A Countrywide Survey of hrp2/3 Deletions and kelch13 Mutations Co-occurrence in Ethiopia

Abstract

Malaria elimination relies on detection of Plasmodium falciparum histidine-rich proteins 2/3 (HRP2/3) through rapid diagnostic tests (RDTs) and treatment with artemisinin combination therapies (ACTs). Data from the Horn of Africa suggest increasing hrp2/3 gene deletions and ACT partial resistance kelch13 (k13) mutations. To assess this, 233 samples collected during a national survey from 7 regions of Ethiopia were studied for hrp2/3 deletions with droplet digital polymerase chain reaction (ddPCR) and k13 mutations with DNA sequencing. Approximately 22% of the study population harbored complete hrp2/3 deletions by ddPCR. Thirty-two of 44 of k13 single-nucleotide polymorphisms identified were R622I associated with ACT partial resistance. Both hrp2/3 deletions and k13 mutations associated with ACT partial resistance appear to be co-occurring, especially in Northwest Ethiopia. Ongoing national surveillance relying on accurate laboratory methods are required to elaborate the genetic diversity of P. falciparum.

Keywords: artemisinin; histidine-rich protein 2; kelch13; malaria; rapid diagnostic tests.

Figure 1.

Study workflow and sample selection. A total of 757 dried blood spot (DBS) samples were received from the malaria surveys form 2017 and 2021 across Afar, Gambella, Oromia, the Southern Nations, Nationalities, and Peoples’ Region (SNNPR), and Somali regions. A total of 233 samples were analyzed with the 3 molecular tools implemented in this study: hrp2/3 end-point polymerase chain reaction (PCR), droplet digital PCR (ddPCR) quantification of hrp2 and hrp3, and k13 sequencing. Abbreviations: P. falciparum, Plasmodium falciparum; RDT, rapid diagnostic test.

Figure 2.

Prevalence of hrp2/3 deletions in relation to polymerase chain reaction (PCR) and droplet digital PCR (ddPCR) and k13 single-nucleotide polymorphism (SNP) sequencing results. A, Differences in hrp2/3 status between PCR and ddPCR. PCR estimated that 30.47% (71 of 233) samples contained hrp2 deletions, compared with 23.61% (55 of 233) for ddPCR. While PCR can only distinguish hrp2 status only by full deletion (hrp2⁻) or no deletion (hrp2⁺), ddPCR can distinguish hrp2 by no deletion, full deletion, mixed (16.74%), or partial (4.29%). B, Among the 233 samples analyzed, a total of 13 unique k13 SNPs were identified across 44 samples (all nonsynonymous). The most prevalent k13 SNP detected was R622I 70.45% [31 of 44], Only 1 other SNP, W518C, was detected in ≥1 sample 4.54% [2 of 44].

Figure 3.

Regional distribution of hrp2/3 and k13 genotypes. Plots demonstrate distribution of samples by region in Ethiopia and their associated k13 and hrp2/3 status. A, Presence of k13 single-nucleotide polymorphisms in the propellor domain, with red highlighting the presence of the R622I mutation. Abbreviations: SNNPR, Southern Nations, Nationalities, and Peoples’ Region; WT, wild type. B, hrp2/3 status of each region obtained with digital droplet polymerase chain reaction, with red highlighting samples that contain both hrp2 and hrp3 deletions (hrp2⁻ and hrp3⁻), representing complete loss of hrp2/3.

Figure 4.

Principal component analysis (PCA) of pfhrp2/3 and pfk13 status. The constructed dissimilarity matrix and the principal components (PCs) in the graph display the 233 samples analyzed for pfk13 single-nucleotide polymorphisms (SNP) (wild type in blue, R622I in green, and other SNPs in red). The hrp2 status assessed by droplet digital polymerase chain reaction is represented by shapes; green circles represent samples that were negative for pfhrp2 and encoded an R622I SNP in the pfk13 propellor domain. PCA explains that the sequence dissimilarity of the pfk13 propellor domain does not differ significantly by pfhrp2 status or R622I.