Decreased susceptibility of Plasmodium falciparum to both dihydroartemisinin and lumefantrine in northern Uganda

Abstract

Artemisinin partial resistance may facilitate selection of Plasmodium falciparum resistant to combination therapy partner drugs. We evaluated 99 P. falciparum isolates collected in 2021 from northern Uganda, where resistance-associated PfK13 C469Y and A675V mutations have emerged, and eastern Uganda, where these mutations are uncommon. With the ex vivo ring survival assay, isolates with the 469Y mutation (median survival 7.3% for mutant, 2.5% mixed, and 1.4% wild type) and/or mutations in Pfcoronin or falcipain-2a, had significantly greater survival; all isolates with survival >5% had mutations in at least one of these proteins. With ex vivo growth inhibition assays, susceptibility to lumefantrine (median IC₅₀ 14.6 vs. 6.9 nM, p < 0.0001) and dihydroartemisinin (2.3 vs. 1.5 nM, p = 0.003) was decreased in northern vs. eastern Uganda; 14/49 northern vs. 0/38 eastern isolates had lumefantrine IC₅₀ > 20 nM (p = 0.0002). Targeted sequencing of 819 isolates from 2015-21 identified multiple polymorphisms associated with altered drug susceptibility, notably PfK13 469Y with decreased susceptibility to lumefantrine (p = 6 × 10^-8) and PfCRT mutations with chloroquine resistance (p = 1 × 10^-20). Our results raise concern regarding activity of artemether-lumefantrine, the first-line antimalarial in Uganda.

Fig. 1. Map of Uganda.

Study sites are labeled. The combined prevalence of the PfK13 469Y and 675V mutations in districts with available surveillance data from 2019 is shown with the color scale. Source data are provided as a Source Data file.

Fig. 2. Ex vivo susceptibilities.

Susceptibilities to lumefantrine (A growth inhibition assay) and DHA (B RSA) in parasites from northern and eastern Uganda. Each point represents the result for a single isolate (A n = 49 for northern and 38 for eastern Uganda; B n = 52 for northern and 29 for eastern Uganda). P-values were determined using the two-sided Mann–Whitney Wilcoxon test. Center bounds of boxes correspond to the median, and minimal and maximal bounds correspond to 25th and 75th percentiles, respectively. Whiskers extend to extreme values no further than 1.5x the IQR from the 25th or 75th percentiles. Source data are provided as a Source Data file.

Fig. 3. Ex vivo ring survival in parasites with different PfK13 genotypes.

Each point represents the result for a single isolate (A n = 50 WT, 18 C469Y, 6 A675V; B n = 50 WT, 8 C469Y, 10 469Y, 4 A675V, 2 675V). Results are shown with mixed and pure mutant isolates combined (A) or shown separately (B). P-values were determined using the two-sided Mann–Whitney Wilcoxon test. Center bounds of boxes correspond to the median, and minimal and maximal bounds correspond to 25th and 75th percentiles, respectively. Whiskers extend to extreme values no further than 1.5x the IQR from the 25th or 75th percentiles. WT, wild type. Source data are provided as a Source Data file.

Fig. 4. Ex vivo susceptibilities in parasites with different genotypes.

Susceptibilities to DHA by RSA in parasites with different PfK13, Pfcoronin (CRN; A, B), and falcipain-2a (FP2a; C, D) genotypes are shown. Each point represents the result for a single isolate (A n = 38 WT, 18 Mut; B n = 26 C469 + CRN-WT, 9 C469 + CRN-Mut, 12 469Y + CRN-WT, 5 469Y + CRN-Mut; C n = 17 WT, 47 Mut; D n = 11 C469 + FP2a-WT, 30 C469 + FP2a-Mut, 3 469Y + FP2a -WT, 15 469Y + FP2a-Mut). PfK13 genotypes at codon 469 are indicated; mutant 469Y includes mixed isolates. P-values were determined using the two-sided Mann–Whitney Wilcoxon test. Center bounds of boxes correspond to the median, and minimal and maximal bounds correspond to the 25th and 75th percentiles, respectively. Whiskers extend to extreme values no further than 1.5x the IQR from the 25th or 75th percentiles. WT, wild type, Mut, any Pfcoronin or falcipain 2a polymorphism. Source data are provided as a Source Data file.

Fig. 5. Lumefantrine susceptibility of parasites with different PfK13 genotypes.

Each point represents the result for a single isolate (A n = 61 WT, 17 C469Y, 6 A675V; B n = 61 WT, 7 C469Y, 10 469Y, 5 A675V, 1 675V). Results are shown with mixed and pure mutant isolates combined (A) or shown separately (B). P-values were determined using the two sided Mann–Whitney Wilcoxon test. Center bounds of boxes correspond to the median, and minimal and maximal bounds correspond to the 25th and 75th percentiles, respectively. Whiskers extend to extreme values no further than 1.5x the IQR from the 25th or 75th percentiles. WT, wild type. Source data are provided as a Source Data file.