Background
Malaria remains a major public health problem in tropical and sub-tropical regions of the world. According to the World Malaria Report 2019, it is estimated that there were 228 million malaria cases and 405,000 malaria-related deaths worldwide in 2018 [
1]. Currently, malaria control relies primarily on measures targeting vectors (insecticide-treated bed nets and indoor residual spraying) and effective anti-malarial treatment of clinical cases [
2]. Since 2001, artemisinin-based combination therapy (ACT) has been recommended as the first-line treatment for
Plasmodium falciparum [
3], and its widespread adoption in malaria treatment policies of endemic nations has played an important role in reducing malaria-related mortality and morbidity. The development of resistance in
P. falciparum to artemisinins and partner drugs is a major threat to malaria control and elimination [
4].
Artemisinin resistance first emerged in western Cambodia in 2007 [
5,
6], and has since been detected in all countries of the Greater Mekong Sub-region (GMS), due to spread and/or independent emergence [
7,
8]. ACT includes artemisinin or one of its derivatives and a partner drug such as lumefantrine, piperaquine, mefloquine, amodiaquine, and pyronaridine. Evolution of resistance in parasites to the artemisinins and the partner drugs would lead to clinical failures of ACT. In Cambodia, clinical resistance to two ACT, artesunate/mefloquine [
9] and dihydroartemisinin/piperaquine (DP) [
10‐
13], has already been identified. To halt the spread of artemisinin resistance in the GMS, ACT efficacy has been monitored in multiple sentinel sites [
14‐
20]. Furthermore, to effectively contain artemisinin resistance in the GMS, countries within the GMS aim to eliminate
P. falciparum malaria from this region by 2025 [
21].
Clinically, artemisinin resistance manifests as delayed parasite clearance with parasite clearance half-life (PC
1/2) exceeding 5 h, resulting in lingering parasitaemia 3 days after initiation of the treatment [
17]. Accurate determination of parasite PC
1/2 requires sampling of peripheral parasitaemia every 6 h after administration of the artemisinin drug [
22]. In resource-limited settings, the day-3 parasite-positive rate can be used as a proxy measure of delayed parasite clearance [
23]. Artemisinin resistance affects the ring stage, and dormant ring-stage parasites are able to endure the onslaught of artemisinins and later cause recrudescence of the disease [
24]. To capture the ring stage-associated resistance phenotype, an in vitro or ex vivo ring-stage survival assay (RSA) measuring the proportion of the 0–3 h ring-stage parasites surviving 6 h of 700 nM dihydroartemisinin treatment was developed [
25,
26]. In 2014, mutations in the propeller domain of the
P. falciparum kelch13 (
pfk13) gene were identified to be associated with artemisinin resistance [
27], providing a molecular marker for surveillance of artemisinin resistance. A large-scale survey of
P. falciparum populations identified as many as 108 non-synonymous
pfk13 mutations, with wide variation in geographical distribution worldwide; mutations associated with delayed parasite clearance were identified only in Southeast Asia [
28]. Likewise, within the GMS,
P. falciparum populations showed striking disparity in the prevalence and distribution of
pfk13 mutations, with the C580Y and F446I being the predominant
pfk13 mutations in east and west GMS, respectively [
27,
29‐
31]. The NN insertion between amino acids 136 and 137 was associated with artemisinin resistance and its prevalence has increased dramatically over the years along the China-Myanmar border [
20,
32].
Molecular markers associated with anti-malarial resistance are useful for resistance surveillance and elucidation of evolution of resistance in parasite populations [
33]. Point mutations in the
P. falciparum chloroquine resistance transporter (
pfcrt) and the
P. falciparum multidrug resistance 1 (
pfmdr1) genes are associated with resistance to chloroquine (CQ) and certain 4-amino-quinoline drugs [
34]. In Africa, the extensive deployment of artemether-lumefantrine (AL) has selected parasites with the wild-type N86 and
pfmdr1 haplotype N86/184F/D1246 [
35‐
39]. In the folate biosynthesis pathway, mutations in
P. falciparum dihydrofolate reductase (
pfdhfr) and
P. falciparum dihydropteroate synthase (
pfdhps) genes as well as amplification of the
GTP-
cyclohydrolase gene are associated with resistance to the antifolate drugs sulfadoxine-pyrimethamine (SP) [
40,
41].
From 2002, ACT has been deployed for the treatment of falciparum malaria in Myanmar and three ACT, AL, DP and artesunate-mefloquine are recommended [
42]. In the GMS, Myanmar has the heaviest malaria burden and its geographical position bridging Southeast Asia and South Asia highlights the need to monitor potential westward spread of resistance. To date, clinical studies to monitor the efficacies of artemisinins or ACT detected artemisinin-resistant
P. falciparum only in southern and eastern Myanmar [
43,
44]. In comparison, ACT remained highly efficacious in northern, northeastern (at the China-Myanmar border) and western Myanmar [
19,
45‐
49]. Molecular surveillance also detected disparate distributions and prevalence of
pfk13 mutations in different regions of Myanmar [
29,
30,
46‐
48,
50], providing a quick assessment of the artemisinin resistance situation. This study evaluated the clinical efficacy of AL for treating falciparum malaria in a western township of Myanmar bordering Bangladesh and India and studied the genetic polymorphisms in genes associated with resistance to AL (
pfk13,
pfcrt and
pfmdr1). Given the extensive use of artesunate-SP in India, this study also genotyped the mutations in the
pfdhfr and
pfdhps genes.
Discussion
The emergence and spread of
P. falciparum resistance to artemisinin in GMS is of great concern and demands the monitoring of clinical efficacy of ACT in malaria-endemic areas of the region. Myanmar occupies an important position in artemisinin resistance containment, because it was among the highest malaria burden countries in the GMS and is geographically linked to the Indian sub-continent [
57]. Since the detection of artemisinin resistance in Cambodia [
6], delayed parasite clearance in patients after ACT or artesunate treatment was first detected in southern Myanmar in 2010 [
42,
44]. One study conducted in northern Myanmar reported 30% of day-3 parasite positivity after treatment with DP in 2013 [
48]. The artemisinin resistance phenotype was also documented in eastern (37.1%) [
43] and northeastern (23.1%) [
20] Myanmar after treatment with artesunate. In southeastern Myanmar, 20% of the cases were still parasitaemic on day 3 after treatment with AL [
49]. Despite the presence of artemisinin resistance, ACT still demonstrated high therapeutic efficacies (95.9–100%) in the above areas. In western Myanmar, artemisinin resistance has not been detected. This study confirmed the absence of clinical artemisinin resistance in western Myanmar, with AL demonstrating 100% therapeutic efficacy with no recrudescence within 28 days of follow-up. Although the number of patients tested here was relatively small, the day-28 therapeutic efficacy of AL was consistent with previous studies conducted in the same area [
46,
47]. However, the day-3 parasite-positive cases (12.2%) just exceeded the 10% threshold recommended by WHO for suspected emergence of artemisinin resistance.
Artemisinin resistance has been associated with mutations in the propeller domain of
pfk13 [
27]. Several mutations including N458Y, Y493H, R539T, I543T, and C580Y have been genetically validated to confer artemisinin resistance [
58]. The NN insert outside of the propeller domain has also been reported to be correlated with artemisinin resistance, initially in China-Myanmar border [
20]. This insert has increased in prevalence over the years and reached 100% in samples collected in 2014–2016 [
32]. No mutations in the propeller domain of the
pfk13 gene were identified in the present study, whereas the NN insert was present in 56.7% patients. This is consistent with a recent study of asymptomatic
P. falciparum infections in this region showing NN insert as the most popular mutation [
59]. Although all of the day-3 parasite-positive samples in the present study harboured NN insert compared to 50% among the day-3 parasite-negative cases, the sample size was too small to perform a robust assessment of the potential association of the NN insert with day-3 parasitaemia. Further investigations are needed to explore the functions of this mutation. The K189T mutation was identified in Myanmar for the first time. This mutation was previously observed in northeast India near Myanmar [
60,
61], but it was not associated with increased clearance half-life [
14]. The study findings suggest that continuous monitoring of
pfk13 gene mutations and RSA in western Myanmar is warranted.
Several studies investigated the relationship between AL treatment and selection of molecular markers associated with treatment failures. Whereas there was no indication of artemisinin resistance-associated
pfk13 mutations, markedly increased prevalence of
pfmdr1 N86 and
pfcrt K76 wild-type alleles was associated with extensive use of AL [
62]. An in vitro study linked the wild-type
pfmdr1 N86 with reduced lumefantrine activity [
36], consistent with the selection of wild-type K76 by lumefantrine [
63]. In
pfmdr1, AL results in the selection of the N86/184F/D1246 haplotype [
37‐
39]. The present study showed that all samples were fixed at K76T and A220S mutations in
pfcrt, but remained wild type at the
pfmdr1 N86 and D1246. The high prevalence of mutations in
pfcrt gene may be the result of continued drug pressure of CQ for treating
P. vivax infections in Myanmar [
64]. In
pfmdr1 gene, Y184F had a frequency of 23.3% and there was no statistically significant association between Y184F and the day-3 parasite-positive and -negative phenotypes (
P = 1.000, Fisher’s exact test). These results were similar to a recent study, which showed the extremely low frequency of N86Y and a moderate prevalence of Y184F in asymptomatic malaria carriers in western Myanmar [
59]. The moderate prevalence of the N86/184F/D1246 haplotype associated with AL selection desires further monitoring.
In recognition of the extensive deployment of the artesunate-SP in India, this study also evaluated
pfdhfr and
pfdhps mutations and detected high prevalence of
pfdhfr (N51I, C59R, S108 N and I164L) and
pfdhps (S436A, A437G and K540E) mutations. Interestingly, these mutations were even more prevalent than previously reported from central Myanmar [
59]. The quintuple mutant of
pfdhps gene (437G and 540E) and
pfdhfr gene (51I, 59R and 108N) was the significant predictor of clinical treatment failure [
56]. Four combined haplotypes containing these quintuple mutations exceeded 70%. In addition, the
pfhdfr I164L mutation associated with SP failures in Asia [
65] also had > 70% prevalence, indicating high-degree SP resistance in this region.
While this study constitutes continued efforts of monitoring the efficacy of anti-malarial drugs in the GMS, it has several limitations. The study reflects the situation that was 5 years ago, and an update is highly desired. The number of patients recruited to this study was small, and an expanded sample size is needed to obtain more accurate estimates of the resistance phenotype. In addition, future studies should extend the follow-up period to 42 days. Furthermore, future studies should also include larger areas along the western Myanmar border to better capture the broad picture of ACT efficacy.
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