resistance was first documented to chloroquine in the 1950s (8), and subsequently extended to many other drug classes, most recently to ACTs in Southeast Asia in 2004 (9–11). There, resistance against the components of ACTs was characterized by resistance to the artemisinin component, which resulted in delayed parasite clearance and subsequent resistance to the partner drug (10, 12). Molecular markers for monitoring resistance against ACT components have been described in the Greater Mekong subregion (9, 13). To characterize and mitigate the risk associated with drug resistance, the World Health Organization (WHO) recommends antimalarial resistance monitoring, inclusive of surveillance for molecular markers of resistance, in endemic countries using a standardized therapeutic efficacy study (TES) protocol (14).

The TES is designed to monitor antimalarial efficacy to inform the Ministry of Health (MoH) on whether a change in the recommended therapy should be considered. In settings where ACTs are recommended, data from TES can be used to evaluate resistance to the artemisinin derivative and the partner drug. The U.S. President’s Malaria Initiative (PMI) and the WorldWide Antimalarial Resistance Network have developed repositories of standardized TES data to provide a global perspective on ACT efficacy and antimalarial resistance to prevent and slow the spread of resistant parasites (15).

The Kenya MoH adopted artemether-lumefantrine (AL) as the first-line treatment for uncomplicated malaria in 2004 (16). Dihydroartemisinin-piperaquine (DP) was adopted as the second-line antimalarial in 2010. AL and DP have been evaluated in efficacy studies in Kenya since 2005 (17) and 2007 (18), respectively. AL and DP were found to be highly efficacious in the most recently published TESs (2010 to 2011) evaluating AL and DP in western Kenya (19, 20). We present the results of a TES conducted in western Kenya to measure the efficacy of AL and DP, 12 and 6 years after their introduction, respectively.

(The results of the study have been previously presented as a poster presentation at the American Society of Tropical Medicine and Hygiene 67th meeting, October 2018, New Orleans, LA, USA; abstract #2105.)

RESULTS

Enrolment and baseline characteristics.

A total of 1,716 children with febrile illness were screened for eligibility (Fig. 1). Among these, parents of 323 (19%) children declined study participation; 365 (21%) children were negative for malaria by microscopy; and 687 (40%) were not eligible for other reasons. A total of 341 children were enrolled; however, one child was subsequently excluded for hyperparasitemia after evaluation microscopy. Among the 340 children, 166 were randomized to AL and 174 to DP arms. There were no differences in losses to follow-up (LTFUs) between the two arms on day 28 (P = 0.31) or 42 (P = 0.59). Age, gender, parasite density, and hemoglobin (Hb) levels were similar between arms at baseline (Table 1).

FIG 1.

Trial enrollment by arm.

TABLE 1.

Baseline characteristics and treatment response outcomes, by study arm^a

Characteristic	AL arm (n = 166)% (95% CI) (n/N)	DP arm (n = 174)% (95% CI) (n/N)	P value
Baseline characteristics
Age (mo), mean (range)	31.3 (6–59)	32.9 (6–59)	0.28
Gender (% female)	48.8	47.1	0.76
Weight (kg), mean (SD)	12.7 (3.0)	12.6 (3.0)	0.97
Hemoglobin (g/dL), mean (SD)	9.8 (1.6)	10.0 (1.6)	0.24
Temp (°C), mean (SD)	38.0 (1.2)	37.8 (1.1)	0.08
Parasite densityb, mean (95%CI)	76,376 (67,829–84,923)	80,663 (71,781–89,544)	0.49
Treatment response rates
Early treatment failure	0.0% (0.0%–2.9%) 0/166	0.0% (0.0%–2.9%) 0/174
Day 3 parasite clearance	99.3% (97.0%–100%) 150/151	100.0% (97.0%–100%) 161/161
Per protocol analyses
Day 28 PCR-uncorrected ACPR	68.2% (59.9%–75.4%) 92/135	96.0% (91.4%–98.1%) 142/148
Day 28 PCR-corrected ACPR	88.5% (80.9%–93.3%) 92/104	98.6% (95.1%–99.6%) 142/144
Day 42 PCR-uncorrected ACPR	43.9% (35.6%–52.4%) 57/130	76.4% (68.8%–82.7%) 107/140
Day 42 PCR-corrected ACPR	78.1% (67.3%–86.0%) 57/73	93.0% (86.9%–96.4%) 107/115
Intention-to-treat analyses (n = 340)
Day 28 PCR-uncorrected ACPR	68.3% (60.5%–76.2%)	96.0% (92.9%–99.1%)
Day 28 PCR-corrected ACPR	90.6% (85.5%–95.7%)	98.7% (96.8%–100.0%)
Day 42 PCR-uncorrected ACPR	45.4% (36.9%–53.9%)	77.0% (70.1%–83.9%)
Day 42 PCR-corrected ACPR	86.5% (80.3%–92.7%)	93.8% (89.6%–98.0%)

^aAL, artemether-lumefantrine; DP, dihydroartemisinin-piperaquine; ACPR, adequate clinical and parasitological response.

^bMean geometric density expressed in parasites/μL.

Adequate clinical and parasitological response (ACPR) outcomes.

(i) Per protocol analyses. Early treatment failure (ETF) was not observed in either treatment arm (Table 1), and both treatments resulted in rapid parasite clearance. Only 4 (3%) and 6 (4%) children were parasitemic on day 2 in the AL and DP arms, respectively. One child in the AL arm remained parasitemic on day 3 but did not meet ETF criteria.

A total of 135 (81.3%) and 148 (85.1%) children completed 28-day follow-up in the AL and DP arms, respectively. The per-protocol (PP) PCR-uncorrected ACPR at day 28 was 68.2% (95% CI: 59.9 to 75.4%) and 96.0% (95% CI: 91.4 to 98.1%) in the AL and DP arms, respectively. There were 14/135 (10.4%) late clinical failures (LCFs) and 29/135 (21.5%) late parasitological failures (LPFs) in the AL arm, and 2/148 (1.4%) LCFs and 4/148 (2.7%) LPFs in the DP arm. Twelve (27.9%) of the AL and 2 (33.3%) of the DP failures were found to be recrudescent infections by PCR. The PP PCR-corrected ACPR at day 28 was 88.5% (95% CI: 80.9 to 93.3%) for AL and 98.6% (95% CI: 95.1 to 99.6%) for DP.

A total of 130 (78.3%) and 140 children (80.5%) completed follow-up through day 42 for AL and DP, respectively. The PP PCR-uncorrected ACPR in the AL arm was 43.9% (95% CI: 35.6 to 52.4%) and in the DP arm was 76.4% (95% CI: 68.8 to 82.7%). By day 42, there were cumulatively 20/130 (15.4%) LCFs and 53/130 (40.8%) LPFs in the AL arm, and 10/140 (7.1%) LCFs and 23/140 (16.4%) LPFs in the DP arm, respectively. Of these, 21.9% (16/73) and 24.2% (8/33) of the AL and DP failures, respectively, were recrudescent infections. The PP PCR-corrected ACPR at day 42 for the AL arm was 78.1% (95% CI: 67.3 to 86.0%) and for DP was 93.0% (95% CI: 86.9 to 96.4%). Fig. 2 shows the cumulative number of reinfection and recrudescence cases over time by arm.

FIG 2.

Changes in reinfection, recrudescence, and hemoglobin over time: cumulative reinfection and recrudescence in artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DP) arms, excluding late treatment failures with missing or inconclusive PCR results.

(ii) Intention-to-treat analyses (ITT). At day 28, the ITT PCR-uncorrected cure rate calculated by survival analysis was 68.3% (95% CI: 60.5 to 76.2%) for AL and 96.0% (92.9% CI: 93 to 99.1%) for DP (Fig. 3). After PCR correction, the ITT cure rate at day 28 was 90.6% (95% CI: 85.5 to 95.7%) for AL and 98.7% (95% CI: 96.8 to 100.0%) for DP. At day 42, the ITT PCR-uncorrected cure rate was 45.4% (95% CI: 36.9 to 53.9) for AL and 77.0% (95% CI: 70.1 to 83.9%) for DP. The ITT PCR-corrected cure rate at day 42 was 86.5% (95% CI: 80.3 to 92.7%) for AL and 93.8% (95% CI: 89.6 to 98.0%) for DP.

FIG 3.

Intention-to-treat analysis: Kaplan-Meier curves for PCR-uncorrected and PCR-corrected ACPR survival function for time until failure at day 28 and 42, by drug.

(iii) Sensitivity analyses. In sensitivity analyses recoding missing PCR as recrudescent, the PCR-corrected ACPR decreased for AL on day 28 to 86.8% (95% CI: 80.9 to 92.6%) in the ITT and 84.4% (95% CI: 76.2 to 90.2%) in the PP analyses. For DP, the 42-day PCR-corrected ACPR was 93.0% (95% CI: 88.6 to 97.4%) in ITT and 92.2% (95% CI: 85.9 to 95.9%) in PP analyses.

Secondary analyses.

Over 65% of children were afebrile by day 1 in both arms, and 99% resolved fevers by day 3. There was no difference in mean hemoglobin at baseline (Table 1) or during follow-up by study arm. While the differences in hemoglobin over time were not statistically significant, children experiencing treatment failure consistently had lower hemoglobin values (Fig. 4).

FIG 4.

Mean change in hemoglobin over time, by outcome. “Failure” included late clinical failure (LCF) and late parasitological failure (LPF); “no failure” included ACPR and loss to follow-up. P values at each time point: P = 0.31 for day 0; P = 0.81 for day 7; P = 0.31 for day 14; P = 0.09 for day 28; and P = 0.06 for day 42.

Baseline parasite density, stratified at 50,000/μL and 100,000/μL, was not associated with late treatment failure at day 28, nor in the subgroup analyses restricted to recrudescent cases (Table 2).

TABLE 2.

Association between baseline parasite density and late treatment failure during the 28-day follow-up period, overall and by drug (artemether-lumefantrine [AL] and dihydroartemisinin-piperaquine [DP])

Baseline parasite density	Failure n (%)	P value	AL (n = 166)	P value	DP (n = 174)	P value
Any failure
≤50,000 (n = 128)	15 (11.7%)	0.339	13 (20.0%)	0.205	2 (3.2%)	0.998
>50,000 (n = 212)	34 (16.0%)		30 (29.7%)		4 (3.6%)
≤100,000 (n = 228)	31 (13.6%)	0.106	29 (25.9%)	0.998	2 (1.7%)	0.096
>100,000 (n = 112)	18 (16.1%)		14 (25.9%)		4 (6.9%)
Recrudescence
≤50,000 (n = 128)	3 (2.3%)	0.106	2 (3.1%)	0.066	1 (1.6%)	0.998
>50,000 (n = 212)	11 (5.19%)		10 (9.9%)		1 (0.9%)
≤100,000 (n = 228)	10 (4.4%)	0.221	9 (8.0%)	0.753	1 (0.9%)	0.998
>100,000 (n = 112)	4 (3.6%)		3 (5.6%)		1 (1.7%)

The reinfection rate was 26% in the AL arm and 3% in the DP arm over the 28-day period, and 51% in the AL arm and 17% in the DP arm over the 42-day period. Accounting for person-time, there were 9.6 times (95% CI: 3.4 to 27.2, P < 0.0001) more reinfections in the AL arm compared to the DP arm at day 28, and 3.1 times (95% CI: 1.9 to 4.9, P < 0.0001) more reinfections at day 42. Thus, had participants received DP instead of AL, 89.6% (95% CI: 70.5 to 96.3%) and 67.2% (95% CI: 47.5 to 79.5%) of the reinfections could have been averted within 28 days and 42 days posttreatment, respectively. For the study sample, this translates to 28 of 31 reinfections at day 28 and 38 of 57 reinfections at day 42 that could have been averted in the AL arm, if children had received DP instead of AL. The comparison of reinfections at the time points of AL and DP primary efficacy outcomes (28 days for AL; 42 days for DP) shows that the number of reinfections was not statistically significantly different (reinfection rate ratio [RR] = 1.68, 95% CI 0.98 to 2.81, P = 0.06; 39.7% infections averted with DP) between the two drugs arms, indicating that the differences seen are largely due to pharmacokinetics.

Adverse events.

The proportion of children who vomited the first dose of medication was similar between the AL (1.4%) and DP (1.9%) arms. One child in the DP arm vomited the drug twice and was referred for rescue treatment. No children developed severe malaria during follow-up. One severe adverse event was recorded in a participant in the AL arm hospitalized with severe dehydration due to a diarrheal illness; the child recovered fully. This event was deemed unrelated to the study drug.

DISCUSSION

The study found that both AL and DP were well-tolerated and efficacious. However, the 28-day PCR-corrected ACPR for AL in both the PP (88.5%, 95% CI 80.9 to 93.3%) and ITT (90.6%, 95% CI 85.5 to 95.7%) analyses are at the cusp of the WHO-recommended threshold of 90% efficacy, considering confidence intervals (14). WHO cautions that if treatment failure exceeds 10%, or in the setting of a failure rate <10% but with a declining trend in efficacy over time, there is sufficient cause for programs to start considering a policy change (21). While the confidence intervals overlap, rates from our study are lower than the 28-day PCR-corrected ACPRs for AL from the four most recent published studies from the same geographic area: 98%, 97% (96% when recalculated per WHO guidelines), 99.6% (98.6% when recalculated), and 96% in 2011, 2011, 2009, and 2006, respectively (Table S2); similarly, the day-42 PCR-corrected ACPR for AL is lower than in previous studies (19, 20). These findings may represent an early indication of waning AL efficacy, as seen in other recent studies in East Africa (22).

In countries with documented artemisinin resistance, delayed parasite clearance has preceded ETF and resistance (10, 12). We did not observe ETFs in this study, and there were no overt signs of delayed parasite clearance—parasitemia resolved in 96% of participants by day 2, and in all but one by day 3. Additionally, a separately published analysis based on this study did not identify mutations associated with artemisinin resistance (23). Specifically, the molecular analysis found no Pfk13 mutations associated with parasite resistance to artemisinin in the study area but indicated a high proportion of wild-type parasites for the Plasmodium falciparum chloroquine resistance transporter gene (Pfcrt). Although the frequency of Pfmdr1 184F mutations was high in the study samples, their association with treatment failure was not statistically significant. These findings, as well as the high efficacy of DP (PCR-corrected ACPR of 98.6% and 93.0% at day 28 and 42, respectively), suggest that sensitivity to the artemisinin component remains, though several recent studies reported potential emergence of artemisinin-resistant malaria in other African countries (24, 25).

In Southeast Asia, artemisinin resistance preceded partner drug resistance, which manifested as increased late-treatment failures (26). If early waning efficacy is being observed in Kenya, our findings suggest this is more likely attributed to the partner drug. We observed high rates of LTF in the AL arm, with high reinfection rates and twice as many recrudescent cases in the AL arm compared to the DP arm. Additionally, we observed increasing levels of Pfmdr1 N86 wild-type alleles (23) compared to prior studies, which have been associated with decreased sensitivity to lumefantrine (27).

Similar to prior studies in Kenya (19, 20, 28) and other studies in East Africa (19, 29–31), DP was highly efficacious at both day 28 and 42 with lower risk of recurrent infections compared to AL. While DP is more expensive than AL, it has been shown to be both clinically and economically superior to AL in cost-effectiveness analyses (32). While we observed high level of reinfections in both arms, likely attributable to the study taking place in a high malaria transmission setting, we estimated that most reinfections at 28 and 42 days could have been averted by using DP instead of AL, largely driven by the differential half-life of the partner drug and the associated difference in the duration of protection. That said, the use of DP instead of AL reduces the time at risk of infection, and thus the frequency of malaria infection. Additionally, in cases resulting in severe anemia, additional chemoprophylaxis against malaria has been shown to reduce mortality in high transmission settings (33). These findings, combined with the simpler dosing regimen and a similar tolerability profile, make DP an attractive treatment option for areas of high malaria endemicity like western Kenya.

However, there are concerns that the widespread adoption of DP in sub-Saharan Africa may lead to the development of resistance as seen in areas of Southeast Asia. There, piperaquine resistance was associated with increased copy numbers of the Pfpm2 gene (26). More recent analyses suggest that subsequently acquired Pfcrt mutations confer higher-level DP resistance (34). Additionally, it has been suggested that other genes may be implicated for artemisinin and piperaquine resistance in Africa, and the presence or absence of established markers of resistance should be corroborated with clinical results (35, 36). For example, in Mali, only 7 of the 65 samples from individuals with clinical failure with DP harbored multiple copies of the Pfpm2 gene, suggesting that other genes may be involved in resistance there (37). Until we have a better understanding of the relevance of these markers, we must be careful not to overinterpret the results of molecular testing performed in the absence of clinical correlation. While it is concerning that 33.9% of samples collected from a Ugandan study between 2014 and 2015 harbored multiple Pfpm2 gene copies, the significance of these findings is unclear as the study was not able to evaluate their association with clinical resistance (38). In our study, geographically close to the Uganda site, none of the recrudescent samples harbored multiple copies of the Pfpm2 gene, and >95% of the parasites harbored the Pfcrt wild-type allele (23). Most importantly, DP was highly efficacious.

There are a few limitations to this study. First, the true efficacy of AL may be underestimated if not all doses were consumed. Though we made efforts to ensure treatment adherence, we were unable to directly observe the evening doses of AL or to adequately assess drug concentration levels. However, prior research has not found a difference in treatment efficacy between fully supervised and unsupervised dosing (39–41). Additionally, the study was not explicitly powered to compare the treatment arms, and the comparative analyses were done on a post hoc basis.

In conclusion, we found that the efficacy of AL is on the cusp of the WHO-recommended threshold of 90%, but we observed more recrudescences and reinfections in the AL arm compared to prior studies in the same area, which might be an early indication of waning AL efficacy in western Kenya. The greater number of LTFs that we observed suggests that waning efficacy is probably due to the partner drug, lumefantrine. While there are concerns that resistance to DP might arise in Africa, considering the greater efficacy compared to AL, simpler dosing schedule, and longer posttreatment prophylactic effect, there may be a benefit in considering DP as an alternative first-line antimalarial in Kenya in the future. At this time, further efficacy monitoring for AL, including pharmacokinetic studies, is recommended.

MATERIALS AND METHODS

Study site.

The study was conducted in Siaya County Referral Hospital and Bar Agulu and Mulaha dispensaries in Siaya County, Kenya between June 2016 and March 2017. Here, malaria transmission is year-round with seasonal peaks in May–July and November–December. Plasmodium falciparum mono-infection is responsible for >85% of Plasmodium infections in this region (42), and in 2015 the prevalence of P. falciparum infection in children <5 years by microscopy was 43.7% (43).

Patient screening and recruitment.

We adapted the 2009 WHO Therapeutic Efficacy Study protocol to our setting (14). Study staff approached parents or legal guardians (referred to collectively as “parents”) of children aged 6 to 59 months who had been triaged by health care staff for malaria testing. Staff collected 200 μL of blood by finger prick from eligible children (Table 3) whose parents provided consent to measure hemoglobin level (Hemocue AB, Ängelholm, Sweden) and prepare blood smears for microscopy. If the thick smear was positive, a thin smear was prepared to estimate parasite count for study eligibility. Children with P. falciparum mono-infection with a parasite density of 2,000 to 200,000 per μL and Hb ≥7.0 g/dL were enrolled.

TABLE 3.

Study inclusion and exclusion criteria

Inclusion criteria

Exclusion criteria

1.Age 6–59 mo2.Wt ≥ 5.0 kg3.Axillary temp ≥ 37.5°C or history of fever in the past 24 hours4.Hemoglobin ≥7 grams/deciliter at enrolment5.Slide-confirmed mono-infection with Plasmodium falciparum and asexual parasite density between 2,000 and 200,000 parasites/μL6. Live within the catchment boundaries of the study site (10 km radius)7. Able to swallow oral medication8.Able and willing to comply with the protocol for the duration of the study9.Able and willing to comply with the study visit schedule on days 2, 3, 7, 14, 21, 28, 35, and 42 10.Parent or caregiver has access to a phone and agrees to have study staff contact them for visit reminders during study period11.Written informed consent provided by parent/guardian

1. Presence of severe malaria or danger signs, including prostration, alteration in level of consciousness, respiratory distress, convulsions, or jaundice2. Severe malnutrition according to WHO child growth standards (wt for age <3 standard deviations)3. Known hypersensitivity to AL or DP4. Use of antimalarials or other drugs with antimalarial activity in the last 2 wks5. General clinical condition necessitates hospitalization6. Evidence of concomitant infections at the time of presentation7. Plan to travel or leave the area within the next 3 mo8. Previously enrolled in this study

Study design, randomization, treatment, and follow-up.

This was an open-label, parallel-arm, randomized, controlled trial with 42 days of follow-up for both arms. Children were block-randomized in a 1:1 ratio to the manufacturers’ recommended weight-based dosing regimens of AL (Coartem; Novartis, Basel, Switzerland) or DP (DuoCotexin; Holley-Cotec Pharmaceuticals, Beijing, China).

Upon enrollment (day 0), and at each subsequent scheduled visit (days 1, 2, 3, 7, 14, 21, 28, 35, and 42), a medical history was obtained from the participants and a physical examination was performed, including measurement of axillary temperature. Parents were asked to bring their children to the clinic for evaluation whenever they were ill. At each visit, except the scheduled day 1 visit, a finger prick blood sample was obtained to prepare malaria blood smears, measure Hb level, and prepare dried blood spots on filter paper (Table S1). Adverse events and severe adverse events were assessed during medical history and examination and addressed at each visit.

Two oral AL doses were administered to children in the AL arm per day with food or milk for 3 days (0, 8, 24, 36, 48, and 60 h). DP was administered orally once a day for 3 days (at 0, 24, and 48 h). All morning, doses of AL and DP were administered under direct observation by study staff at the clinic on days 0, 1, and 2. The evening doses of AL were provided to parents for home administration with food or milk. Study staff called parents in the evening to remind them to give the AL dose to the child and to bring the blister pack to the clinic the next day for adherence confirmation. Full and half doses were readministered to children who vomited within 30 and 31 to 60 min of administration, respectively. Those who vomited within 30 min of redosing were referred for rescue treatment and withdrawn from the study. Children with treatment failure were treated with quinine per MoH guidelines.

Outcomes and assessments.

Antimalarial efficacy was assessed by standardized, predefined clinical and parasitological outcomes (Table 4) (14). The primary outcome for each arm, adequate clinical and parasitological response, was classified at 28 days for AL and 42 days for DP. Follow-up was complete when a participant met one of the four classification criteria in Table 4, withdrew from the study, or was lost to follow up.

TABLE 4.

Classification of responses to treatment (14)

Classification	Definition
Early treatment failure (ETF)	• Danger signs or severe malaria on day 1, 2 or 3, in the presence of parasitemia;• Parasitemia on day 2 higher than on day 0, irrespective of axillary temp;• Parasitemia on day 3 with axillary temp ≥ 37.5°C; and• Parasitemia on day 3 ≥ 25% of count on day 0.
Late clinical failure (LCF)	• Danger signs or severe malaria in the presence of parasitemia on any day between day 4 and day 28 for AL (day 42 for DP) in participants who did not previously meet any of the criteria of early treatment failure; and• Presence of parasitemia on any day between day 4 and day 28 for AL (day 42 for DP) with axillary temp ≥37.5°C in patients who did not previously meet any of the criteria of early treatment failure.
Late parasitological failure (LPF)	• Presence of parasitemia on any day between day 7 and day 28 for AL (day 42 for DP) with axillary temp <37.5°C in patients who did not previously meet any of the criteria of early treatment failure or late clinical failure.
Adequate clinical and parasitological response (ACPR)	• Absence of parasitemia on day 28 for AL (day 42 for DP), irrespective of axillary temp, in patients who did not previously meet any of the criteria of early treatment failure, late clinical failure, or late parasitological failure.

Laboratory procedures.

Thick and thin blood smears stained with 10% Giemsa were used for participant screening; blood smears were stained with 3% Giemsa for evaluation of parasitemia (identification to the species level, and density). Study slides were independently read by two microscopists certified as expert readers, blinded to the treatment arm (44). Microscopists confirmed the presence of asexual parasites and gametocytes after examining 100 microscopic fields (44 to ,46). When positive, parasites were counted against 500 white blood cells (WBCs) and expressed per μL of blood using an assumed 8,000 WBCs. Slides with discordant qualitative results or parasite densities discordant by more than 50% were reexamined by a third microscopist; the arithmetic mean parasite density of the two most concordant reads was considered final.

Samples of AL and DP used in this study were sent to the Centers for Disease Control and Prevention, Atlanta, GA, USA, for quality testing using high performance liquid chromatography (Agilent Technologies, Waldbronn, Germany) (47). All drug samples tested for quality assurance contained adequate concentrations of active ingredients.

Molecular analysis.

Per WHO recommendation, to differentiate between recrudescence and reinfection, genotype analyses based on merozoite surface protein-1 (msp1), merozoite surface protein-2 (msp2), and glutamate-rich protein (glurp) were performed using PCR on dried blood spot samples by gel electrophoresis-based methods (48). A serial algorithm starting with msp2 followed by glurp and finally msp1 was used. Only the samples provisionally classified as recrudescence were further analyzed for the next marker; reinfections were not analyzed further. Infections were classified as recrudescent when at least one identical allele (≤20 bp) for each of the three markers (msp1, msp2, and glurp) was present in the pre- and posttreatment blood samples; all other infections were classified as reinfections. Nested mutation-specific PCR sequencing was performed to analyze mutations associated with drug resistance (i.e., mutations in the P. falciparum K-13 propeller [Pfk13], P. falciparum chloroquine resistance transporter [Pfcrt], P. falciparum multidrug resistant 1 [Pfmdr1], and P. falciparum plasmepsin-2 [Pfpm2] genes); these results are reported elsewhere (23). Genotyping results are included in Table S3.

Sample size.

The study was powered to have a 95% confidence interval (95% CI) with 4% precision around an assumed ACPR of 95%. To allow for a 20% loss to follow-up, we increased our sample size from 140 to 175 children in each arm. The study was not powered to detect a difference in efficacy between treatment arms.

Statistical analysis.

The coprimary outcomes were the per protocol and intention-to-treat PCR-corrected ACPRs on day 28 for AL and day 42 for DP. Secondary outcomes included PCR-uncorrected ACPR rates for both AL and DP groups, hematologic response, parasite clearance by day 3, rates of early treatment failure, late parasitological failure, late clinical failure, and the effect of baseline parasitemia on treatment failure. Post hoc secondary analyses included differences in reinfection rates by study drug and the potential number of reinfections averted by use of DP.

Children who withdrew or were LTFU were excluded from PP analyses. Those with reinfections and missing or failed PCR tests were excluded from PP PCR-corrected analyses, for the respective period, per WHO guidance (14). In the 8 instances where PCR results were missing or failed, we recoded their results as recrudescent and conducted a sensitivity analysis. The Kaplan-Meier method was used for ITT analyses to estimate the survival function and time-to-failure metrics. The log-rank test was used to estimate statistical significance; all treatment failures, withdrawals, and LTFUs were censored on the day of the event or the last day of follow-up. PCR-corrected and -uncorrected cumulative success rates were calculated using Kaplan-Meier estimator. Reinfection rate ratios (RR = reinfection incidence rate in AL arm/reinfection incidence rate in DP arm) were calculated by Poisson regression, with person-time at risk offset. The preventable fraction of reinfections in the AL arm was calculated ([RR–1]/RR) to estimate the proportion of reinfections averted had DP been preferentially administered. Geometric mean parasite densities were calculated to evaluate differences between arms at baseline. We used a χ² test for categorical variables and Student's t test or Wilcoxon rank-sum test for nonparametric continuous variables. Data were collected on tablets with surveys programmed with CommCare software (Dimagi, Cambridge, MA, USA). Statistical analyses were performed using SAS 9.4 (SAS Institute, Cary, NC, USA).

Ethical statement.

The Kenya Medical Research Institute (KEMRI, Nairobi, Kenya) Scientific and Ethics Review Unit approved this study. The U.S. Centers for Disease Control and Prevention (CDC, Atlanta, GA, USA) relied on KEMRI for approval. Written informed consent was obtained from parents of enrolled study participants, and a long-lasting insecticidal net was provided to all children enrolled in the study. Trial registration number: NCT05060198, 29 September 2021.