Association between Plasmodium falciparum Kelch13 mutations and malaria parasite clearance half-life after artemisinin-based therapy: an updated WWARN systematic review and individual patient data meta-analysis
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Background
Artemisinin-based combination therapies remain the first-line treatment for uncomplicated Plasmodium falciparum malaria. Plasmodium falciparum Kelch13 mutations emerge and spread within distinct malaria epidemiological and immunological contexts, shaping the expression of artemisinin resistance (ART-R). Understanding the clinical phenotypes of these mutations requires evaluation across diverse transmission settings and geographic regions.
Methods
A systematic review (SR) and individual patient data meta-analysis (IPDMA) were conducted (PROSPERO: CRD42019133366) to identify studies that included serial parasite density measurements and Kelch13 genotyping. Associations between Kelch13 mutations and parasite clearance half-lives (PC1/2) and Day 3 parasite positivity were assessed. Receiver operating characteristic (ROC) analyses identified PC1/2 thresholds most strongly associated with relevant Kelch13 mutations.
Findings
The SR identified 86 eligible studies, and individual patient data were obtained from 45 studies (n=16,823 patients from 539 study sites in 33 countries). After excluding hyperparasitaemia, Day 3 parasite positivity exceeded 10% overall across all WHO-validated, candidate, and potential mutations, but not for other Kelch13 mutations. Parasite clearance rates were consistently shorter in moderate-to-high-transmission than in lower-transmission areas. The WHO threshold PC1/2>5h had a sensitivity of 36% (27.9-44.6) for detecting WHO-validated mutations in moderate-to-high transmission areas and 81% (78.5-82.8) in lower transmission areas. In moderate-to-high transmission settings, a PC1/2 threshold of 3.1h optimally discriminated WHO-validated mutations (ROC AUC 0.78; 95% CI 0.74–0.82; sensitivity 75% (95% CI:67%-82%); specificity 71%, 95% CI: 69%-73%). Additional emerging Kelch13 mutations associated with delayed parasite clearance were identified in relatively small African and Asian sample sets.
Interpretation
Compared with WT parasites, parasites carrying WHO-validated ART-R mutations were associated with a 34%-59% longer mean PC1/2, regardless of endemicity, treatment regimen, or age. Given the low sensitivity of the PC1/2>5h threshold for identifying WHO-validated ART-R parasites in moderate-to-high transmission settings, where its recalibration is indicated. Broader genotyping is warranted for identifying emerging Kelch13 mutations associated with delayed parasite clearance.
Research in Context: Panel
Evidence before this study
Artemisinin-based combination therapies (ACTs) remain the first-line treatment for Plasmodium falciparum malaria. The emergence and spread of artemisinin partial resistance (ART-R), characterised by delayed parasite clearance after treatment and mediated primarily by non-synonymous mutations in the Kelch13 propeller domain, pose a major threat to global malaria control. Previous individual-patient data meta-analyses (IPDMAs) have established an association between Kelch13 mutations and delayed clearance, but these analyses were largely restricted to low-transmission settings in Southeast Asia (SEA). Comprehensive evaluation of these relationships in Africa, which bears 95% of the global falciparum malaria burden and where transmission intensity, host immunity, and infection complexity differ, is needed.
Added value of this study
This study represents the largest and most geographically comprehensive IPDMA of Kelch13 -associated ART-R to date, integrating published and unpublished data from 16,823 patients at 539 sites across 33 countries, including 22 in Africa. Extending on the previous analysis and incorporating the newly updated WHO compendium of relevant Kelch13 mutations, this study evaluated parasite clearance phenotypes across diverse transmission intensities, including underrepresented African populations. WHO-listed mutations of ART-R were strongly associated with Day 3 positivity and prolonged parasite clearance half-life (PC1/2) after treatment, while additional Kelch13 mutants were also associated with delayed clearance, thereby expanding the contemporary molecular landscape of ART-R. The WHO-recommended PC1/2 threshold of >5h showed low sensitivity for detecting ART-R in moderate-to-high transmission settings. Our findings support transmission-specific interpretation of PC1/2 and indicate that a threshold of 3.1 h more accurately identifies WHO-validated ART-R mutations in moderate-to-high transmission settings, which bear the greatest global malaria burden.
Implications of all the available evidence
ART-R is now established across much of SEA and is emerging independently in multiple African parasite populations. Building on the previous WorldWide Antimalarial Resistance Network (WWARN) IPDMA and the expanding WHO evidence base on drug-resistance molecular markers, this study strengthens the evidence linking Kelch13 mutations to delayed parasite clearance following treatment in Africa, while demonstrating that the phenotypic expression of ART-R varies with transmission intensity and epidemiological context. These findings support context-specific interpretation of parasite clearance metrics, integrated with molecular surveillance and therapeutic efficacy studies, to improve early detection of emerging ART-R and guide timely public health responses.
