Expansion of artemisinin partial resistance mutations and lack of histidine rich protein-2 and -3 deletions in Plasmodium falciparum infections from Rukara, Rwanda

Abstract

Background: Emerging artemisinin partial resistance and diagnostic resistance are a threat to malaria control in Africa. Plasmodium falciparum kelch13 (k13) propeller-domain mutations that confer artemisinin partial resistance have emerged in Africa. k13-561H was initially described at a frequency of 7.4% from Masaka in 2014-2015, but not present in nearby Rukara. By 2018, 19.6% of isolates in Masaka and 22% of isolates in Rukara contained the mutation. Longitudinal monitoring is essential to inform control efforts. In Rukara, an assessment was conducted to evaluate recent k13-561H prevalence changes, as well as other key mutations. Prevalence of hrp2/3 deletions was also assessed.

Methods: Samples collected in Rukara in 2021 were genotyped for key artemisinin and partner drug resistance mutations using molecular inversion probe assays and for hrp2/3 deletions using qPCR.

Results: Clinically validated k13 artemisinin partial resistance mutations continue to increase in prevalence with the overall level of mutant infections reaching 32% in Rwanda. The increase appears to be due to the rapid emergence of k13-675V (6.4%, 6/94 infections), previously not observed, rather than continued expansion of 561H (23.5% 20/85). Mutations to partner drugs and other anti-malarials were variable, with high levels of multidrug resistance 1 (mdr1) N86 (95.5%) associated with lumefantrine decreased susceptibility and dihydrofolate reductase (dhfr) 164L (24.7%) associated with a high level of antifolate resistance, but low levels of amodiaquine resistance polymorphisms with chloroquine resistance transporter (crt) 76T: at 6.1% prevalence. No hrp2 or hrp3 gene deletions associated with diagnostic resistance were found.

Conclusions: Increasing prevalence of artemisinin partial resistance due to k13-561H and the rapid expansion of k13-675V is concerning for the longevity of artemisinin effectiveness in the region. False negative RDT results do not appear to be an issue with no hrp2 or hpr3 deletions detected. Continued molecular surveillance in this region and surrounding areas is needed to follow artemisinin partial resistance and provide early detection of partner drug resistance, which would likely compromise control and increase malaria morbidity and mortality in East Africa.

Keywords: Plasmodium falciparum; Artemisinin; Drug resistance; K13; Malaria; R561H; Rukara; Rwanda; kelch13.

Fig. 1

Distribution of Previous reports of K13 Mutations in Rwanda. The map illustrates the geographic distribution of research sites that have reported on K13 mutations, including Kirehe, Ngoma, Huye, Masaka, and Rukara

Fig. 2

Upset plot of MDR1 and K13 mutations. A total of 82 samples had genotype calls at K13 R561H, MDR1 N86Y, MDR1 Y184F and MDR1 D1246Y. The majority of the parasites had the NFD haplotype in MDR1, with 31.7% (19/60) having the K13 561H mutation

Fig. 3

Upset plot of DHPS mutations A total of 83 samples had genotype calls at DHPS A437G, K540E, and A581G and 45.8% (38/83) of the infections had all three mutations, while 88.0% (73/83) had both DHPS 437G and 540E

Fig. 4

Proportional Euler Venn Diagram Comparing the Three Utilized Diagnostic Methods 130 of the 274 samples (H) were negative for all three tests and are excluded from the Venn diagram [29]