Background
Although global malaria incidence and death rates have decreased in recent years, malaria remains one of the biggest threats to populations living within the tropical and sub-tropical world. In the Greater Mekong Sub-region (GMS) of Southeast Asia, a regional malaria elimination plan has been endorsed, aiming to eliminate
Plasmodium falciparum malaria by 2025 and all malaria by 2030 [
1]. This ambitious goal is met with challenges, especially the recently developed artemisinin resistance among
P. falciparum populations in this region [
2‐
4]. Further complication of the situation results from the increasing prevalence of
Plasmodium vivax in this region, a parasite that is resilient to current control measures [
5]. Although chloroquine (CQ) remains the primary treatment option for
P. vivax infections, the emergence of CQ resistance has led to the use of artemisinin-based combination therapy (ACT) for the treatment of vivax malaria in Indonesia [
6]. Monitoring drug resistance in
P. vivax populations is important for eliminating all malaria from the GMS.
In regions co-endemic for
P. falciparum and
P. vivax, both species can share the same vectors and human hosts, and are often subject to similar forces of natural selection [
7,
8]. Several studies showed that anti-malarial drug pressure induces strong selection on both parasite species [
9,
10]. It is thus assumed that the widespread use of ACT for treating
P. falciparum infection may exert similar collateral selective pressure on
P. vivax populations. Recently, mutations in the propeller domain of the
Pfk13 gene have been incriminated as important determinants of artemisinin resistance in
P. falciparum [
11‐
13]. Although ACT is still highly efficacious for treating vivax malaria [
14,
15], vigilance is required on potential emergence of resistance in this parasite [
16]. A recent study aiming to determine whether similar mutations mediating artemisinin resistance are also present in the
Pfk13 orthologue in
P. vivax (here named
Pvk12 gene as this gene is located on chromosome 12) found that parasites with a
Pvk12 mutation (V552I) was circulating in Cambodia at a very low frequency [
17]. To further examine the genetic diversity of this gene in the GMS, recent collections of clinical isolates of
P. vivax from both sides of the China-Myanmar border were examined. This region has a very long history of artemisinin deployment and there is an indication that CQ efficacy for treating
P. vivax malaria is decreasing [
18]. Here
Pvk12 genes in 262
P. vivax parasite isolates were sequenced and molecular evolution analyses were performed to determine whether this gene is subject to potential selection.
Discussion
Mutations in the propeller domain of
Pfk13 gene have been associated with artemisinin resistance in
P. falciparum, which is manifested as delayed parasite clearance after artemisinin treatment [
11,
12]. A recent analysis of worldwide
P. falciparum populations identified numerous non-synonymous mutations with marked geographic disparity in their frequency and distribution, which may reflect the demographic history of the parasite populations and different drug use histories [
30]. In sharp contrast, studies conducted to date in the GMS, where artemisinin resistance has emerged in
P. falciparum, showed a highly conserved
Pvk12 gene [
17,
31]. In Cambodia where
Pfk13 mutations were highly prevalent, analysis of 284
P. vivax isolates identified only two isolates with a non-synonymous mutation, V552I [
17], although these parasites may have been exposed to higher drug selection pressure. Similarly, analysis of 66
P. vivax samples from central China identified a G581R mutation in one sample (Wang et al. unpublished). In comparison, this analysis of 262 full-length
Pvk12 sequences only identified that two parasite isolates (2%) collected from Yunnan’s Tenchong County contained a non-synonymous mutation, M124I, which is located near the N-terminal region. Thus, mutations identified here, being outside of the propeller domain, are unlikely involved in artemisinin resistance even if
Pvk12 is presumed to play an analogous role to
Pfk13 in resistance. Furthermore, although the recent genomic studies of world
P. vivax populations identified five non-synonymous mutations in
Pvk12 (N25I, S253T, V552I, V652L, and A710E) in 228 samples, all of these mutations were rare mutations [
31]. Specifically, the N25I mutation was identified only in 2% of the western Thai samples (N = 89), whereas the S253T mutation was found only in 8% of the Papua Indonesia samples (N = 55), and each of the three other mutations was present only in one sample. Though some of these mutations are located in the propeller domain, their functions still need to be evaluated individually as mutations within the propeller domains such as A578S in
Pfk13 may not necessarily be associated with artemisinin resistance [
12].
The observed deficiency of mutations in the
Pvk12 gene is in sharp contrast to
Pfk13 from the sympatric
P. falciparum populations [
11,
30]. While it is unknown whether
Pvk12 and
Pfk13 are functionally equivalent, the high level of conservation of
Pvk12 suggests that (1)
Pvk12 may play a different role in parasite biology even if
Pvk12 has a similar function to
Pfk13 and its conservation is maintained by functional constraints (purifying selection) and, (2) if
Pvk12 is involved in artemisinin resistance, the different mutation rates in
Pfk13 and
Pvk12 may suggest different drug selection pressures on these two parasites. Likewise, there is evidence that
pfk13 is under natural selection as non-synonymous mutations exceed synonymous mutations [
32]. In contrast,
Pvk12 appears to be under purifying selection, though the limited polymorphisms at four positions of
Pvk12 in 11 of 262 samples analyzed here preclude a robust evolutionary analysis. To date the functions of
Plasmodium
k13 orthologues are still unknown, but their similarity in sequence to the human KEAP1 protein, which is involved in the regulation of the antioxidant responses [
33], suggest that they might have similar functions. In
P. falciparum, it was found that the
Pfk13 is linked to the phosphotidylinositol-3-phosphate kinase (
PI3K) pathway and artemisinin-conferring
Pfk13 mutations are associated with increased
PI3K activity and higher levels of the product phosphatidylinositol-3-phosphate [
34]. If a similar pathway linking
k13 and
PI3K works in both parasite species, drug selections may be imposed on different enzymes within the same pathway. Interestingly, divergent selections have been identified on the phosphoinositide-dependent kinase PDK-1 among
P. vivax populations, an enzyme participating in the
PI3K pathway [
28]. It is unknown whether this observation suggests that artemisinin family drugs might have acted on the
PvPDK-
1 gene instead of
Pvk12 gene in
P. vivax. It is also possible that the highly conserved
Pvk12 gene among worldwide
P. vivax populations may suggest that
Pvk12 may have nothing to do with artemisinin resistance, like that the
pvcrt-o gene is not a major player in CQ resistance in
P. vivax as compared to the critical role of its orthologue
pfcrt in mediating CQ resistance in
P. falciparum.
Mutations within
Pvk12, though rare, displayed quite significant geographical disparities. Three different non-synonymous mutations have been identified in parasite populations from different parts of the GMS [
17,
31]. Considering evidence of independent emergence of artemisinin resistance-conferring
Pfk13 mutations in different areas of the GMS [
35], this difference in
Pvk12 may indicate different demographic histories and the presence of gene flow barriers among these parasite populations. It could also reflect different drug use histories for
P. falciparum malaria, which may have exerted collateral selection on
P. vivax, given that the front-line treatment for
P. vivax malaria in most GMS countries remains as CQ and primaquine. Nevertheless, the collateral selection by artemisinins in the case of mixed
P. falciparum/
P. vivax infections that are treated by ACT should be minimal.
Plasmodium vivax parasites reappearing after ACT treatment may be the relapsing parasites resulted from the awakening hypnozoites, and thus further transmission probably will be from these unselected parasites. In this regard, future surveillance on artemisinin resistance in
P. vivax may need to focus on areas such as Indonesia where ACT has replaced CQ for treatment of vivax malaria, though recent analysis of a limited number of samples did not detect mutations in the propeller domain of
Pvk12 in the Papua Indonesia samples [
31].
Authors’ contributions
DS, BY, HY and DZ carried out the experimental work and data analysis. RR, HY and WY participated in data analysis. DS, YZ and CL performed manuscript writing. YR, YZ and CL conceived the study and participated in the design of the study. All authors read and approved the final manuscript.