Absence of putative artemisinin resistance mutations among Plasmodium falciparum in Sub-Saharan Africa: a molecular epidemiologic study

Abstract

Plasmodium falciparum parasites that are resistant to artemisinins have been detected in Southeast Asia. Resistance is associated with several polymorphisms in the parasite's K13-propeller gene. The molecular epidemiology of these artemisinin resistance genotypes in African parasite populations is unknown. We developed an assay to quantify rare polymorphisms in parasite populations that uses a pooled deep-sequencing approach to score allele frequencies, validated it by evaluating mixtures of laboratory parasite strains, and then used it to screen P. falciparum parasites from >1100 African infections collected since 2002 from 14 sites across sub-Saharan Africa. We found no mutations in African parasite populations that are associated with artemisinin resistance in Southeast Asian parasites. However, we observed 15 coding mutations, including 12 novel mutations, and limited allele sharing between parasite populations, consistent with a large reservoir of naturally occurring K13-propeller variation. Although polymorphisms associated with artemisinin resistance in P. falciparum in Southeast Asia are not prevalent in sub-Saharan Africa, numerous K13-propeller coding polymorphisms circulate in Africa. Although their distributions do not support a widespread selective sweep for an artemisinin-resistant phenotype, the impact of these mutations on artemisinin susceptibility is unknown and will require further characterization. Rapid, scalable molecular surveillance offers a useful adjunct in tracking and containing artemisinin resistance.

Keywords: artemisinin resistance; drug resistance; falciparum malaria; molecular epidemiology.

Figure 1.

Sequencing results of a mixture of parasite strains 3D7 and Cam91. A, Schematic approach and output of quality-filtering program applied to sequencing reads. B, Kernel density plot of the number of false-positive nucleotides that would be detected across the length of our amplicon, using 3 different minimum Phred base quality cutoffs (q33, q34, and q35) at various minimum percentage minor allele frequency cutoffs. C, Venn diagram of the number of bases censored by the 5 main quality metrics (using the q34 filter) applied to the sequencing reads in panel A.

Figure 2.

Expected and observed frequencies of the C580Y substitution in mixtures of parasite strains 3D7 (C580) and Cam91 (580Y). Gray diamonds are proportions of alleles in 2 independent replicates for each mixture; the solid line is a linear regression line fitted to these points, with the shaded area indicating the error estimate for the line (R² = 0.98). The horizontal lines indicate the minimum percentage minor allele cutoffs that were ultimately used for known (dotted) and novel (dashed) loci.

Figure 3.

Epidemiology of Plasmodium falciparum K13-propeller mutations in 14 sub-Saharan African sites. Proportions within each geographic site of wild-type alleles, coding mutations, and silent mutations. Proportions calculated with the assumption that each K13-propeller haplotype contained only a single mutation, as observed here and in Cambodia [6]. Abbreviation: DRC, Democratic Republic of the Congo.