Prevalence of antifolate drug resistance markers in Plasmodium vivax in China

Abstract

The dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes of Plasmodium vivax, as antifolate resistance-associated genes were used for drug resistance surveillance. A total of 375 P. vivax isolates collected from different geographical locations in China in 2009–2019 were used to sequence Pvdhfr and Pvdhps. The majority of the isolates harbored a mutant type allele for Pvdhfr (94.5%) and Pvdhps (68.2%). The most predominant point mutations were S117T/N (77.7%) in Pvdhfr and A383G (66.8%) in Pvdhps. Amino acid changes were identified at nine residues in Pvdhfr. A quadruple-mutant haplotype at 57, 58, 61, and 117 was the most frequent (57.4%) among 16 distinct Pvdhfr haplotypes. Mutations in Pvdhps were detected at six codons, and the double-mutant A383G/A553G was the most prevalent (39.3%). Pvdhfr exhibited a higher mutation prevalence and greater diversity than Pvdhps in China. Most isolates from Yunnan carried multiple mutant haplotypes, while the majority of samples from temperate regions and Hainan Island harbored the wild type or single mutant type. This study indicated that the antifolate resistance levels of P. vivax parasites were different across China and molecular markers could be used to rapidly monitor drug resistance. Results provided evidence for updating national drug policy and treatment guidelines.

Keywords

drug resistance / antifolates / molecular markers / Plasmodium vivax / China

1 Introduction

Plasmodium vivax is the most geographically widespread among the five Plasmodium species that cause human malaria [1]. According to the latest world malaria report, about 229 million malaria cases were reported in 2019 in 87 malaria-endemic countries and approximately a quarter of the cases were due to P. vivax [2], which was mainly distributed in Southeast Asia. In China, P. vivax has been the major Plasmodium spp. for several decades. In particular, an epidemic re-emergence and outbreak of P. vivax occurred in 2006 on the Huanghuai Plain in Central China, where Anopheles sinensis mosquitoes were prevalent [3,4]. Malaria cases have declined to a low level due to targeted strategies and measures, including timely diagnosis and standard treatment; integrated measures consisting of medication for risk groups, vector control, and health education were implemented from 2010 to 2015 [5].

Antifolate drugs comprise compounds that bind to enzymes involved in parasite folate biosynthesis, and the most widely used drug combination within this class is sulfadoxine–pyrimethamine (SP) [6]. To date, SP has been mainly used as an intermittent preventive treatment for infants and pregnant women in Africa [7,8] and has been rarely used for P. vivax treatment. However, SP remains in use to treat patients with uncomplicated P. falciparum infections in few counties where P. vivax is co-endemic with P. falciparum [9]. For example, SP plus artesunate serves as first-line treatment for uncomplicated chloroquine-resistant falciparum in most areas in India as well as in Iran, Saudi Arabia, Yemen, and Sudan [10]; as such, the P. vivax parasite remains under SP drug selection pressure. In China, pyrimethamine was used for prophylaxis [11] and was combined with primaquine (PQ) for radical treatment of P. vivax [12]. According to the latest national antimalarial drug policy in China [13], chloroquine (CQ) plus PQ is used to treat P. vivax; piperaquine (PPQ), pyronaridine, or artemisinin combination therapies (ACTs) plus PQ are used to treat patients infected with CQ-resistant P. vivax.

Clinical, epidemiological, molecular, and biochemical studies have shown the genetic basis of SP resistance, which is caused by mutations in two genes, namely, the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes. Point mutations associated with pyrimethamine and sulfadoxine resistance have been identified in P. vivax dhfr (Pvdhfr) at codons 51, 58, 117, and 173 and in P. vivax dhps (Pvdhps) at codons 382, 383, and 553 [14,15].

A high prevalence of mutations in Pvdhfr and Pvdhps has been reported in Asia [16–21], Africa [22], and Oceania [23]. Studies involving molecular surveillance of P. vivax resistance are limited in China. The present study investigated the prevalence of Pvdhfr and Pvdhps in field isolates of P. vivax from Liaoning, Jiangsu, Tibet, Yunnan, and Hainan provinces in China. Results provide evidence of the level of SP resistance in P. vivax in China.

2 Materials and methods

2.1 Study site

Blood samples containing P. vivax were collected from Dandong, Suining, Nyingchi, Yingjiang, Tengchong, and Hainan. Dandong City in Liaoning Province, which is located in the northeastern part of China, ranges in latitude from 39°43′ to 41°09′ N and in longitude from 123°22′ to 125°41′ E. Dandong City is the largest border city in China, facing DPR Korea across the Yalu River. Suining County in Jiangsu Province is located in the Huanghuai Plain of Central China at 33°56′27.6″ N and 117°51′10.8″ E and has a typical temperate continental climate. Nyingchi, known as Linzhi, is a prefecture-level city in the southeastern part of Tibet at coordinates of 29°38′55.68″ N and 94°21′41.04″ E and has climate defined as subtropical highland. Yingjiang and Tengchong counties in Yunnan Province are located in the southwestern part of China, which border Kachin State, Myanmar. These two counties are mountainous with several alluvial plains and have various climate types. The border has no barrier, and a large number of immigrants cross the border frequently. Hainan is an island province in China and the nation’s southernmost point; the climate in Hainan varies from subtropical to tropical.

2.2 Sample collection and DNA extraction

Blood was collected from P. vivax-infected patients from different geographical locations in China in 2009–2019 prior to antimalarial drug treatment. The blood samples were spotted on filter paper (Whatman™ 903, GE Healthcare, USA) and dried. Microscopic examination of Giemsa-stained thick smears was used to diagnose malaria. Parasite DNA was extracted using a QIAamp DNA Mini Kit (Valencia, CA, USA) as described by the manufacturer. A nested polymerase chain reaction (PCR) for amplifying the small-subunit rRNA gene of Plasmodium spp. [24] was performed to confirm the positive samples and identify the species before sequencing Pvdhfr and Pvdhps genes.

2.3 Nested PCR and sequencing

The primers and cycling conditions for nested PCR and the sizes of the PCR products are shown in Table S1. The PCR products were purified using filter plates (Edge Biosystems, Gaithersburg, MD, USA), directly sequenced, and analyzed on an ABI 3730XL automatic sequencer. The amplification products were analyzed by 1.5% agarose gel electrophoresis before sequencing. Bidirectional sequencing was performed, and all independently amplified PCR products were sequenced twice.

2.4 Data analysis

The output sequence data were assembled, edited, aligned using Geneious (version 2021.0.3) software, and compared with the reference sequences of Pvdhps (accession number: XM001617159) and Pvdhfr (accession number: X98123) from GenBank. The mixed alleles were determined according to the emergence of two chromatogram peaks at one nucleotide site through Mutation Surveyor (Soft Genetics LLC., version 5.1, State College, PA, USA). All the detected single-nucleotide polymorphisms (SNPs) of Pvdhps and Pvdhfr were combined for principal component analysis (PCA) using the EIGENSOFT program [25]. SAS software (SAS Institute Inc, Version 9.2, Cary, NC, USA) was used for data processing and statistical analysis. Chi-square test and Fisher’s exact test were used to evaluate differences among subgroups. A P value<0.05 was used to identify differences with statistical significance.

3 Results

3.1 Pvdhfr haplotype

With respect to the Pvdhfr gene, 291 P. vivax isolates were sequenced successfully. Amino acid changes in Pvdhfr at residues 13, 15, 57, 58, 61, 76, 99, 117, and 173 were detected in 2.1%, 0.3%, 61.5%, 76.3%, 59.5%, 0.3%, 7.9%, 77.7%, and 1.0% of the sequences, respectively (Table 1). The majority of the isolates carried at least one mutation in Pvdhfr (94.5%, 275/291), whereas 16 of the 291 isolates were identified as containing wild-type Pvdhfr. The most predominant point mutation was S117T/N (77.7%), followed by S58R (76.3%), F57L/I (61.5%), and T61M (59.5%). However, the I13L and I173L mutant alleles were rare and detected in only six and three isolates, respectively. The prevalence of wild-type Pvdhfr varied significantly in different geographical sites (P<0.001). Most samples from Suining (10/16) harbored the wild type, while only few isolates harbored the wild type in Yingjiang (3/58) and Tengchong (2/204) counties in Yunnan Province. Additionally, two novel point mutations, namely, A15V and R76G, which have not been reported, were detected.

The haplotype analysis revealed 16 distinct allelic forms from all samples (Table 2). The single-mutant haplotypes S58R and S117N and the double-mutant alleles S58R/S117N and F57L/S58R had relatively low prevalence and were identified in two, one, four, and four isolates, respectively. Another single-mutant haplotype, namely, H99S, which is not associated with pyrimethamine resistance, was observed in 21 P. vivax isolates. A quadruple-mutant haplotype, I₁₃A₁₅L₅₇R₅₈M₆₁R₇₆H₉₉T₁₁₇I₁₇₃ (29.2%) was the most common mutant form, followed by the quintuple-mutant haplotype I₁₃A₁₅L₅₇R₅₈M₆₁R₇₆S₉₉T₁₁₇I₁₇₃ (28.2%). Triple-mutant haplotypes were not observed in this study.

3.2 Tandem repeat variation in Pvdhfr

Variations in the central tandem repeat region between the amino acid positions 88 and 103 of Pvdhfr were identified (Fig. 1). Three types of tandem repeat variation were observed. Type 1 contained three copies of GGDN repeats, the same as the reference strain (accession number: X98123.1), and was designated as the wild type. Type 2 also had three copies of GGDN repeats but showed a mutant allele at codon 99. Type 3 lacked six amino acids from positions 98 to 103. Approximately 40% (118/291) of samples were Type 1, and 35.4% (103/291) were Type 2. Except for one isolate from Dandong, which carried a single mutant H99S, Types 1 and 2 were detected only in samples from Yunnan Province. By contrast, Type 3 was present in 70 P. vivax samples with a deletion of 18 nucleotides from amino acid positions 98 to 103. Interestingly, when combined with other point mutants, Type 3 comprised various mutant alleles. The majority were I₁₃A₁₅F₅₇R₅₈T₆₁R₇₆_T₁₁₇I₁₇₃ (51.4%, 36/70), whereas I₁₃A₁₅F₅₇S₅₈T₆₁R₇₆_S₁₁₇I₁₇₃, I₁₃A₁₅F₅₇S₅₈T₆₁R₇₆_N₁₁₇I₁₇₃, I₁₃A₁₅F₅₇R₅₈T₆₁R₇₆_N₁₁₇L₁₇₃, I₁₃A₁₅L₅₇S₅₈T₆₁R₇₆_S₁₁₇I₁₇₃, and I₁₃V₁₅F₅₇S₅₈T₆₁R₇₆_T₁₁₇I₁₇₃ were found in 20, eight, three, two, and one isolates, respectively. Most samples (85.6%) from Dandong were Type 3 and differed significantly from the samples from the other regions.

3.3 Pvdhps haplotype

Polymorphisms were successfully assessed in the Pvdhps gene from 349 P. vivax samples. Nonsynonymous mutations of the Pvdhps gene were detected at codons 382, 383, 399, 512, 553, and 571; point mutations at codons 580 and 585 were not observed. Four synonymous mutations (codons 402, 495, 554, and 561) were detected. The wild-type Pvdhps was found in all sites, with a total number of 111, the majority of which were found in Suining (16/16), Nyingchi (23/25), Hainan (24/26), and Dandong (5/8) and few in Tengchong (14/216). Nine different mutant haplotypes were observed, consisting of two single-, three double-, three triple-, and one quadruple-mutant haplotypes (Table 2). In comparison with the wild-type Pvdhps (31.8%), the most prevalent haplotype was the double-mutant allele S₃₈₂G₃₈₃M₃₉₉K₅₁₂G₅₅₃E₅₇₁ (39.3%, 137/349), which was detected only in Yunnan Province, mostly in Tengchong County (129/137). The single-mutant allele A383G was common in Yingjiang (20/58) but rare in Tengchong, with only one isolate each from Nyingchi and Hainan Island. M399I, a novel mutation in the Pvdhps gene detected in this study, was observed in three, one, and one isolates from Dandong, Nyingchi, and Hainan Island, respectively. Multiple-mutation haplotypes were mostly observed in Yunnan Province and comprised double mutants (S382C/A383G, A383G/E571Q, A383G/A553G), triple mutants, and quadruple mutants at codons 382, 383, 512, and 553.

In addition, two isolates with the mixed allele A553A/G were identified, and the mutated alleles at codon 512 showed three different types, K512T/M/E, each of which carried the double-mutation A383G/A553G.

3.4 Analysis of allelic combinations of Pvdhfr and Pvdhps

Allelic combinations of Pvdhfr and Pvdhps genes are responsible for the biosynthesis of folate and are potentially under similar drug group pressure. A total of 280 P. vivax isolates were sequenced successfully for both genes. The combinations of point mutations in Pvdhfr and Pvdhps were analyzed at codons 57, 58, 61, 117, and 173 and at codons 382, 383, and 553, respectively. Thirty-three haplotypes were identified, whereas 13.2% harbored the wild type. The hexaploidy-mutant alleles I₅₇R₅₈M₆₁T₁₁₇/G₃₈₃G₅₅₃ and L₅₇R₅₈M₆₁T₁₁₇/G₃₈₃G₅₅₃ were the two most common haplotypes, with frequencies of 18.2% and 17.5%, respectively. The quintuple-mutant haplotype L₅₇R₅₈M₆₁T₁₁₇/G₃₈₃ and the quadruple-mutant R₅₈N₁₁₇/G₃₈₃G₅₅₃ were present at a low prevalence rate of 5%. The single-mutant A383G in Pvdhps, the single-mutant S117N, and the double-mutant S58R and S117N in Pvdhfr and wild-type Pvdhps were detected in 6.1% (17/280), 2.5% (7/280), and 4.3% (12/280) of the samples, respectively. Rare haplotypes included single or double mutations at positions 382 and 383 of Pvdhps in combination with multiple Pvdhfr mutations at positions 57, 58, 61, 117, and 173, which were found in few isolates. One P. vivax sample from Yingjiang County showed the haploid-mutant allele I₅₇R₅₈M₆₁T₁₁₇/C₃₈₂G₃₈₃T₅₁₂G₅₅₃.

3.5 Geographical distribution of Pvdhfr and Pvdhps haplotypes

With regard to the different locations of isolates, Dandong, Suining, and Nyingchi, which are located in northeastern, central, and southwestern China, respectively, are classified as temperate regions, whereas Yingjiang and Tengchong in southwest of China and Hainan Island in southern China are subtropical regions. Isolates from temperate regions were dominated by wild-type Pvdhfr and Pvdhps, whereas multiple-mutation haplotypes, consisting of quadruple and quintuple mutants of Pvdhfr as well as double, triple, and quadruple mutants of Pvdhps, were all from subtropical regions. The geographical distributions of various haplotypes observed in Pvdhfr and Pvdhps are shown in Table 2. When the haplotypes of Pvdhfr and Pvdhps were combined, isolates from Yingjiang and Tengchong counties in Yunnan Province revealed great diversity and 30 distinct mutant alleles were observed. Among them, the hexaploidy-mutant alleles I₅₇R₅₈M₆₁T₁₁₇/G₃₈₃G₅₅₃ (8.2%) and L₅₇R₅₈M₆₁T₁₁₇/G₃₈₃G₅₅₃ (17.5%) were all from the two counties. The single-mutant Pvdhfr at positions 57 and 117 combined with the wild-type Pvdhps was observed only in isolates from Dandong. Only one isolate from Nyingchi was successfully sequenced and showed the wild type, while three samples from Hainan Island carried double mutations of Pvdhfr and wild-type Pvdhps R₅₈N₁₁₇/WT (Table 3).

The use of PCA on all the detected SNPs in Pvdhfr and Pvdhps showed the first PC on the horizontal axis and the second and third PCs on the vertical axis. The samples were colored according to their geographical location. The PCA results indicated a distinction among the isolates. Parasites from Yunnan Province were clustered and separated from the isolates from Hainan Island and from temperate regions, including Liaoning, Jiangsu, and Tibet (Fig. 2).

4 Discussion

The combination SP is the most commonly used antimalarial treatment in almost all malaria-endemic areas between the 1960s and the 1980s [26] and remains in use to treat patients with P. falciparum infections in few counties [10] and as an intermittent prophylactic treatment for infants and pregnant women in Africa [7,8]. Prior to the introduction of SP combination therapy, pyrimethamine resistance in 1953 and sulfadoxine resistance in 1957 had already been reported [27]. By the early 1990s, SP resistance-related mutations in dhps and dhfr were documented [28] and point mutations in Pvdhps and Pvdhfr, which are associated with SP resistance, were well known [14,18,19,29]. Molecular markers for drug resistance have been recommended for antimalarial drug resistance surveillance by the WHO [30].

Soft and hard selective sweeps have occurred with striking differences in the Pvdhfr haplotype among P. vivax isolates [31]. Six point mutations in Pvdhfr associated with pyrimethamine resistance were identified, namely, P33L, F57L/I, S58R, T61M, S117N/T, and I173F/L, corresponding to residues A16V, C50R, N51I, C59R, S108N, and I164L in Pfdhfr [32]. Additionally, mutations in Pvdhfr at codons 57 and 61 in combination with those at codons 58 and 117 have been reported to be correlated with SP treatment failure [29]. Indeed, we found that the frequencies of the single-mutant haplotype at S58R or S117N and the double-mutant S58R/S117N were relatively low in all sites, whereas the quadruple-mutant haplotype L₅₇R₅₈M₆₁T₁₁₇ (29.2%) and quintuple-mutant haplotype L₅₇R₅₈M₆₁R₇₆S₉₉T₁₁₇ (28.2%) associated with SP treatment failure were highly prevalent in two sites in Yunnan Province. Moreover, most samples from the three sites in temperate regions were revealed as wild-type or single-mutant haplotypes. The only three isolates from Hainan Island all carried the double-mutation S58R/S117N, indicating high resistance, moderate resistance, and sensitivity to pyrimethamine in Yunnan, Hainan and intemperate regions, respectively. When compared with reports from other countries, the prevalence of double or quadruple mutations in China is lower than that in Myanmar [33], Thailand [16], and Cambodia [18]. This difference is related to the duration and dosage of pyrimethamine usage in different countries. Pyrimethamine was widely used for malaria prophylaxis in all malaria-endemic areas in China in the late 1950s and the early 1960s [34] and was added to salt for prophylaxis in the 1980s. Pyrimethamine combined with PQ was used for radical treatment of P. vivax in Central China 40 years ago [11]. Tandem repeat variants were identified in the Pvdhfr gene. The majority of the samples (221/291) had three copies of tandem repeats (Types 1 and 2) (Table 2), which commonly occurred with quadruple-mutant Pvdhfr alleles. Type 3 with a deletion of six amino acids usually coincided with the double-mutation S58R/S117N. However, the mechanism of the tandem repeat variants in pyrimethamine resistance remains unclear [17,35] because these variants are not present in the Pfdhfr gene in P. falciparum [36,37].

Mutations in the dhfr gene correlated with sulfadoxine resistance include S382A, A383G, K512E, A553G, and V585A in P. vivax and the corresponding mutations S436A/F, A437G, K540E, A581G, and A613S/T in P. falciparum [38]. In the present study, wild-type Pvdhps was predominant in temperate regions and in Hainan Island, with frequencies of 62.5% (5/8), 100% (16/16), 92.0% (23/25) and 92.3% (24/26) in Dandong, Suining, Nyingchi, and Hainan Island, respectively. Nevertheless, half of the samples (29/58) from Yingjiang and few isolates (14/216) from Tengchong showed wild-type Pvdhps. A large proportion of samples (233/349) carried the A383G mutation, followed by A553G (164/349). A383G and A553G are the two key mutations in Pvdhps and are strongly linked to sulfadoxine resistance. With respect to Pvdhps haplotypes, P. vivax isolates from temperate regions and Hainan Island showed only wild-type or single-mutant alleles, whereas the majority of the isolates from subtropical regions carried multiple mutations. The double-mutant A383G/A553G (39.3%, 137/349) was the most frequent haplotype; all mutant samples were detected in Yingjiang and Tengchong counties in Yunnan Province. These findings are in agreement with previous studies [11,39], which indicated that the level of pyrimethamine resistance was significantly different between temperate and subtropical regions in China. Compared with Pvdhfr, Pvdhps mutations exhibited less diversity and lower prevalence in China mainly due to the relatively limited use of sulfadoxine in China. According to published and unpublished data, sulfadoxine was not introduced in Central China [40,41], while the SP combination was widely used in Yunnan Province and some areas in Hainan Island [42]. This finding was fully verified by the present results. Some studies have shown that mutations in the Pvdhps gene were more than twice as likely to emerge in isolates with multiple mutations in the Pvdhfr gene [43]. In the present work, the double-mutation A383G/A553G with S58R/T61M/S117T was dominant among the combined mutations in Pvdhfr and Pvdhps [44,45]. These double mutations cause the disruption in the sulfadoxine binding site in P. vivax, similar to that in P. falciparum [38].

The prevalence and patterns of SP resistance varied among different regions in China, which may have been caused by the difference in genomic populations in different geographical areas [46–49] or drug pressure of sulfadoxine and pyrimethamine [39,50,51]. The PCA revealed that parasites from Yunnan Province were separated from those in the other sites, indicating limited barriers to gene flow in China. Moreover, behavior or gene changes in Anopheles strains or species could favor the subpopulation of P. vivax parasites and lead to changes in the parasite population structure [4,52], which should be considered. SP was used in Yunnan Province for over 20 years, starting in the early 1960s [42]. By contrast, pyrimethamine alone was used as prophylaxis in Central China during the same period [40,41] and sulfadoxine was not introduced in this region. In the 1980s, pyrimethamine is also added to salt for prophylaxis in China. In Hainan Island, SP in combination with artemisinin or PQ is used to treat infections caused by P. falciparum and CQ-resistant P. falciparum [51]. This wide usage of SP may have placed P. vivax under drug selection pressure, especially in subtropical regions (Yunnan Province and Hainan Island), where P. vivax and P. falciparum mixed-species infections are common. Therefore, the history of usage of the antifolate drugs sulfadoxine and pyrimethamine in different regions may be related to differences in drug pressure and resistance levels.

This study has limitations. First, a certain number of samples were not sequenced successfully because of DNA degradation, which caused the sample size to be small at some sites. Second, information or published data on the history of SP usage in China are very limited. Although SP was used widely in the last century in China, many data and results were not published or available, and the development of pyrimethamine resistance in P. vivax under natural field conditions in China may not be well described.

Antimalarial drug resistance has been the primary obstacle to global malaria elimination [53]. This study determined the molecular prevalence and patterns of Pvdhfr and Pvdhps mutations in different geographical regions and provided essential information on the level of resistance to SP in P. vivax in China. Another point of concern is that, although SP is not used in China, many malaria cases are imported from Africa and Southeast Asia, where SP is still used. Molecular markers for antimalarial drug resistance could be used to rapidly monitor the emergence and spread of drug resistance in imported malaria cases in China. In addition, this study provides evidence of antifolate drug resistance to inform national drug policy in China and update treatment guidelines.

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