The spread of a validated molecular marker of artemisinin partial resistance pfkelch13 R622I and association with pfhrp2/3 deletions in Eritrea

Eritrea is the first African country to switch away from exclusive use of HRP2-based RDTs for the detection of P. falciparum due to high prevalence of pfhrp2/3-deleted P. falciparum parasites causing false-negative RDT results. While heavy reliance on malaria RDTs played a significant role in the rapid expansion of pfhrp2/3-deleted parasites in Eritrea, we hypothesize that the use of antimalarial (artesunate-amodiaquine) may have also contributed to their spread. We conducted a retrospective investigation of mutations in the propeller domain of the P. falciparum kelch13 gene in samples collected in 2016 (n=50) from the Northern Red Sea Zone before the RDT switch away from HRP2-RDTs and in samples collected in 2018-2020 (n=587) from the Gash Barka, Anseba and Debub Zones after the RDT switch. No mutations were identified in the 2016 samples. However, in 2018-2019 samples, we detected five different single non-synonymous mutations. The most prevalent mutation was pfk13 R622I, which was detected in samples collected from all health centres, with an overall prevalence of 11.9% (ranging from 5.9% to 28%). Parasites carrying the R622I mutation have diverse microsatellite marker haplotypes, indicating that they had evolved multiple times from different genetic backgrounds. The prevalence pfk13 R622I was significantly higher in single pfhrp3-deleted parasites (18.0%) compared to parasites without pfhrp2/3 deletions (6.2%) and dual pfhrp2/3-deleted parasites (9.0%), suggesting association between the pfk13 R622I mutation and the pfhrp2/3 deletions in Eritrea. Continuous monitoring the trends in pfhrp2/3 and pfk13 mutants is needed to inform effective malaria management strategies in Eritrea.