Background
Historically, malaria was one of the most serious infectious diseases in China. China has made great contributions towards global malaria control in the past 40 years. In 2010, China launched the National Malaria Elimination Programme (NMEP) 2010–2020 with the goal to interrupt local malaria transmission by 2020. Over the following five years, malaria cases decreased dramatically and there has been no indigenous malaria case reported since 2017 [
1]. Now, China has achieved malaria elimination nationwide and is ready for World Health Organization (WHO) certification. However, with increasing globalization, larger numbers of people entering or returning from malaria-endemic areas present challenges to malaria elimination in China [
2]. According to the national malaria report, there were more than 2500 imported cases annually, including over 100 patients with severe symptoms and approximately 10 deaths in 2017 and 2018 [
3].
Over the past 50 years,
Plasmodium falciparum has developed resistance to all anti-malarial drugs that have been used, including chloroquine (CQ), amodiaquine, sulfadoxine-pyrimethamine (SP), quinine, piperaquine, and mefloquine. Recently, the emergence and spread of multidrug resistance, including artemisinin and partner drug resistance of
P. falciparum in Southeast Asia, poses a significant risk to malaria control and eradication goals in the world. The WHO had implemented a strategy to eliminate
P. falciparum from the six countries in the Greater Mekong Sub-region (GMS) by 2025 to respond to the threat of an untreatable multidrug-resistant parasite [
4]. Several mutations in the
P. falciparum gene encoding a kelch protein on chromosome 13 (
pfk13) are associated with artemisinin resistance [
5] and have arisen multiple times and spread in the GMS. Over 200 non-synonymous
pfk13 mutations have been reported to date, of which nine validated variants (F446I, N458Y, M476I, Y493H, R539T, I543T, P553L, R561H, C580Y) and over 20
pfk13 variants are considered as candidate mutations [
6].
Pfk13 mutations were detected predominantly in the GMS and were rare in Africa, but their profile was highly heterogeneous [
5‐
7].
Mutations in
P. falciparum CQ resistance transporter (
pfcrt), located on the digestive vacuole membrane, were responsible for CQ resistance or treatment failure [
8,
9]. Polymorphisms affecting amino acids at
pfcrt residues 72–76 were observed in CQ-resistant field isolates, whereas
pfcrt CVMNK haplotype was regarded as CQ-sensitive isolates [
10,
11]. Polymorphisms in the
P. falciparum multidrug-resistant 1 (
pfmdr1) gene, encoding the plasmodial homologue of mammalian multidrug-resistant transporters, have previously been linked with anti-malarial drug resistance [
12‐
15]
. The mutations involving
pfmdr1 codons N86Y, Y184F, S1034C, N1042D, and D1246Y have been proven to be associated with mefloquine, lumefantrine, amodiaquine, CQ, and artemisinin, as well [
16,
17].
Artemisinin-based combination therapy (ACT), which combines a fast-acting, rapidly eliminated artemisinin derivative with another slower-acting partner drug with a longer half-life, has been integral to the recent success of global malaria control. According to the current national malaria treatment policy in China, the first-line drugs to treat P. falciparum include three ACT (dyhidroartemisinin-piperaquine, artesunate-amadiquine, artesunate-piperaquine). Molecular surveillance of anti-malarial drug resistance markers is one of the tools to monitor and track the emergence and spread of drug resistance in imported malaria cases in China. This study collected the data of reported malaria cases from the national malaria case report system between 2012 and 2015, which were used to analyse malaria epidemiology in China. Dried blood spots were collected from P. falciparum-infected individuals returning from Africa in 2012–2015. The haplotypes of pfcrt, pfmdr1 and pfk13 genes were estimated by nested PCR and sequencing. The prevalence of different haplotypes of each gene was evaluated. The geographical distribution of the haplotypes of pfcrt, pfmrd1 and pfk13 genes in imported P. falciparum isolates from Africa were mapped.
Discussion
This study was part of the national anti-malarial drug surveillance network and supported by the National Malaria Diagnosis Reference Laboratory Network and NMEP. China has set up a well-organized network for malaria diagnosis, treatment and surveillance covering national, provincial and county levels. Nevertheless, there are several challenges in the post-malaria elimination phase in China. One big challenge is how to maintain strong surveillance and response capacity after malaria elimination because thousands of imported malaria cases are reported in China annually.
Plasmodium falciparum has developed resistance to all anti-malarial drugs, including ACT [
22]. This study evaluated the prevalence of
pfcrt,
pfmdr1 and
pfk13-propeller mutations of
P. falciparum isolates imported from Africa and the geographical distribution of the prevalence of these three genes in imported African
P. falciparum isolates was mapped as well.
CQ was a first-line anti-malarial drug to treat uncomplicated falciparum malaria in Africa from the 1940s, and was widely used because of its high efficacy, safety and low cost [
23]. CQ resistance was first identified along the Thai-Cambodian border in the late 1950s [
24,
25], and first reported in Africa in the 1970s [
26]. In Africa, CQ was replaced by SP and ACT for uncomplicated malaria treatment between the late 1990s and early 2000s. The
pfcrt mutations in codons 72–76 were considered to be the most reliable molecular marker for CQ resistance [
19]. The prevalence of
pfcrt mutations in Africa decreased significantly in contrast to the late 1990s. The reduction of prevalence of the
pfcrt mutation and return of CQ sensitivity was also found in other studies in several malaria-endemic countries in Africa [
27‐
29]. The termination of CQ use resulted in recovery of its efficacy. The most common haplotype of
pfcrt was the wild type CVMNK with the prevalence of 62.8%, which was higher compared with that in the 1990s. Although only a few isolates were detected with single mutation at codon 76, the prevalence of triple mutant haplotype CVIET was 29.4%. In addition, 52 isolates with mixed triple mutant haplotypes CV M
/I N
/E K
/T were identified. According to the published study, CQ resistance may have been caused by selective drug pressure, and multiple genomic background of the strains. Resistant mutations selected by anti-malarial drugs remove linked neutral variation as they sweep (increase in frequency) through a parasite population [
30].
The
pfmdr1 gene was associated with resistance to multiple anti-malarial drugs [
12‐
14]. The
pfmdr1 N86Y and
pfcrt K76T variants have been shown to be in strong linkage disequilibrium, which is associated with CQ, mefloquine, lumefantrine, quinine, and dihydroartimisinin resistance in vitro [
31‐
33]. This study identified
pfmdr1 mutations in only codons 86, 184 and 1246 and total 12 haplotypes, including six mixed mutant haplotypes, were detected. The predominant mutation of Y184F had prevalence of 24.7% (107/434). The single mutant haplotype of
pfmdr1 N86Y was at low prevalence of 5.3% (23/434), lower than another study with the prevalence of 31.0% in 2012 and 8.2% in 2016 [
34]. The single mutant type NY
Y was not detected in this study, suggesting that NY
Y was rare in Africa compared with previous data [
35]. In addition, the single mutant haplotype
YYD and N
YD was common in Africa while prevalence of the double mutant haplotype
YFD,
YY
Y and N
FY was not significantly different among the different sub-regions (
P > 0.05). This difference might be caused by the diversity of drug pressure and transmission intensity among the countries or regions in Africa.
Mutations in
pfk13-propeller domain were first confirmed to be associated with artemisinin resistance in 2014 [
20]. Until now, nine validated variants and over 20 candidates or associated mutations of
pfk13 have been identified [
6]. Forty-nine out of 1,357 isolates showed
pfk13-propeller mutations with prevalence of 3.6% (49/1,357) in this study. The non-synonymous mutations in
pfk13 are rare in Africa and their profile is diverse [
6,
36‐
38]. A total of 22 non-synonymous and four synonymous polymorphic sites were identified in this study (Table
3). C580Y and F446I mutations, which are the most common mutations in GMS, and the predominant mutation in southern China, respectively [
39], were not detected in imported African isolates in this study. Three mutations in
pfk13-propeller domain, including M476I, R539T and P553L associated with artemisinin resistance, were observed in three isolates in this study. Another
pfk13 mutation, M579I was identified from one isolate from Equatorial Guinea, which was reported to be associated with artemisinin resistance in Africa [
40]. Nevertheless, this mutation was not observed in this study. The presence of C580Y mutation was detected in three patients (2.7%, 3/113) from migrant Chinese workers returning from Ghana in 2013, but this needed further characterization [
41]. Previous studies reported that R539T mutation was identified from a population returning to China from Africa [
42]. In this study, although one isolate carried the R539T variant, there was no evidence to prove this was an artemisinin-resistant isolate because there was no treatment failure outcome associated with the variant. The A578S variant, which is the most common mutation in
pfk13 in Africa, was identified from 10 isolates (four from Equatorial Guinea, two from Angola, and one each from the Republic of Congo, Ghana, Guinea, and Uganda). A578S is comprised of two tightly linked SNPs and might be involved in artemisinin resistance in Africa [
43]. Recently, the de novo emergence and clonal expansion of
pfk13 R561H lineage has been reported in Rwanda and this mutation has been confirmed as a mediator of artemisinin resistance in vitro [
44]. Another more recent study reported that
pfk13 R561H occurred in 4.5% (3/66) of the isolates collected in southern Rwanda in 2019 [
45]. Interestingly, an imported malaria case from Rwanda to China was detected with R561H mutation [
46] and one isolate from southeast Tanzania carried this mutation too [
47]. Therefore, molecular marker surveillance could provide early warning and evidence for efficacy of anti-malarial drugs to treat imported cases. China has set up an anti-malarial drug surveillance network that is responsible for implementing an integrated drug efficacy study (iDES) of anti-malarial drugs for national policy and molecular surveillance in the entire country.
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