Malaria has been scourging human beings for millennia, and remains responsible for over 430,000 child deaths in Africa every year [
1]. The world has made remarkable strides in battling against this ancient enemy during the past decade, reducing by an impressive 47% in mortality rate globally between 2000 and 2013 [
1]. However, parasite resistance to anti-malarials remains an ever-present obstacle to eliminate malaria. Chloroquine and sulphadoxine-pyrimethamine have failed as crucial medicines in the treatment of the deadly malaria parasite
Plasmodium falciparum due to the emergence and rapid spread of drug resistance. More worryingly, resistance to artemisinin (ART) family drugs has been detected and is spreading in Southeast Asia [
2-
4], posing a major threat to the implementation of artemisinin-based combination therapy (ACT) as a defensive line against
P. falciparum.
ART resistance is manifested clinically as delayed parasite clearance half-life (>5 hours)
in vivo [
4,
5]. An
in vitro ring-stage survival assay (RSA
0-3h), which measures the percentage of early ring-stage parasites (0–3 hrs post-invasion of red blood cells (RBCs)) that survive exposure to a pharmacologically relevant concentration of dihydroartemisinin, has been developed to reflect this ART resistance phenotype [
6]. Recent work has associated ART resistance with mutations in the propeller domain of a kelch gene on chromosome 13 (
PF3D7_1343700,
K13 gene) [
5]. The
K13 mutation M476I was initially identified in a Tanzanian
P. falciparum strain that had undergone
in vitro ART selection for five years. Research on parasite isolates from Cambodia, where ART resistance was first observed, identified
K13 mutations Y493H, R539T and C580Y to be associated with delayed clearance [
5]. These mutations were confirmed to contribute to
in vitro ART resistance through genetic manipulations of the
K13 gene [
7,
8]. A large, multicentre, clinical study further indicates that ART resistance is spreading in the Greater Mekong Subregion (GMS), where single-point mutations in the propeller domain of
K13 after the position 440 are collectively associated with ART resistance [
4]. Surveys conducted in different regions showed that
K13 mutations associated with ART resistance were restricted to certain areas of the GMS, including Cambodia, Thailand, Myanmar, and Vietnam. The C580Y mutation is the predominant one approaching fixation in Western Cambodia [
5,
9-
11]. These mutations have not been detected in Bangladesh and Laos [
4,
10,
12]. Surveys of African parasite populations, while having found a diverse array of mutations within the
K13 gene, did not detect those mutations associated with ART resistance [
13-
17].
ART family drugs have been used in China’s Yunnan Province since the late 1970s [
18]. In recent years, clinical efficacy studies conducted in this region showed that artemisinin drugs for treating falciparum malaria remain highly effective [
19,
20]. However, the proportion of day 3 parasite-positive cases in one study reached 18.5% [
20], well above the 10% threshold set by the World Health Organization as a proxy indicator of suspected ART resistance [
21], suggesting possible emergence of ART resistance in this area. In this study, the polymorphisms of
K13 genes in parasite populations along the China-Myanmar border were investigated, and the presence of
K13 mutations that are associated with clinical ART resistance in this region was demonstrated using longitudinally archived parasite samples. More importantly, parasite strains carrying wild-type
K13 alleles have been declining through the six years of sample collection.