Examining the Early Distribution of the Artemisinin-Resistant Plasmodium falciparum kelch13 R561H Mutation in Areas of Higher Transmission in Rwanda

Abstract

Background: Artemisinin resistance mutations in Plasmodium falciparum kelch13 (Pfk13) have begun to emerge in Africa, with Pfk13-R561H being the first reported in Rwanda in 2014, but limited sampling left questions about its early distribution and origin.

Methods: We genotyped P. falciparum positive dried blood spot (DBS) samples from a nationally representative 2014-2015 Rwanda Demographic Health Surveys (DHS) HIV study. DBS were subsampled from DHS sampling clusters with >15% P. falciparum prevalence, as determined by rapid testing or microscopy done during the DHS study (n clusters = 67, n samples = 1873).

Results: We detected 476 parasitemias among 1873 residual blood spots from a 2014-2015 Rwanda Demographic Health Survey. We sequenced 351 samples: 341/351 were wild-type (97.03% weighted), and 4 samples (1.34% weighted) harbored R561H that were significantly spatially clustered. Other nonsynonymous mutations found were V555A (3), C532W (1), and G533A (1).

Conclusions: Our study better defines the early distribution of R561H in Rwanda. Previous studies only observed the mutation in Masaka as of 2014, but our study indicates its presence in higher-transmission regions in the southeast of the country at that time.

Keywords: antimalarial resistance; artemisinin; drug resistance; k13; kelch13; r561H.

Figure 1.

Previous studies and our study design. A, Sampling sites of P. falciparum genotyping studies done in Rwanda, 2010–2015. Sites included Masaka, Ruhuha, Bugarama, Kibirizi, Nyarurema and Rukara [1], and the Huye district, 2010–2015 [10]. B, DHS 2014 sampling clusters. We sequenced samples from clusters with >15% malaria prevalence. C, Study design. We used DBS samples from the HIV arm of a 2014–2015 DHS study, selecting 2255 samples taken from 67 clusters with >15% malaria, as calculated in the malaria/anemia arm. Abbreviations: DBS, dried blood spot; DHS, Rwanda Demographic Health Surveys; PCR, polymerase chain reaction; RDT, rapid diagnostic test.

Figure 2.

Geographic distribution of Pfk13 mutations in high–malaria prevalence demographic health survey clusters in Rwanda. A, Mutations confirmed by sequencing amplicons corresponding to Pfk13 amino acids 516–572 are presented as pie charts, the center of each corresponding to the center of the DHS survey district. Mutation presence at the DHS cluster level shown for the Kirehe and Ngoma districts. B, The 97.5% confidence interval of inferred R561H prevalence using a spatial binomial logistic model. Abbreviation: DHS, Rwanda Demographic Health Surveys.