Local emergence in Amazonia of Plasmodium falciparum k13 C580Y mutants associated with in vitro artemisinin resistance

Abstract

Antimalarial drug resistance has historically arisen through convergent de novo mutations in Plasmodium falciparum parasite populations in Southeast Asia and South America. For the past decade in Southeast Asia, artemisinins, the core component of first-line antimalarial therapies, have experienced delayed parasite clearance associated with several pfk13 mutations, primarily C580Y. We report that mutant pfk13 has emerged independently in Guyana, with genome analysis indicating an evolutionary origin distinct from Southeast Asia. Pfk13 C580Y parasites were observed in 1.6% (14/854) of samples collected in Guyana in 2016-2017. Introducing pfk13 C580Y or R539T mutations by gene editing into local parasites conferred high levels of in vitro artemisinin resistance. In vitro growth competition assays revealed a fitness cost associated with these pfk13 variants, potentially explaining why these resistance alleles have not increased in frequency more quickly in South America. These data place local malaria control efforts at risk in the Guiana Shield.

Keywords: Guyana; P. falciparum; South America; artemisinin resistance; epidemiology; evolution; global health; infectious disease; kelch 13; malaria; microbiology.

Figure 1.. Distribution of the pfk13 C580Y mutant parasites among Guyana regions.

Pie charts represent the total number of isolates analyzed per region. Mutants are represented in red.

Figure 2.. Whole-genome sequence analysis of pfk13 C580Y mutant parasites in Guyana.

(a) Comparison of the haplotypic background of pfk13 C580Y mutant parasites from Guyana, 2016, and Southeast Asia, 2010–2012. Across Pf3k samples from Cambodia, Thailand, and Vietnam, 45 unique C580Y-coding haplotypic backgrounds were identified and compared to haplotypes from Guyana. Columns represent 149 sites containing non-singleton single nucleotide polymorphisms (SNPs) found within a 150 kb segment surrounding the pfk13 C580Y-coding allele. At a given site, the more common allele is marked blue, the less common allele is orange, and missing calls are grey. The Y-coding variant for codon 580 of pfk13 is represented by the red blocks; wild-type is blue. Only the five pfk13 C580Y mutant samples with fewer than 15% missing calls are depicted here. (b) Analysis of relatedness at the whole-genome level among Guyana clones. Pairwise identity-by-descent (IBD) was estimated for all pairs of Guyana samples with high quality whole-genome sequence data (<70% missing calls). Pairwise comparisons between samples exhibiting the pfk13 C580Y allele are indicated in red, and show uniformly high levels of relatedness, suggesting a single clonal lineage harboring the resistance mutation. (c, d) Principal components analysis of parasites from Guyana or other geographic regions using SNP calls from whole-genome sequence data. (c) The parasites from Guyana and French Guiana form a single cluster when compared with parasites from Africa. (d) The two edited parasite lines from French Guiana are highly similar to the sequenced parasite samples from Guyana including a pfk13 mutant.

Figure 2—figure supplement 1.. Number of single nucleotide differences between pairs of parasites within different geographic locations.

The median number of nucleotide differences between the two edited French Guianan lines and Guyanese parasites (FrGu-Guy; 0.23 per thousand nucleotides) is lower than the median nucleotide differences between parasite pairs drawn from within any of the other analyzed populations. To account for potential differences in sequencing depth and quality across populations, calculations were made using a set of high quality SNP calls (GATK quality score >20;<80% missing calls for the given population). FrGu: French Guiana, Guy: Guyana, DRC: Democratic Republic of Congo.

Figure 3.. Temporal distribution of pfk13 C580Y mutants in Region 1 of Guyana per month of collection, 2016–2017.

The percentage of pfk13 C580Y mutants for each month of identification is represented above each bar.

Figure 4.. Ring-stage Survival Assays in parasites from French Guiana.

Data show survival rates of ring-stage parasites (0–3 hr post invasion of human erythrocytes) after a 6 hr pulse of 700 nM DHA, as measured by microscopy 66 hr later. Data illustrate mean ± SEM percent survival from three independent repeats compared with dimethyl sulfoxide (DMSO)-treated parasites as a control for two isolates from French Guiana (O141-A, R086). Parents harbored wild-type pfk13 allele, and for zinc-finger nuclease edited isogenic parasites, control (ctrl) isolates harbored wild-type pfk13 allele with silent mutations or pfk13 mutations (C580Y or R539T). IPC4912, a Cambodian reference strain harboring the I543T pfk13 mutation was used as a control. A parasite line is considered resistant when the survival rate is greater than 1%. Student’s t-test was used to assess significant differences between survival rates of parental and pfk13-edited parasites. *p<0.05; **p<0.01; ns: not significant.

Figure 5.. Competition growth assays of pfk13 mutant and wild-type parasites.

(a) Frequency of wild-type and mutant parasites in co-culture, as measured by TaqMan allelic discrimination qPCR. Data show the percentage of pfk13 mutant parasites in the culture over 60 days with sampling every two days. Error bars represent the SEM of pfk13 mutant allele frequency between the two biological replicates (including two technical replicates for qPCR). A percentage below 50% indicates the mutant was less fit than the isogenic pfk13 wild-type line. (b) Percentage change per generation of pfk13 mutant allele frequency relative to wild-type. Data show that pfk13 mutations confer an in vitro fitness cost in both parasite lines. Differences in growth rates were calculated as the percent change in pfk13 mutant allele frequency averaged over 30 generations. Error bars represent the SEM of percentage growth change between the two biological sampling experiments calculated for every generation in each co-culture. Significance was calculated using the Wilcoxon signed-rank test in every generation across the two biological replicate experiments. **p<0.01, ***p<0.001; ns: not significant.

Figure 5—figure supplement 1.. Representative standard curves for qPCR reactions targeting pfk13 C580/C580Y or pfk13 R539/R539T allele.

(a, b) Standard curves showing good amplification efficiency (between 88% and 95%) and high sensitivity using 10-fold serially diluted genomic DNA obtained from wild-type pfk13 C580 and R539 or mutant pfk13 C580Y or R539T parasites. (c, d) Scatter plots for percent wild-type and mutant alleles in multiplexed qPCR assays using pre-defined mixtures of plasmids. We used mixtures comprising of pfk13 C580 and C580Y or R539 and R539T expressing plasmids in fixed molar ratios of wild-type: mutant alleles (0:100, 20:80, 40:60, 50:50, 60:40, 80:20, 100:0) to validate the specificity of using TaqMan qPCR assays to determine the pfk13 allele frequency.

Figure 5—figure supplement 2.. Reproducibility of TaqMan allelic discrimination qPCR performed on R086^R539T and R086 parasites.

(a) Scatter plots show the percentage of pfk13 R539T parasites in two separate qPCR technical replicate runs which correlate perfectly. (b) Scatter plots show the percentage of pfk13 R539T parasites in two independent sampling replicates over 60 days in culture, which showed consistent trends.