Background
Plasmodium vivax infects approximately 100 million people annually and endangers 40% of the world’s population [
1]. Malaria has been a significant public health problem in China until recently, predominantly in two areas of China: subtropical zone (Yunnan and Hainan provinces) and temperate zone (such as Anhui, Henan, Hunan, Hubei, and Jiangsu provinces). Due to China’s national malaria control programme launched in 1978, the intensity and scale of vivax malaria outbreaks have decreased dramatically through vector control and drug treatment of febrile individuals [
2]; however, malaria incidence rose steadily between 2000 and 2006 [
3]. In subtropical China, Yunnan is one of the two provinces with year-round local transmission of
P. vivax and
Plasmodium falciparum and has suffered one of the highest malaria morbidity and mortality rates in China. In temperate zone of Central China, outbreaks of malaria have been reported in Anhui and Henan provinces. In 2006, malaria prevalence increased considerably, with the highest numbers of malaria cases reported in Anhui province [
4]. Although malaria incidence is low in China, the areas and populations at risk are large.
P. vivax malaria accounted for > 95% of all malaria cases reported in China in 2007 [
5], having become the sole parasite species and responsible for more than 90% of re-emerged malaria cases reported in temperate zone of China in 2009 [
6]. In 2010, a countrywide malaria elimination policy was launched by the Ministry of Health of P. R. China with the goal of eliminating malaria by 2015 in China—with the exception of the border region in Yunnan province, and to completely eliminate malaria from China by 2020. Therefore, there is an urgent need to monitor vivax malaria transmission and drug resistance in subtropical and temperate zones of China.
Resistance to common anti-malarial drugs, such as chloroquine (CQ), has been reported for
P. vivax around the world, including Indonesia [
7], Myanmar [
8], India [
9], Vietnam [
10], Turkey [
11], and Ethiopia [
12]. CQ and primaquine have been used as first-line therapies for radical cure of vivax malaria in China for the past 60 years, and clinical failures or reduced efficacy of CQ-primaquine drug combinations for treating vivax malaria had been reported at the Yunnan-Myanmar border [
13], in Yunnan [
14] and Anhui [
15] provinces. Although sulphadoxine-pyrimethamine (SP) is rarely used to treat patients with
P. vivax malaria, pyrimethamine, a component of the two combination regimens [Maloprim® (plus dapsone) and Fansidar® (plus sulphadoxine)], has been widely used for malaria prophylaxis in China between the mid-1960s and early 1990s [
16]. This may place
P. vivax under the selection of SP drug stress in China, especially in the subtropical zone where
P. vivax and
P. falciparum mixed-species infections were common.
P. vivax malaria re-emerged in many counties of China in recent years, possibly caused by social and environmental changes and intrinsic differences in parasites with long or short relapse patterns [
17‐
20]. Even changes in mosquito strains or species could favour a subpopulation of vivax parasites and lead to changes in the parasite population structure [
21], and the emergence and spread of drug-resistant malaria should, of course, be held accountable. Studies in other
P. vivax-endemic regions such as Vietnam highlight the geographic heterogeneity and temporal dynamics of drug resistance in
P. vivax[
10], raising further concerns about the presence of clinical resistance to SP and CQ in these areas of China and highlighting the importance of continued surveillance of CQ drug efficacy.
Molecular genetic markers of resistance are useful for monitoring the emergence and spread of anti-malarial drug resistance; a better understanding of the mechanisms of drug action and resistance are essential for fulfilling the promise of eradicating malaria [
22]. Currently, several genetic markers, including dihydrofolate reductase (
dhfr), dihydropteroate synthase (
dhps), and multidrug resistance (
mdr-1), have been used to study the prevalence and spread of SP- and CQ-resistant
P. vivax[
23‐
26]; however, molecular epidemiologic information on
P. vivax parasite resistance to CQ and primaquine in China is limited. This study investigated the frequencies and patterns of mutations in
pvdhfr,
pvdhps, and
pvmdr-1 linked to SP and CQ resistance in
P. vivax isolates from Yunnan and Anhui provinces of China, and the results provide important information for molecular surveillance of drug-resistant
P. vivax in these areas.
Discussion
Emergence and spread of CQ- or SP-resistant
P. vivax strains has been reported in many malarial regions around the world, especially in Southeast Asia, which is responsible for the increasing morbidity and mortality of
P. vivax[
29]. The use of anti-malarial drugs exhibits enormous geographic heterogeneity in subtropical and temperate zones of China. With increasing movement of human populations, drug-resistant
P. vivax populations and the parasite transmission pattern in the regions are also changing constantly. The emergence and spread of drug-resistant strains of human
Plasmodium species may be partly responsible for re-emergence of malaria in China. SP was widely used for malaria prophylaxis between the mid-1960s and early 1990s, and CQ-primaquine is still the first-line anti-malarial drug for treating
P. vivax malaria in China [
16]. Molecular markers have been validated as tools for surveillance of resistance. Analysis of point mutations in these marker genes thus serves as a valuable molecular approach for mapping drug resistance and monitoring malaria-control measures [
30]. In this study, the profile of mutations in marker genes associated with CQ and antifolate drug resistance among the
P. vivax parasites obtained from patients of the subtropical (Xishuangbanna in Yunnan) and temperate (Bozhou in Anhui) zones of China were determined to better understand the current and changing patterns of CQ- and SP-resistant
P. vivax in different malaria-endemic areas. The
P. vivax parasite populations in subtropical and temperate zones were shown to differ dramatically in
pvdhfr,
pvdhps, and
pvmdr-1 allele frequencies, i.e.,
P. vivax populations in subtropical zone are mostly resistant to SP and are likely more tolerant to CQ, whereas the majority of
P. vivax populations in the temperate zone are still effectively susceptible to SP and CQ.
The long history of CQ use has exposed
P. vivax to this drug pressure continually in China. Clinical failures after standard CQ treatment were reported in four cases of
P. vivax malaria in Yunnan province in 1996 [
14]. A trend of gradual decline of
in vitro sensitivity to CQ had also been documented between 2005 and 2008 in some areas of China, especially at the Yunnan-Myanmar border and in central China [
31], and reduced efficacy of CQ combinations for treating vivax malaria patients from 2007 to 2008 was reported at the Yunnan-Myanmar border [
13]. The relatively high frequency (9%) of
pvmdr-1 Y976F mutation among the Xishuangbanna isolates is consistent with the previous reports of declining sensitivity to CQ. The Y976F mutation had been reported in
P. vivax isolates from many malaria-endemic regions around the world, including Indonesia [
26], Thailand [
32], Myanmar [
32], and Mauritania [33, and is associated with an increase in CQ IC
50 values
in vitro[
15]. Cases of CQ-resistant
P. vivax malaria have been reported in the areas of Yunnan bordering Myanmar, Laos, and Vietnam [
8,
10]. The long history of CQ use, as well as frequent population movement across the borders, may contribute to the CQ-resistant
P. vivax detected in Xishuangbanna. However, the Y976F mutation was not detected in any Bozhou isolates, which is consistent with the results showing that CQ is still effective in killing
P. vivax parasites from the temperate zone in an
in vitro schizont maturation inhibition assay [
15]. The presence of Y976F in
pvmdr-1 in Xishuangbanna isolates suggests a trend toward decreased CQ sensitivity.
This study detected the F1076L mutation in
pvmdr-1 in 100% of Bozhou isolates, which is consistent with previous reports from Central China (100%) [
26], the Republic of Korea (100%) [
26], Mauritania (100%) [
26], and Madagascar (100%) [
34]; however, only 36% of Xishuangbanna isolates had the F1076L mutation. It has been reported that the F1076L substitution may not be linked to drug resistance but may rather be a geographic variant [
34].
SP inhibits the activities of
dhps and
dhfr in the folate biosynthesis pathway of both
P. falciparum and
P. vivax parasites, resulting in a synergistic anti-malarial effect [
35]. Resistance to antifolate drugs in both parasite species is found to be involved with point mutations in
dhps and
dhfr[
23]. Although
P. vivax infections are not generally treated with SP, the parasite is often exposed to the drug due to mixed infections and misdiagnosis [
36]. In this study, five mutations at amino acid positions 57, 58, 61, 99, and 117 and seven mutated alleles in
pvdhfr were detected in Xishuangbanna isolates, while only mutations at positions 99 and 117 of
pvdhfr and three mutated alleles in
pvdhfr were found in Bozhou isolates. It has been reported that double mutation S58R and S117N in
pvdhfr may first arise under drug pressure and move toward development of resistance to SP [
23]. Interestingly, approximately 62% of Xishuangbanna isolates was found to have double mutations at S58R/S117N, whereas no Bozhou isolates were detected with these mutations. Instead, 39% of Bozhou isolates had a single S117N mutation, indicating that the
P. vivax isolate circulating in Xishuangbanna may be under stronger drug pressure than those in Bozhou. The most prevalent double mutations of S58R/S117N in Xishuangbanna isolates were similar to those in previous reports from East Timor [
37], the Philippines [
37], Vietnam [
37], and Pakistan [
38]. Clinical studies have shown that patients carrying triple and quadruple
pvdhfr mutant vivax parasites were more likely associated with SP treatment failure than those with WT parasites [
39,
40]. The triple and quadruple mutations in
pvdhfr have been detected in Yunnan [
16], Hainan [
16], and Guizhou [
41] provinces of China’s subtropical zone, and in some other countries including Indonesia [
40], Papua New Guinea [
40] and India [
42]. Similarly, 61% of Xishuangbanna isolates had quadruple mutant alleles (57L/58R/61M/117T and 57I/58R/61M/117T), strongly suggesting that many Xishuangbanna
P. vivax isolates are resistant to sulphadoxine drugs. The triple and quadruple mutations were not detected in isolates in Bozhou as well as in other temperate areas of China (such as Wuhe county in Anhui province) [
41], Thailand [
43], Vietnam [
43], Korea [
43], Afghanistan [
44], Pakistan [
45], and Iran [
46], indicating that Bozhou
P. vivax isolates may still be sensitive to antifolate drugs.
Among mutated codons in
pvdhfr, the T61M mutation was mostly linked to the S117T mutation [
23]. In the present study, two mutations at T61M/S117T with quadruple mutated alleles (57L/58R/61M/117T and 57I/58R/61M/117T) were detected in Xishuangbanna isolates. The observation is consistent with the early reports from Thailand [
47], but is in contrast to other reports from the temperate zone of China (Wuhe county in Anhui province) that most T61M mutations were found as single mutations and arose independently [
41]. In the current study, the three-copy repeat type was the predominate type in both Xishuangbanna (79%) and Bozhou (97%) isolates, which was similar to the findings from India [
42], Afghanistan [
44], Iran [
46], and Wuhe county in Anhui province and Luodian county in Guizhou province of China [
41]. The association between
pvdhfr mutations and tandem repeat polymorphisms has been extensively investigated and used to predict the prevalence of drug-resistant malaria around the world [
43]. Therefore, the observations from this study suggest increasing prevalence of antifolate-resistant parasites in the Xishuangbanna region. Additionally, no isolates were detected with the four-copy repeat type from either Xishuangbanna or Bozhou, in contrast to other reports showing that quadruple-mutant
dhfr alleles were exclusively associated with the four-copy repeat type in Thailand [
23], India [
42], and Myanmar [
48]. Whether the four-copy repeat type contributes to drug resistance requires further investigation.
It has been suggested that mutations at codons 382, 383, 512, 553, and 585 in
pvdhps gene are related to reduced sensitivity to sulfadoxine [
46,
49]. In the present study, four
pvdhps mutations at codons S382A (32%), A383G (79%), K512E (2%), and A553G (28%) and five mutant-allelic types were detected in Xishuangbanna isolates, including 25% single-mutant allele (A383G), 47% double-mutant allele (S382A/A383G and A383G/A553G), and 8% triple-mutant allele (S382A/A383G/A553G and A383G/K512E/A553G), strongly suggesting that Xishuangbanna
P. vivax isolates were highly resistant to sulphadoxine. The high prevalence of mutant
pfdhps alleles is similar to a previous report in Yunnan province of China (Nu River) [
16]. In contrast, all isolates from Bozhou carried the WT allele, which is similar to reports from Wuhe county in Anhui province [
31,
41] and Luodian county in Guizhou province [
41] of China, Mauritania [
33], Iran [
38], and Afghanistan [
44]. The absence of mutations in
pvdhps in this study indicated that
P. vivax populations in Bozhou may still be effectively susceptible to sulphadoxine.
Molecular epidemiologic studies in different areas have shown dramatically different mutation rates in
pvdhfr and
pvdhps, which may be attributed to different drug-selection pressures or the intrinsic differences among endemic strains of
P. vivax. It is important to take into account the presence of A383G mutation of
pvdhps along with double
pvdhfr mutations, as SP drug-treatment failure was more frequently associated with multiple mutations in
pvdhfr and
pvdhps[
50]; when the
P. falciparum parasite carries mutant alleles of
dhfr and
dhps, clinical effectiveness of SP is compromised [
51]. In the current study, disregarding the mutations at codon 99 of
pvdhfr, which is not considered to be related to the anti-malarial drugs, 31.6% of 114 isolates (including Types 8, 14 to 19, and 21 to 28) contained the mutations in
pvdhfr and
pvdhps and 4.4% of 114 isolates (including Types 16, 19, and 26) mutated simultaneously in the three drug-resistant genes
pvdhfr,
pvdhps, and
pvmdr-1 were found in the Xishuangbanna isolates but not in Bozhou isolates. Although SP was not recommended to treat
P. vivax malaria in China due to the intensive use of antifolate drugs for treating
P. falciparum infections,
P. vivax has been under SP drugs pressure through mixed infections and/or incorrect diagnosis in Yunnan. In contrast, without the introduction of sulphadoxine in Anhui—where
P. vivax is the only or most predominant malaria parasite,
P. vivax is not under sulphadoxine but rather pyrimethamine drug pressure. Therefore, the different usage of antifolate drug in Yunnan and Anhui may lead to the high prevalence of
pvdhfr and
pvdhps mutated alleles in Xishuangbanna isolates and relatively low prevalence of these mutations in Bozhou isolates. In tropical area, in addition to longer transmission period, short-term relapses are more common (1–2 months), leading to increased expose of
P. vivax parasites to sub-therapeutic concentrations of SP and CQ. Regardless, the results from this study indicates that the high prevalence of multiple mutations of
pvdhfr,
pvdhps, and
pvmdr-1 genes may further reduce the sensitivity to SP and CQ in
P. vivax populations in Xishuangbanna of Yunnan, while the
P. vivax population may still be susceptible to SP and CQ in Bozhou of central China due to the absence of multiple mutations in
pvdhfr,
pvdhps, and
pvmdr-1.
Authors’ contributions
XZS and FL designed the study and wrote the manuscript. BH, SH, and HL carried out the field work. BH and SH performed experiments, analysed data, and wrote the manuscript. XT and JY performed the preliminary data analysis. All authors read and approved the final manuscript.