Artemisinin (ART) is a quick, effective anti-malarial drug that was discovered in China [
1,
2]. Since ART-based combination therapies were recommended by the World Health Organization (WHO) as a first-line treatment of malaria in 2005, the global malaria burden has significantly declined [
3]. However, in 2008, ART resistance was reported in
Plasmodium falciparum parasites in western Cambodia; a delay in clearance time was observed in infected patients who had received the standard 3-day treatment [
4,
5]. The resistance has spread across the mainland of Southeast Asia, including Cambodia, Thailand, Vietnam, Myanmar, and China–Myanmar border areas [
6,
7]. The spread of ART resistance, particularly in Africa, will certainly threaten global malaria control programmes. Therefore, efficient tools, such as molecular markers, for the detection and surveillance of ART-resistant parasites are urgently required to contain the spread of the resistance.
Currently, tools available for the detection and surveillance of ART-resistant malaria parasites include measurement of in vivo parasite clearance rates and ring-stage survival assay of 0–3 h (RSA
0–3 h) that is well correlated with in vivo parasite clearance rates [
8]. The K13 propeller gene of
P. falciparum (
k13; PF3D7_1343700) has been identified as a molecular marker for monitoring ART-resistant parasites [
9]. A single mutation after position 440 in
k13 is associated with slow parasite clearance rates in vivo and high parasite survival rates in vitro [
7,
10]. The prevalent mutant alleles of
k13 in the Thai–Cambodian/Thai–Myanmar border areas are C580Y, Y493H, R539T, and I543T, while that in the northern Myanmar and China–Myanmar border areas is F446I [
7,
9,
11]. Mutant alleles such as C580Y and R539T have been confirmed to be correlated to ART resistance characterized by long in vivo parasite clearance half-lives after ART treatment and high RSA
0–3 h survival rates [
9,
12]. In addition, the association of the C580Y mutation with ART resistance was also confirmed through introduction of the mutation into
k13 [
13]. However, F446I is the most prevalent mutant allele of
k13 at the China–Myanmar border and in northern Myanmar. Previous study reported that of 392 isolates from the China–Myanmar border, 128 had the F446I mutation with a prevalence of 32.7% [
11]. Two other studies showed the F446I mutation with a prevalence of 36.5 and 27.2% in this area [
14,
15]. It is unclear whether this mutation is associated with ART resistance. Therefore, homology recombinant technology was used to introduce the F446I mutation in
k13 gene of 2 distinct laboratory strains, 3D7 and FCC1/HN, of
P. falciparum and investigate the impact of the mutation on ART susceptibility.