Association of Plasmodium falciparum kelch13 R561H genotypes with delayed parasite clearance in Rwanda: an open-label, single-arm, multicentre, therapeutic efficacy study

Abstract

Background: Partial artemisinin resistance is suspected if delayed parasite clearance (ie, persistence of parasitaemia on day 3 after treatment initiation) is observed. Validated markers of artemisinin partial resistance in southeast Asia, Plasmodium falciparum kelch13 (Pfkelch13) R561H and P574L, have been reported in Rwanda but no association with parasite clearance has been observed. We aimed to establish the efficacy of artemether-lumefantrine and genetic characterisation of Pfkelch13 alleles and their association with treatment outcomes.

Methods: This open-label, single-arm, multicentre, therapeutic efficacy study was done in 2018 in three Rwandan sites: Masaka, Rukara, and Bugarama. Children aged 6-59 months with P falciparum monoinfection and fever were eligible and treated with a 3-day course of artemether-lumefantrine. Treatment response was monitored for 28 days using weekly microscopy screenings of blood samples for P falciparum. Mutations in Pfkelch13 and P falciparum multidrug resistance-1 (Pfmdr1) genes were characterised in parasites collected from enrolled participants. Analysis of flanking microsatellites surrounding Pfkelch13 was done to define the origins of the R561H mutations. The primary endpoint was PCR-corrected parasitological cure on day 28, as per WHO protocol.

Findings: 228 participants were enrolled and 224 (98·2%) reached the study endpoint. PCR-corrected efficacies were 97·0% (95% CI 88-100) in Masaka, 93·8% (85-98) in Rukara, and 97·2% (91-100) in Bugarama. Pfkelch13 R561H mutations were present in 28 (13%) of 218 pre-treatment samples and P574L mutations were present in two (1%) pre-treatment samples. 217 (90%) of the 240 Pfmdr1 haplotypes observed in the pretreatment samples, had either the NFD (N86Y, Y184F, D1246Y) or NYD haplotype. Eight (16%) of 51 participants in Masaka and 12 (15%) of 82 participants in Rukara were microscopically positive 3 days after treatment initiation, which was associated with pre-treatment presence of Pfkelch13 R561H in Masaka (p=0·0005). Genetic analysis of Pfkelch13 R561H mutations suggest their common ancestry and local origin in Rwanda.

Interpretation: We confirm evidence of emerging artemisinin partial resistance in Rwanda. Although artemether-lumefantrine remains efficacious, vigilance for decreasing efficacy, further characterisation of artemisinin partial resistance, and evaluation of additional antimalarials in Rwanda should be considered.

Funding: The US President's Malaria Initiative.

Translation: For the French translation of the abstract see Supplementary Materials section.

Figure 1:. Study sites and prevalence of Pfkelch13 mutations in pre-treatment samples by study site

This study was done in three Rwandan health centres: Rukara (Kayonza Distric), Masaka (Kicukiro District) and Bugarama (Rusizi District). The prevalence of the Pfkelch13 R561H mutation was found in ten (20%) of 51 samples in Masaka, and eight (22%) of 82 in Rukara; that of the P574L marker was two (1%) of 82 samples in Rukara. Prevalence of the candidate artemisinin partial resistance markers was one (2%) of 51 samples in Masaka, three (4%) of 82 samples in Rukara, and one (1%) of 85 samples in Bugarama.

Figure 2:. Reduced heterozygosity on the sampled loci around the pfk13 gene in R561H isolates

A total of 33 R561H mutations(nine from Masaka and 24 from Rukara) and 49 wildtype isolates (24 from Masaka and 25 Rukara) are shown (samples with mixed infections or missing data at any loci were excluded). We observed distinct haplotypes with the R561H mutation(compared with the wildtype (blue and green lines), with a reduction in the heterozygosity in those with the R561H mutation. The heterozygosity of the wildtype genotypes was significantly different and higher than the one observed in the sympatric R561H mutant haplotypes in the two populations.