Malaria is a major public health problem in the Greater Mekong Subregion (GMS), including Cambodia, China, Laos, Myanmar, Thailand, and Vietnam [
1]. Malaria in these GMS countries is concentrated along the international borders. Since 2001, artemisinin (ART) combination therapy (ACT) has been recommended as the first-line treatment in the national treatment guidelines of most malaria endemic countries and have played an important role in reducing global malaria-associated mortality and morbidity. However, the recent emergence of ART resistance in
Plasmodium falciparum in the GMS is extremely concerning.
P. falciparum isolates resistant to ARTs were first detected in this region in 2008 [
2]. Since then, ART resistance has spread and/or emerged in other areas of the GMS [
3‐
6]. ART resistance is defined as the parasite clearance half-life of >5 h or presence of parasites in patients 3 days after ART treatment. Recently, mutations in the propeller domain of the
Kelch 13 (K13) gene from
P. falciparum (PF3D7_1343700) were shown to be associated with in vitro resistance to ART as well as in vivo delayed parasite clearance [
7]. Kelch-like proteins consist of a series of four to seven Kelch motifs which interact with different binding partners, thereby mediating a wide variety of cellular functions [
8]. Some Kelch proteins also act as substrate adaptors for the cullin 3 ubiquitin ligases [
9], but their exact functions in
Plasmodium remain to be elucidated. In a transcriptomics study, Mok et al. have shown that the parasites with mutated K13 have an upregulated unfolded protein response pathway [
10]. Another study has shown that ART acts via the parasite’s cell stress response involving the ubiquitin/proteasome system, which is enhanced by certain
k13 mutations [
11]. Furthermore, phosphatidylinositol-3-kinase (PI3K)-mediated signaling has been identified as a probable target of ART, and K13 has been shown to regulate the levels of PI3K in parasites [
12]. More studies are required in order to delineate the underlying molecular mechanism of K13-mediated ART resistance. Following the identification of
k13 gene as a molecular marker for ART resistance [
7,
13], numerous studies have been performed to assess the polymorphism in this gene from various malaria endemic regions [
14‐
21]. Several mutations in the Kelch propeller domain have now been associated with in vitro ring-stage survival assays and delayed parasite clearance rates in patients treated with ARTs [
7,
13,
22]. Consequently, sequencing the Kelch propeller domain of the
k13 gene has become an important tool in the surveillance of ART resistance in
P. falciparum. A total of 186
k13 alleles, including 108 nonsynonymous mutations, have been reported so far in
P. falciparum [
23]. There is significant geographic heterogeneity in both the patterns of the
k13 mutations and their prevalence across the GMS [
23], possibly reflecting different drug histories and evolutionary origins. Fortunately, the resistance associated mutations are still confined to Southeast Asia. Some rare alleles are found in other regions but are not associated with ART resistance [
23‐
26].
While
P. falciparum is responsible for the majority of malaria-related mortality,
Plasmodium vivax is the most prevalent parasite species outside of Africa.
Plasmodium vivax caused an estimated 13.8 million cases globally in 2015, and accounted for about half of all malaria cases outside Africa [
27]. Although chloroquine (CQ) remains the primary treatment option for
P. vivax, ACT is used in places such as Indonesia where CQ resistance is evident in this parasite [
28]. To date, there are no reports of clinical resistance of
P. vivax to ARTs. Yet, in areas with co-endemicity of
P. falciparum and
P. vivax, mixed infections often occur at high prevalence [
29]. ACT is also used to treat mixed-species infections [
30,
31]. As a result,
P. vivax may have been under similar drug selective pressure as
P. falciparum. For example, point mutations in
pvdhfr and copy number variations in
pvmdr1 may reflect past drug histories of pyrimethamine and mefloquine, respectively, which have been used to treat falciparum malaria [
32‐
34]. Therefore, it would be interesting to determine whether ART drugs have imposed similar selection on
PvK12 gene. PvK12 is the ortholog of the PfK13, and is present on chromosome 12 of
P. vivax [
35]. A recent study showed that a nonsynonymous mutation in the PvK12 gene circulates at very low frequencies in Cambodia where ART resistance in
P. falciparum first emerged [
35]. Thus, this study aims to characterize the baseline genetic variability of PvK12 gene in parasite populations from various regions in the GMS collected in 2004–2008, before the first reports of ART resistance in
P. falciparum.