Background
Malaria is one of the important parasitic diseases threatening human beings over many decades. Emergence and spread of drug resistance is an important cause of morbidity and mortality in malaria. Artemisinin derivatives are the most potent drugs against multidrug-resistant
Plasmodium falciparum. Artemisinin-based combination therapy (ACT) has been recommended by the World Health Organization (WHO) to use as the first-line treatment for multidrug-resistant falciparum malaria [
1]. Artesunate-mefloquine (AS-MQ) had been used in Thailand to treat uncomplicated falciparum malaria since 1995 due to the emergence of MQ resistance [
2]. Later AS–MQ was used in other Southeast Asian countries including Cambodia [
2]. Unfortunately, the efficacy of AS–MQ declined along the Thai-Cambodian border after a few years of implementation [
3,
4]. Increased AS–MQ failure rates observed in Thailand and Cambodia were usually associated with MQ resistance [
5,
6]. Recently, a few studies have shown evidence of artemisinin resistance in
P. falciparum, defined as delayed parasite clearance, i.e., presence of parasitaemia on day 3 following treatment with AS monotherapy or ACT [
7,
8]. Subsequently, artemisinin resistance has emerged independently and spread to many areas of the Greater Mekong Subregion (GMS) [
9,
10]. To determine the situation of artemisinin resistance, a novel ring survival assays (RSA) (i.e., in vitro or ex vivo RSA) has been used to represent the delayed parasite clearance phenotype [
11]. Ariey et al. have identified mutations of the Kelch13 (
k13) gene as molecular markers for artemisinin resistance [
12]. Mutations in the
k13 gene were correlated with delayed parasite clearance and also increased RSA (0–3 h) rate [
12,
13]. The mutations were associated with artemisinin resistance in Cambodia and other countries in the GMS [
12,
14‐
16].
Due to the emergence of artemisinin resistance in some areas along the Thai-Cambodian border, an artemisinin resistance containment project has been launched to cease the spread of artemisinin resistance since 2009 [
17]. The combination of AS–MQ has been replaced by a fixed-dose combination of atovaquone–proguanil (ATQ–PG) (Malarone
®) to reduce artemisinin pressure in these areas. ATQ inhibits the mitochondrial electron transport chain at the bc1 complex [
18,
19]. Mutations in the
cytochrome b (
cytb) gene resulted in amino acid changes at codon 268, exchanging tyrosine for serine (Y268S) or, less frequently, asparagine (Y268 N) conferred ATQ resistance and treatment failure [
20,
21]. Monitoring in vitro drug sensitivities and also molecular markers of
P. falciparum isolates is essential to detect the emergence and spread of drug resistance and provide valuable information for a rational drug use policy. Little is known about the characteristics of
P. falciparum after the artemisinin resistance containment project. This study was aimed to determine phenotypes and genotypes of
P. falciparum isolated from the Thai–Cambodian border after the artemisinin resistance containment project compared with those collected before.
Discussion
Due to the emergence of artemisinin resistance along the Thai–Cambodia border especially in Chanthaburi and Trat Provinces, the artemisinin resistance containment project was launched in 2009 by the Ministry of Public Health, Thailand [
17]. Reduction of artemisinin pressure was one of objectives in this project by replacing AS–MQ with a fixed dose combination of ATQ–PG. In this study, both phenotypes and genotypes of
P. falciparum isolates collected after the artemisinin resistance containment project, 2009–2016 were compared with the isolates collected before 2009. The parasites collected from 2009 to 2016 showed significantly higher CQ, QN, MQ, PPQ and AS IC
50s compared with the parasites collected before 2009. The IC
50s of these Thai isolates were in the same range as those reported in Cambodian isolates in 2013 [
33]. Previously described cut-off points for in vitro anti-malarial resistance were used to determine the parasite’s resistant phenotypes [
28‐
32]. Of 101, only one isolate collected before 2009 exhibited CQ sensitive. No CQ-sensitive isolate was detected among the parasites collected from 2009 to 2016. Although CQ was not used to treat falciparum malaria, Thai isolates of
P. falciparum remain resistant to CQ. This may be due to vivax malaria sharing similar endemic areas with falciparum malaria. Thus, CQ, the first-line treatment for vivax malaria could cause a drug pressure for
P. falciparum as well. The cure rate of MQ has rapidly declined soon after using as a monotherapy to treat falciparum malaria in 1991 [
34]. Because MQ is a long half-life drug, drug pressure could cause the emergence of MQ resistance. As a result, AS–MQ combination was used as the first-line treatment of uncomplicated falciparum malaria since 1995. The parasites collected from 2009 to 2016 showed significantly increased MQ IC
50 compared with the parasites before 2009. In addition, approximately 57% of the isolates collected after 2009 exhibited in vitro MQ resistance. Increased MQ resistance after 2009 may be influenced by the delayed parasite clearance phenotype of
P. falciparum against AS in these areas. Slow parasite clearance causes more parasites to be exposed to the partner drug, i.e., MQ, increasing the risk of resistance selection of the partner drug, which in turn increases the risk of treatment failure. In the present study, the parasites with the
pfmdr1 184F allele showed a significantly higher MQ IC
50 than others.
A few cases of ATQ–PG treatment failure have been reported. Treatment failure of ATQ–PG was due to ATQ resistance and has been linked to point mutations in the target gene, the
cytb gene [
20,
32,
35]. Determination of the phenotypes and genotypes related to ATQ–PG response in Thai isolates of
P. falciparum will be useful for rational drug use. According to Musset and colleagues (2006) [
32], the cut-off point for in vitro ATQ resistance was the IC
50 > 1900 nM. None of parasite isolates in this study exhibited ATQ resistance. In addition, they contained no mutations in the
cytb 268 codon, molecular markers for ATQ resistance. The present results are similar to our survey in 2008 showing no evidence of ATQ resistance in Thai isolates of
P. falciparum collected from both Thai–Cambodian and Thai–Myanmar borders [
36]. A recent study in Cambodia also showed similar results indicating that
P. falciparum isolated from Western Cambodia remained sensitive to ATQ in vitro and showed no point mutations in the
cytb gene [
33]. Recently, a successful cure of a multidrug-resistant
falciparum case after artemisinin-based and QN-based treatment failure was reported in a subject that traveled to Cambodia [
37]. These results suggest that a fixed-dose combination of ATQ–PG could be used the artemisinin-resistant areas with careful monitoring.
In this study, the IC
50 of AS but not DHA was increased among the parasites isolated from 2009 to 2016. However, using the IC
50 of > 10.5 nM as the cut-off point for in vitro AS and DHA resistance [
31], no parasite exhibiting AS and DHA resistance was collected in the year before and after the artemisinin resistance containment project. Although no evidence exists of full artemisinin resistance, partial artemisinin resistance defined by delayed parasite clearance following treatment with an AS monotherapy or with an ACT is widespread in the Great Mekong Subregion [
38]. To date, more than 200 nonsynonymous mutations in the
k13 gene have been reported. Several mutations in the
k13 gene were associated with delayed parasite clearance in vivo and in vitro including N458Y, Y493H, R539T, I543T, R561H and C580Y [
38]. In the GMS, mutations in the
k13 gene have spread and are distinctly reported according to their geographical areas [
38,
39]. In the eastern GMS including Thai–Cambodia border, C580Y, R539T, Y493H, I543T, and P553L were commonly identified with the domination of C580Y. In the present study, 7 SNPs were identified including G436S, F483L, Y493H, G538 V, R539T, V568G, and C580Y. The most common SNPs of the isolates collected from 2009 to 2016 were C580Y (63.5%) and R539T (15.4%). Ring survival assay was not performed in the present study, however, reduced in vitro AS sensitivity was identified by in vitro sensitivity assay in the parasites with the
k13 539T allele compared with others. For the newly identified SNPs including G436S and F483L, validation as a resistance marker will be required.
After 2009, ATQ–PG has been used to reduce artemisinin pressure in this area. In the absence of drug pressure, some resistant parasites might be less fit than their sensitive counterparts [
40]. However, after the artemisinin resistance containment project,
k13 mutations had increased significantly from 18.4 to 84.6%. Of these mutations, the
k13 C580Y allele is increasing and replacing other haplotypes along the Thai-Cambodia border indicating a selective sweep in these areas. A study of parasites collected in 2007 found that 50% (11/22) of parasites from Chanthaburi and Trat Provinces contained the
k13 mutations [
41]. Of these, 45.5% (10/22) contained the
k13 580Y allele indicating that parasites with the
k13 580Y allele spread widely before the artemisinin resistance containment project. Recent studies indicated that the parasites with the
k13 580Y allele arose in western Cambodia and then spread to other countries in the western Great Mekong Subregion including Thailand, Lao PDR and Vietnam [
12,
41‐
43]. Thus, sensitive parasites might not compete with the main haplotype, the
k13 580Y allele. One other factor that might influence the spread of artemisinin resistance in these areas is cross-board migration. Approximately one half of malaria cases in Thailand were foreign migrant workers [
2]. As part of the artemisinin resistance containment project in Thailand, AS–MQ has been replaced by ATQ–PG to reduce artemisinin pressure in Chanthaburi and Trat provinces, Thailand which has been implemented since 2009. However, ACT remains the first-line treatment for uncomplicated falciparum malaria in Cambodia [
38]. Thus, artemisinin pressure in these areas might not be effectively reduced. In addition, different policies and implementation of primaquine, as a
P. falciparum gametocytocide, may influence the spreading of artemisinin resistance along Thai–Cambodian border [
44,
45].
DHA-PPQ was used as the first-line drug for multidrug-resistant falciparum malaria in Cambodia [
46]. Unfortunately, treatment failure of DHA-PPQ was promptly reported possibly due to the existing resistant parasites because PPQ monotherapy was used in Cambodia in the 1990s [
47]. A few studies showed a link between the
pfmdr1 copy number and PPQ sensitivity [
48‐
50]. However, no significant difference of PPQ IC
50 between the parasites with one and more than
pfmdr1 copy number was found in the present study (18.8 ± 6.3 and 22.9 ± 13.3,
p = 0.078, Independent
t test) similar to the recent study using parasites collected from both Thai–Myanmar and Thai–Cambodian border areas [
51]. Genetic markers for PPQ resistance including nonsynonymous SNP encoding a Glu415Gly mutation in a putative exonuclease (exo-E415G) and plasmepsin 2–3 amplification have been identified [
52,
53]. Both in vitro PPQ sensitivity and ring survival assay were used to identify the association between PPQ resistance and these genes. Treatment failure of DHA-PPQ in Cambodia has been associated with parasites containing the
k13 mutations and multiple plasmepsin 2 copy [
53]. Recent studies have shown that parasites with the
k13 580Y allele and plasmepsin 2 amplification have emerged and spread widely in the western Mekong Basin Subregion causing DHA-PPQ treatment failure [
42,
43]. In the present study, the parasites containing the
k13 580Y alleles exhibited significant higher PPQ IC
50 compared with the others. This could be explained by parasites with the 580Y allele acquiring reduced PPQ susceptibility in this area. Unfortunately, other genetic markers for PPQ resistance including exo-E415G and plasmepsin 2–3 amplification were not determined in our study.