Background
Artemisinin is a sesquiterpene lactone, containing the peroxide group, extracted and isolated from the leaves of
Artemisia annua. The drug and its derivatives play a role in killing
Plasmodium falciparum by inhibiting the activity of phosphatidylinositol-3-kinase (PfPI3K) [
1], with few side effects [
2,
3]. Therefore, the World Health Organization (WHO) has advocated artemisinin-based combination therapy (ACT) as the first-line anti-malarial treatment of uncomplicated falciparum malaria in malaria-endemic areas to effectively reduce the incidence of the disease and the risk of death, thereby significantly reducing the burden of malaria worldwide [
4]. The first discovery of artemisinin-resistant isolates in Cambodia in 2008 [
5], was followed by a spread to Myanmar and Thailand [
5‐
10]. Presently, artemisinin resistance is primarily observed in Cambodia, Laos, Thailand, Myanmar, and the shared border with Yunnan Province, China [
11‐
14].
The clinical resistance phenotype of
P. falciparum to artemisinin is a prolongation of
Plasmodium clearance time in human circulating blood [
6,
7]. Consequently, the expression of PfPI3K is up-regulated in artemisinin-resistant parasites [
1]. Mbengue et al. [
15] further confirmed that some loci mutations in the
k13 gene in
P. falciparum could lead to this altered expression. In Cambodia [
16], Southeast Asia [
17], and other areas, the prolonged duration for
Plasmodium clearance is associated with the propeller domain loci mutations in the
k13 gene of
P. falciparum isolates. Adams et al. [
18] demonstrated that the propeller area of the
PfK13 protein is associated with a variety of cellular functions, such as ubiquitin-regulating proteins and oxidative stress, and the loci mutations in that area may alter the interaction of these proteins. Although Tun et al. [
19], Wang et al. [
20], and Huang et al. [
21] have found that the F446I locus mutation in the propeller domain of the
k13 gene in
P. falciparum isolates in Myanmar has a stable impact on the artemisinin resistance appearance, the genetic relationship within different populations is yet to be elucidated.
In the early 1980s, the chloroquine resistance of
P. falciparum was monitored systematically in Yunnan Province [
22‐
26]. Based on the clinical curative effect [
27‐
29], artemisinin was used gradually for the treatment of cerebral malaria and chloroquine-resistant falciparum malaria in malaria-endemic areas [
30‐
32]. In 1996, a decreased sensitivity to artemisinin was detected in the treatment of falciparum malaria in Yunnan [
33]. By 2005, the rate of chloroquine resistance in the main falciparum malaria endemic areas of the province was about 70% [
34], while the minimum inhibitory concentration of artemisinin increased four- to eightfold [
35]. Recently, molecular markers of
P. falciparum chloroquine resistance (
Pfcrt gene) and artemisinin resistance (
kelch13 gene) were monitored. The results show that mutations of
Pfcrt gene were identified from 81.3% isolates in Yunnan Province [
14], and the detection rates of chloroquine-resistant
Pfcrt and artemisinin-resistant
k13 P. falciparum, were 85% and 35%, respectively [
36,
37]. The isolates having the two resistant molecules accounted for about 27.1% of the population [
38], demonstrating the complexity of drug resistance of
P. falciparum in Yunnan Province.
However, genetic markers alone might not be sufficient for determining the status of anti-malarial resistance in a region, and hence, it is essential to observe the resistance phenotype in a specific number of
P. falciparum samples. In recent years, the number of Yunnan indigenous falciparum malaria cases has declined to less than 5 per year, along with the cases that meet the conditions of in vivo test for
Plasmodium resistance [
39]. In contrast, the prevalence of falciparum malaria is still serious in Myanmar, which borders Yunnan Province. More than 70% cases diagnosed and reported by Yunnan Province are still infected ‘in the region’, rather than indigenous infection [
37].
Therefore, understanding the artemisinin-resistant phenotype of falciparum malaria cases in Myanmar not only facilitates the selecting of a reasonable scheme for standardized treatment of the falciparum malaria cases infected in Myanmar, but also may provide a solution to the predicament that in vivo testing anti-malarial drug resistance are unable to be carry out in Yunnan Province due to the lack of indigenous infection volunteers. If the genetic similarity of Plasmodium isolates between Yunnan Province and Myanmar could be proved enough high, it would represent that the biological characteristics of the two groups isolates are stable homogeneity, and the artemisinin resistance characteristics observed from one group could be regarded as a common feature of both groups. Consequently, in this study the association between the mutation of k13 and artemisinin resistance of Plasmodium isolates from Yunnan Province was analysed by using population genetics analytic method, while the artemisinin resistant phenotypes of P. falciparum had to be tested in vivo on falciparum malaria cases in Myanmar.
Discussion
The present study was designed to detect the locus mutations at the propeller domain of artemisinin-resistant
PfK13 protein in
Plasmodium in 194 cases of falciparum malaria in Yunnan Province from January 2013 to December 2015 [9, 37, and 43]. A total of 11 single-locus non-synonymous mutations F446I, N458Y, S459L, C469Y, G533A, E556D, P574L, A578S, V581I, E668D, and A676D were detected in the 444–709 aa region at the C-terminal of
PfK13 protein. Among these loci, S459L and E668D were discovered recently [
14,
16,
19‐
21,
43,
49‐
52], while the highest mutation rate of 21.1% at locus 446 (Table
1) was lower than that in the blood sample from Myanmar as reported by Tun et al. [
14,
19]. Moreover, this mutation rate was lower than 73.2%, 27.2%, and 70.8% detected in blood samples from China-Myanmar border areas as reported by Huang et al. [
21] and Wang et al. [
20,
52], which might be associated with the heterogeneity of samples used in various studies. In a majority of the previous studies on the artemisinin resistance markers from blood samples of patients with falciparum malaria in the China-Myanmar border region, the clinical efficacy of anti-malarial drugs was evaluated [
21,
45,
52,
53]. Nevertheless, the blood samples in the present study were collected from falciparum malaria cases from all the geographical areas of the Yunnan Province for consecutive 3 years, without any limitations on the density of
Plasmodium and clinical manifestations. Therefore, the samples were continuous and systematic, allowing the monitoring of artemisinin resistance markers similar to the routine conditions in Yunnan Province.
Notably, the mutants that were not detected at loci 493, 539, 543, and 580 were considered to be closely related to the phenotype of artemisinin resistance in 194 blood samples collected from falciparum malaria cases in Yunnan Province [
54,
55], and the multivariate mutations described by Taylor et al. [
49] and Huang et al. [
51] were not found in each mutation locus. This phenomenon suggested that the locus mutation associated with artemisinin resistance might occur when the
k13 gene mutation is accumulated. A total of 15 non-synonymous mutation loci F446I, G450V, N458Y, C469Y, A481V, F483S, L492S, Y519K, G533A, P553L, E556D, P574L, C580Y, A675V, and A676D were detected in the propeller domain in the
PfK13 protein of
P. falciparum in blood samples collected from 190 falciparum malaria cases in Myanmar. Among them, G450V, Y519K, and A675V were discovered recently [
14,
16,
19‐
21,
43,
49‐
52]. The double mutation consisting of synonymous mutations at locus 449 and non-synonymous mutations at locus 676 was found in one case (Fig.
4). Moreover, 2.1% of the samples presented mutations at locus 580 [
54,
55] that was closely related to the phenotype of artemisinin resistance (Table
1). In addition, the mutation rate at locus 446 was 42.6%, which was higher than that in the isolates from cases in the Yunnan Province, thereby indicating pronounced hyper-mutation of the falciparum malaria cases in Myanmar [
56,
57]. In addition, a lower mutation rate in
k13 in the falciparum malaria cases in the isolates in Yunnan might be attributed to the 15.1% African isolates in the samples. These African isolates are mainly derived from Angola, Cameroon, Congo, Guinea, Nigeria, Tanzania, Mali, Ethiopia, Chad, and Gabon that are still considered as areas with a lower pressure of artemisinin drugs than that in Southeast Asia [
11,
12,
14,
58].
Fst is a critical indicator of the degree of differentiation between subpopulations and populations, which can be used to quantify the genetic relationship between different populations. The value of Fst ranges from 0 to 1, and it refers to a similar genotype in the random mating and a unique genotype in complete isolation, respectively, when used for comparison between the populations [
59]. In the present study, Fst was used to evaluate the degree of differentiation of
k13 between the population of
P. falciparum isolates in Yunnan and Myanmar. The results demonstrated that although the type of mutations and the types and number of haplotypes in the
k13 gene of two
P. falciparum from falciparum malaria cases in Yunnan and Myanmar were different (Table
1), the genetic differentiation coefficient between the two groups was small (Fst = 0.0410,
P < 0.05). Furthermore, the intra-population and the inter-population variation accounted for 95.9% and 4.1%, respectively. Hence, in the present study, similar genetic backgrounds were detected in the populations of
P. falciparum isolates from cases in Yunnan and Myanmar. Therefore, the degree of risk of artemisinin resistance in the
k13 gene mutation obtained in the isolates from Myanmar (Table
2) could also be reported in the falciparum malaria cases in Yunnan. This phenomenon indicated that the risks of artemisinin treatment failure in Yunnan cases infected with
P. falciparum with 446I mutations or that in any locus of 446I, 469Y, 676D, 458Y, and 574L in the
k13 gene were 1.640-fold (95% CI 1.284–2.095) and 1.840-fold (95% CI 1.412–2.398) of the cases infected with wild-type
P. falciparum, respectively. Unlike the evaluation of the genetic association of artemisinin resistance in the China-Myanmar border region reported by Huang et al. [
21] and Wang et al. [
53], the results of the current study could be utilized to deduce the hazards of
k13 mutation in the Yunnan Province. Nonetheless, no correlation was detected between the
k13 gene mutation and artemisinin resistance in the isolates in Yunnan cases. However, the polymorphism mutation loci, especially the mutation at locus F446I in
k13can be used as a molecular marker for monitoring the artemisinin resistance in
P. falciparum in Yunnan Province.
Recently, the stellar layout of the mediatory network of haplotypes has been considered as evidence of population expansion [
60‐
63]. Herein, both the evolution networks of haplotypes in the
k13 gene in
P. falciparum isolates from Yunnan and Myanmar cases were stellar, and the low-frequency haplotypes accounted for a large proportion in the population. These results demonstrated a continuous expansion of the
P. falciparum population in the two groups, which is affected by the external environment screening. Together with the Ka/Ks ratio > 1 in both groups (Table
1), the “positive diversified selection” from the two populations indicated that the
P. falciparum escapes the pressure of artemisinin.
The non-parametric correlation analysis is the simplest associative analysis method used in the case–control study for the direct comparison between the two groups with respect to the alleles and gene frequencies of genetic markers. A significant correlation between diseases with some alleles can guide the development of causal relationship study, and ultimately could provide the direction for finding the genetic causes of disease susceptibility. Nevertheless, the present study has limitations. First, the sample size was small for the genetic association study, and the degree of risk of k13 mutation needs to be elucidated further. Second, the subjects undergoing a phenotype test are foreign ethnicity in Myanmar, which might cause race-related genetic heterogeneity in Yunnan cases. In addition, the area for the phenotypic study, Lazan Myanmar, shows a high prevalence of malaria. Therefore, the use of microcosmic evaluation indicators obtained in this study should be employed cautiously in different falciparum malaria endemic areas. The expansion of the sample size of the homogenous study and the relevant systematic analysis of the genetic relationship between Artemisinin resistance phenotype and k13 gene mutation in P. falciparum are imperative for future investigations.
In this study, the successful rates of PCR amplification and sequencing were not near 100%, which may be related to the different preservation durations of and the different cryopreservation conditions of blood samples during last many years. In addition, whether the density of plasmodium and the concentration of genomic DNA extracted from falciparum malaria cases blood effect on the efficiency of PCR amplification and the successful rate of sequencing of PCR products? They will be verified in another study.
Finally, the original intention using these blood samples for research should be explained further. In China, diagnosis, reporting and management of the malaria cases are carried out in administrative regions, such as in a province and a county. In the management measures to malaria cases, the identification of the Yunnan indigenous infection cases or cases imported is mainly to facilitate statistics of malaria elimination evaluation indicators and this identification has no special guiding role whether selecting some control and protection measures in appearing malaria epidemic situation area. On the contrary, because the potential epidemic hazards both indigenous and imported cases are the same, so the epidemic interdiction measures adopted are almost as same as comprehensive and systematic, for example, these measures must be carried out such as screening
Plasmodium infection for health residents, protection of susceptible population from malarial interruption, reducing vectors density for malaria transmission. Therefore, with the
Plasmodium isolates of falciparum malaria cases reported by Yunnan Province as the research samples, it was not only helpful to reflect the continuity and integrity of management for falciparum malaria cases in Yunnan Province, but also necessary to understand the biological characteristics of the special malaria case isolates population for Yunnan Province. In previous studies, some genes of the
Plasmodium population of falciparum malaria cases isolates found in Yunnan Province, which included almost 80% of the infection cases imported from Myanmar and few other parts from Yunnan indigenous infection cases and infection cases imported from Africa, had only existed a very weak genetic differentiation between these and pure Myanmar cases isolates populations [
37,
38,
64]. This study also showed that there was no significant differentiation of
k13 gene between the two populations. These results suggest that the characteristic of the population imported from Myanmar are confounded by a small amount of Yunnan indigenous isolates or isolates imported from Africa in all of Yunnan falciparum cases isolates.
Authors’ contributions
YiD was responsible for the coordination of all project, study design, statistics and analysis of the data, and wrote the manuscript, JW performed the microscopy examination, AS, YaD and MC carried out the gene testing, YX administered the data, JX guided making the geographical map. All authors read and approved the final manuscript.