Background
Malaria is a mosquito-borne infectious disease that seriously threatens human health, among which falciparum malaria caused by
Plasmodium falciparum is the most serious, mainly in tropical and subtropical regions in sub-Saharan Africa and Southeast Asia (SEA) [
1]. In 2019, there was an estimated 229 million malaria cases from 87 malaria-endemic countries. Furthermore, approximately 94% of estimated cases were detected in Africa. The countries of Nigeria, Congo, Uganda, Mozambique, and Niger account for 51% of malaria cases (117 million). Additionally, it estimated approximately 409,000 deaths are estimated globally [
1]. Although there have been no indigenous malaria cases reported in China for three consecutive years since 2017 [
2], potential challenges remain in imported malaria cases. In recent years, with globalization, the number of migrant workers, tourists, and businesspeople in China has increased gradually, especially those returning from Africa and SEA [
3], which has brought severe pressure for malaria eradication in China. Thus, it is necessary to strengthen surveillance for imported malaria.
Anti-malarial drugs are considered the major measure for malaria control [
4]. However, with the continuous use of anti-malarial drugs,
P. falciparum gradually achieves drug resistance and spreads rapidly [
5]. Chloroquine (CQ) is a safe, inexpensive, and effective anti-malarial drug for malaria therapy. However, in the 1940s,
P. falciparum parasites developed resistance to CQ. Since then, CQ-resistant (CQR) strains have begun to spread rapidly around the world [
6]. After the discovery of artemisinin (ART) in the 1970s, malaria control was temporarily eased. To improve clinical efficacy and delay the emergence of parasite drug resistance, artemisinin-based combination therapy (ACT) have been recommended by the World Health Organization (WHO) since 2001 [
7]. Unfortunately, ART resistance of
P. falciparum isolates was reported in SEA [
8‐
10]. Recently, dihydroartemisinin-piperaquine (DHA-PPQ) resistance has been detected in western Cambodia [
11‐
14]. Although ACT remains effective in Africa and SEA, prolonged use of ART would lead to anti-malarial drug resistance. Anti-malarial drug resistance (ADR) would be disastrous for global malaria control. Therefore, in the absence of more choices, it is urgent to monitor the ADR status of
P. falciparum parasites.
Mutations detected in
P. falciparum essential genes including
pfcrt,
pfmdr1,
pfdhfr,
pfdhps,
pfk13, and
pfpm2 have been used as molecular markers of drug resistance. The
pfk13 polymorphism has been considered to be related to ART resistance [
9]. However, previous studies demonstrated that the distribution of alleles for
pfk13 varies according to the mutations [
15]. In SEA, the alleles of the 580
Y mutation account for the vast majority [
9]. In Africa, the mutation rate of
pfk13 remained relatively low. In 2016, the newly discovered local ART resistance mutation 561
H of
pfk13 was reported from Rwanda, Africa [
16]. The 72–76 amino acid mutation in
pfcrt, especially the 76
T mutation, was the primary marker of CQR [
17‐
19]. Several mutations in
pfmdr1 are related to the resistance of
P. falciparum to CQ, amodiaquine (AQ) and mefloquine (MQ) [
20,
21]. At present, several newly detected mutations in
pfcrt, including 93
S, 97
Y, 101
F, 145
I, 343
L, 353
V and 356
T, have been identified to be associated to with PPQ with a decreasing trend for the susceptibility of
P. falciparum strains in South America [
13]. However, there was limited information on the effects of these alleles on PPQ in Africa, where malaria is endemic.
In the present study, polymorphisms of pfcrt, pfmdr1, and pfk13 for P. falciparum isolates imported from Africa in Wuhan, China were surveyed. This survey will provide valuable information for rational medication for malaria patients in clinical practice, preventing the spread of ADR P. falciparum in Africa and China.
Discussion
For the past several decades, the emergence and rapid transmission of
P. falciparum ADR parasites has become a major cause of malaria burden globally [
24]. In China, the continuous influx of imported malaria increases the possibility of malaria respreading [
25]. The malaria-endemic area, including Africa and SEA, was the primary source of imported malaria in China including in Wuhan [
16]. Thus, continuous surveillance of imported malaria and ADR profiles is essential for malaria eradication in the non-malarial regions, particularly Wuhan, China.
The mutation of 76
T in
pfcrt was related to CQR [
17]. For
pfcrt, CV
IET and
SVMN
T were the dominant mutant haplotypes. In Africa, mutant haplotype CV
IET occurs more frequently [
26]. CVIET (9.62%) was the most common mutant haplotype in the current study and was mainly distributed in West Africa (5.77%).
SVMN
T is mainly detected in South America and SEA and is rarely found in Africa [
27]. The presence of
SVMN
T was not found in this survey. However,
SVMN
T was observed in Tanzania and Angola [
28,
29]. In these regions, AQ was considered as the driving factor for haplotype selection of
S VMN
T [
28,
29]. The CQ treatment resulted in high failure rates in southern Cameroon between 1999 and 2004 [
30]. However, after an interval of 9 years, the frequency of CVMNK in southeastern Cameroon nearly doubled; Conversely, the CV
IET decreased significantly [
31]. Drug pressure caused by CQ declined during the period as a result of the cessation of drug imports to these countries. In the present study, haplotypes of CVMNK, CV
IET, CV M/
I N/
E K/
T with proportions of 87.50, 9.62, and 2.88% were observed during 2017–2019, respectively. Compared with the previous study [
16], all current observed data indicate that the wild-type haplotype is increased and haplotypes of the mutation type and mixed type are decreased. In recent years, the frequency of CVMNK has increased in several regions of Africa [
32,
33], which is consistent with this survey results. In the present data, CVMNK is mainly concentrated in West Africa (36.54%) and Central Africa (28.85%), especially in the Congo (21.15%) and Nigeria (15.38%). After CQ was discontinued in most countries in sub-Saharan Africa in the 1990s, the investigated isolates regained all or part of their sensitivity to anti-malarial drugs [
34,
35]. It will offer the possibility for these areas to reintroduce CQ in the future for malaria control. Therefore, continuous monitoring of
pfcrt to evaluate CQ resistance dynamics in a certain area is urgent.
DHA/PPQ is one of the ACT, effective against simple malaria. Thus, the effect of PPQ cannot be ignored. However, long-term use of anti-malarial drugs particularly PPQ induced ADR [
14]. Previous studies indicated that several mutations of
pfcrt (93
S, 97
Y, 101
F, 145
I, 343
L, 353
V, and 356
T) were related to reducing parasite sensitivity to PPQ [
13,
14,
36‐
39]. In this study, no mutations were detected at loci 93, 97, 101, 145, 343, 350, and 353. In an investigation of African isolates, consistent with the results of this study, no mutations at these sites were reported [
13]. In this study, 5 isolates carried the mutant allele, and 3 isolates were mixed type at loci 356. In 2011–2012, the 356
T in Gambia and Congo were 78.7 and 36.5%, respectively [
40]. The 356
T mutation was found in 54.7% of
P. falciparum detected in Africa in 2017–2018. However, they also reported that the 356
T mutation was not associated with in vitro reduced susceptibility to PPQ [
13]. Therefore, continuous observations of
pfcrt mutations and susceptibility tests in vitro related to PPQ are necessary.
The
pfmdr1 gene has been reported to be involved in regulating drug sensitivity or tolerance to several anti-malarial drugs, such as CQ, MQ, quinine (QN), artemether-lumefantrine (AL), and even ART [
41]. The
pfmdr1 gene 86
Y mutation is a potential marker for CQR, while 184
F may play a role in resistance to multiple anti-malarial drugs [
41]. The previously reported 86
Y and 184
F mutations in
pfmdr1 are most prevalent in Asia and Africa [
42]. The frequencies of 86
Y (4.72%) and 184
F (47.17%) were monitored in this study, of which 184
F was more prevalent. The results were similar to previous results in Nigeria and Senegal [
43,
44]. In addition, compared with this previous survey in 2011–2016 [
16], allele 86
Y was significantly reduced. This is consistent with the results discussed above regarding the sensitivity of
pfcrt gene recovery to CQ in recent years. Among the six observed haplotypes in this study, N
F (43.40%) and NY (34.91%) were also the most frequent, mainly found in West Africa and Central Africa, especially in Congo and Nigeria, which could be a result of selective pressure by resistance to different drugs. In Nigeria, a previous study showed that N
F was closely related to the sensitivity of AL [
45]. It may be that the first-line drug CQ is replaced by AL, leading to an increased incidence of N
F in these countries.
The
pfk13 gene was crucial in the molecular surveillance of ADR for falciparum malaria parasites. To date, more than 200 nonsynonymous mutations of
pfk13 have been reported [
46]. In SEA and, more recently, South America, a number of these mutations have been associated with delayed parasite clearance following ACT, including mutations at loci 446, 458, 474, 476, 493, 508, 527, 533, 537 543, 553, 568, 574, 580 and so forth [
46]. In Africa, a number of nonsynonymous mutations in
pfk13 have been identified, including mutations at loci149, 189, 189, 561, 575, 579, 589, 578, 592, 637, 641, 656 and so forth [
46,
47]. In this survey, no mutation was found in
pfk13. Because these sample size was insufficient, it was not sufficient to say that the African plasmodium isolates were still highly sensitive to ART; it is necessary to carry out relevant tests with a larger sample size in the future. Although there is no mutation in the
pfk13 gene to indicate ART resistance, it cannot be ignored that
pfk13 is no longer the only biomarker of ART resistance, and there may be other genes as markers of ART resistance [
48,
49]. Thus, genetic markers of ADR are urgently required. Previous studies have demonstrated that the new candidates
pfubp-1 and
pfap2mu are implicated in ART resistance in the
P. falciparum [
48,
49]. Alarming the high morbidity and mortality rates in Africa and the increased status of ADR in Africa could hamper malaria prevention, control, elimination, and even eradication. Therefore, it is critical to monitor mutations associated with ART resistance globally, especially in Africa, by delaying parasite clearance.
It is worth noting that several shortcomings of the current study cannot be neglected. First, affected by the epidemic of COVID-19, the sample size remains small. Thus, valuable information for the molecular surveillance of ADR is limited. Second, 17 samples failed SNPs analysis of
pfk13 because of the failure of amplification and sequencing. In a further study, advanced gene-editing tools, particularly the CRISPR/Cas9 technique, should be considered using
P. falciparum drug resistance genes [
50,
51]. The CRISPR/Cas9 technique can rapidly locate the key sites related to ADR in targeted genes. Compared to natural mutation under long-term drug pressure, artificially introduced mutation by CRISPR/Cas9 can effectively shorten the process of discovering drug resistance sites. CRISPR/Cas9 will offer a useful measure for the discovery of novel mutations in drug resistance genes.
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