• View in gallery

    Diagram of Plasmodium vivax pyrophosphokinase–dihydropteroate synthase (pppk-dhps) region. Boxes represent exons and lines represent introns. Numerals above each box are the exon number, and numerals below each box are the nucleotide coordinates of the exon. In the case of exon 2, pppk occupies nucleotides 272–1241, and dhps occupies nucleotides 1242–2283.

  • View in gallery

    Network diagram of P. vivax dhps and flanking sequences. Note that only haplotypes with more than one isolate are included in the diagram. In the online version, colored circles represent dhps and flanking haplotypes; lines represent mutational steps connecting haplotypes, and black nodes represent hypothetical ancestral haplotypes or haplotypes present in the population but not sampled. Color coding in the online version is as follows: wild type, blue; 383G, purple; 383A/383G, green; 383G/553G, yellow; 382C/383G/553G, orange, 382A/383G/553G, red. The number in parentheses below the country of origin and genotype label refers to the row occupied by the selected haplotype in Table 2. This figure appears in color at www.ajtmh.org.

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Assessment of the Origins and Spread of Putative Resistance-Conferring Mutations in Plasmodium vivax Dihydropteroate Synthase

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  • 1 Department of Genome Sciences, University of Washington, Seattle, Washington; College of Public Health Sciences, Chulalongkorn University, Bangkok, Thailand; Faculty of Medicine, University of Kelaniya, Ragama, Sri Lanka; Grupo Salud y Comunidad, Universidad de Antioquia, Medellín, Colombia; Faculty of Allied Health Sciences, Thammasat University, Pathumthanee, Thailand

Infection with Plasmodium vivax is usually treated with chloroquine, but parasites are often exposed inadvertently to sulfadoxine-pyrimethamine. To infer patterns of selection and spread of resistant parasites in natural populations, we determined haplotypes of P. vivax dihydropteroate synthase (dhps) alleles that could confer resistance to sulfadoxine. We amplified the P. vivax pyrophosphokinase (pppk)–dhps region and its flanking intergenic regions from 92 contemporary global isolates. Introns and exons of pppk-dhps were highly polymorphic, as were the flanking intergenic regions. Eighteen haplotypes were associated with wild-type alleles, but several different putatively sulfadoxine-resistant alleles have arisen in areas of intensive sulfadoxine-pyrimethamine use. Even when they encoded changes to the same amino acid, these mutant alleles were associated with multiple different haplotypes. Two main conclusions can be drawn from these data. First, dhps alleles resistant to sulfadoxine have arisen multiple times under drug pressure. Second, there has been convergent evolution of a variety of alleles that could confer resistance to sulfa drugs.

INTRODUCTION

Infections with Plasmodium vivax are generally treated with chloroquine, but incorrect species diagnosis, presumptive treatment of infections with P. falciparum, and mixed species infection frequently expose P. vivax populations to the antifolate sulfadoxine-pyrimethamine. Early studies suggested that P. vivax may be less susceptible to sulfa drugs than is P. falciparum, although sample sizes were generally extremely small and the sulfa drug was often not paired with an inhibitor of dihydrofolate reductase (DHFR).1 Reports of chloroquine resistance in some P. vivax populations have increased interest in alternative drug treatments for persons with P. vivax malaria, and several recent studies have shown that sulfadoxine-pyrimethamine is effective against P. vivax at least in some regions.2 Notably, a recent randomized clinical trial found that sulfadoxine-pyrimethamine was as effective as chloroquine against P. vivax in Pakistan and Afghanistan.3 These observations increase the potential importance of understanding the mechanisms and patterns of resistance to sulfadoxine-pyrimethamine in P. vivax populations.

The mechanisms of sulfadoxine-pyrimethamine have been intensively studied in P. falciparum and P. vivax. In both species, point mutations in the gene that encodes DHFR confer resistance to pyrimethamine. 2 , 4 6 In P. falciparum, non-synonymous single nucleotide polymorphisms (SNPs) that alter the amino acids within the active site of dihydropteroate synthase (DHPS) have been demonstrated to confer resistance to sulfadoxine.7 9 There is no system to directly test whether non-synonymous SNPs in the P. vivax dhps gene also confer resistance to sulfa drugs. However, Korsinczky and others 10 noted that in every P. vivax isolate assessed to date there is a valine at amino acid 585, 10 16 and suggested that this residue may be responsible for steric hindrance and concomitant reduction of sulfadoxine sensitivity. Based on a homology model, Korsinczky and others predicted additional amino acids in P. vivax DHPS that when mutated may play a role in sulfadoxine resistance. 10 It was proposed that polymorphisms in P. vivax codons 382, 383, 512, 553, and 585, homologous to P. falciparum codons 436, 437, 540, 581, and 613, respectively, may be associated with sulfadoxine resistance in P. vivax.

Polymorphisms in the P. vivax dhps gene have been assessed from a global sampling of isolates, 10 15 and mutations in codons 382, 383, 512, and 553 are more prevalent in regions with high versus low use of sulfadoxine-pyrimethamine. 15 These codons are homologous to those associated with sulfa drug resistance in P. falciparum and they have been observed exclusively in isolates carrying pyrimethamine-resistant dhfr alleles.10 , 11 , 14 , 15 These observations suggest that although valine at residue 585 may reduce the sulfadoxine sensitivity of P. vivax parasites relative to that of P. falciparum, mutations in additional P. vivax dhps residues further compromise sulfadoxine efficacy.

By assessing the emergence of putative sulfadoxine resistance-conferring mutations in the P. vivax dhps gene, it is possible to infer the mechanisms by which parasites resistant to sulfadoxine-pyrimethamine are selected and spread in natural populations. 17 This information is crucial to predict how resistance to other drugs, for which molecular markers have not yet been identified, may spread. Several groups have investigated the relatedness of resistance-conferring alleles of the P. falciparum dhps gene.18 20 Roper and others assessed three micro-satellites flanking the P. falciparum dhps gene and found that several haplotypes were associated with the single-mutant and double-mutant dhps alleles in Africa. 20 A more recent study has shown that there were five apparent origins of these sulfadoxine-resistant lineages in Africa. 21 A study from the Amazon region of South America found that the P. falciparum dhps triple mutant was associated with a single haplotype, 18 and a more recent study in Venezuela corroborated this observation. 19

To study this question in P. vivax, we determined the sequence of the P. vivax pyrophosphokinase (ppk)–dhps region and the flanking intergenic regions from 92 contemporary isolates from four regions: Colombia, Indonesia, Sri Lanka, and Thailand. We then constructed haplotypes based on the sequence data, and inferred the relatedness of the alleles that were identified. We have demonstrated that there have been multiple origins of P. vivax dhps alleles associated with sulfa drug resistance, and that there has been strong convergent evolution of these alleles that does focus on the codons predicted to confer sulfa drug resistance.

MATERIALS AND METHODS

Orientation of the P. vivax dhps gene.

P. vivax pppk-dhps resides on chromosome 14. A total of 497 basepairs lie between P. vivax pppk-dhps and the nearest upstream open reading frame, and 631 basepairs lie between P. vivax dhps and the nearest downstream open reading frame (www.tigr.org). Nucleotide coordinates in this report are based upon the El Salvadorian Sal I isolate, the reference sequence from The Institute for Genomic Research. There are two introns and three exons (Figure 1). The first exon spans nucleotides 1–126 and encodes PPPK; the first intron spans nucleotides 127–372. The second exon spans nucleotides 373–2283; nucleotides 373–1241 encode PPPK, and nucleotides 1242–2283 encode DHPS. Intron two spans nucleotides 2284–2432, and the third exon, encoding the remainder of the DHPS domain, spans nucleotides 2433–2549.

Sample sources and DNA extraction.

Genomic DNA was extracted from filter paper blotted with the blood of patients infected with P. vivax (Qiagen, Rockville MD). We amplified and sequenced the P. vivax pppk-dhps region and the entire flanking intergenic region (497 basepairs upstream/631 basepairs downstream) from 92 contemporary patient isolates (Colombia, n = 9; Indonesia, n = 35; Sri Lanka, n = 16; Thailand, n = 28) and from MR4 samples Panama (1966), Nicaragua (pre-1986), Pakchong (Thailand, 1972), and ONG (Vietnamese refugee who had spent time in Indonesia, collected 1980). 22 , 23

The dhfr haplotypes of all isolates included in this study have been previously reported. 24 The Colombian samples were collected from Urabá on the western coast of Colombia in 2005. The Indonesian samples are derived from two locations, Papua (n = 22) in 1996–1999 and Java (n = 13) in 2000, which are separated by approximately 3,200 km. The parent study from which these isolates have been derived is discussed elsewhere. 25 The Thai samples are derived from the Thailand/Cambodia border (n = 6) and from the Thailand/Myanmar border (n = 22) in 2005; the parent study has been described elsewhere. 14 , 15 The Sri Lankan samples are derived from the north part of the island (n = 12) and the southern part (n = 3) in 2003. In addition, there is one Sri Lankan isolate whose geographic origin is unknown.

All isolates were derived from studies that had been reviewed and approved by the appropriate local Institutional Review Board. These anonymous samples are in the exempt category of the University of Washington Human Subjects Review Board.

Amplification of P. vivax dhps and flanking region.

A nested polymerase chain reaction (PCR) protocol was used. Primer sequences are shown in Table 1. For the first-round amplification of the P. vivax dhps and flanking regions, 0.2 μL of FailSafe enzyme (Epicentre Technologies, Madison WI), 1 μL each of 20 μM S1120 and S1121, 2 μL of genomic DNA, 10 μL of FailSafe buffer E, and water were combined to give a total volume of 20 μL. The PCR protocol used modified hotstart, in which reactions are transferred to the PCR machine only when the machine has reached 94°C. For the first-round and second-round amplifications, the same protocol was used: 94°C for 3 minutes; five cycles at 94°C for 30 seconds, 67°C for 45 seconds, and 72°C for 4 minutes; five cycles at 94°C for 30 seconds, 65°C for 45 seconds, and 72°C for 4 minutes; five cycles at 94°C for 30 seconds, 63°C for 45 seconds, and 72°C for 4 minutes; three cycles at 94°C for 30 seconds, 61°C for 45 seconds, and 72°C for 4 minutes; three cycles at 94°C for 30 seconds, 59°C for 45 seconds, and 72°C for 4 minutes; three cycles at 94°C for 30 seconds, 57°C for 45 seconds, and 72°C for 4 minutes; five cycles at 94°C for 30 seconds, 55°C for 45 seconds, and 72°C for 4 minutes; with a final extension at 72°C for 5 minutes.

For the second-round amplification of P. vivax dhps and flanking sequences, 1 μL of FailSafe enzyme (Epicentre Technologies), 5 μL each of 20 μM S1122 and S1123, 10 μL of first-nested product (used without any cleanup), 50 μL of FailSafe buffer E, and water were combined to give a total volume of 100 μL. Products were purified by using the GeneClean kit (Qbiogene, Solon OH) following manufacturer’s instructions. Products were quantified by subjecting 5 μL of purified product to electrophoresis on a 0.7% agarose gel stained with ethidium bromide.

Sequencing.

Sequencing was performed using an ABI capillary sequencer (Applied Biosystems, Foster City, CA), and double coverage with independent primers was used (Table 1). Sequences were aligned and analyzed with Sequencher (Gene Codes, Ann Arbor, MI). Insertions and deletions were manually verified. Sequences were aligned with the Sal I reference sequence (www.tigr.org).

Data analysis.

The freely available program Network was used to generate median joining networks. 26 To aid in generation of Network diagrams, sequences were aligned with the commercially available program DNA Alignment (version 1.1.3.0; Fluxus Engineering, Suffolk, United Kingdom). Briefly, the median joining network enables visualization of the mutational paths that may have led to the observed data. Haplotypes are linked based on the assumption that mutations are more likely to derive from a more frequent haplotype and proceed to a less frequent haplotype. In interpreting network diagrams, circles represent haplotypes; the diameter of the circle is proportional to the number of isolates with that allele. The length of lines linking haplotypes is proportional to the number of mutational steps separating haplotypes. Nodes (black circles) represent hypothetical ancestral haplotypes linking the presently extant haplotypes, or may represent haplotypes that, although presently extant, were not sampled.

The Network diagram is especially helpful when trying to determine whether multiple haplotypes of a given P. vivax pppk-dhps allele are the result of independent origins or of mutations accumulating on an already established haplotype. In the case where multiple haplotypes of a given pppk-dhps allele are present as a result of independent mutations, the haplotypes will be widely dispersed on the Network diagram, separated by a large number of mutational steps. In the case where multiple haplotypes of a given P. vivax pppk-dhps allele are present as a result of mutations accruing on one genetic background (i.e., there is one origin of the allele of interest, with additional polymorphisms accumulating stepwise over time), the haplotypes will cluster and will be separated by a limited number of mutational steps.

For the presented Network diagram, sequences of all 96 isolates described in the Materials and Methods were included (92 patient isolates and 4 MR4 isolates). A multi-nucleotide in-frame repeat region, as first described by Menegon and others, was identified within the dhps coding region; a repeat region in this position has not been observed in P. falciparum.13 There were 8 repeat types, as described in Tables 2 and 3. The Network program is designed to assess the relatedness of alleles that differ by SNPs rather than indels. Therefore the repeats were recoded such that each repeat type was represented as only one SNP removed from any other repeat type. This factor is important because repeats are likely to be the result of the appearance or disappearance of large segments of DNA as a unit, rather than an addition or subtraction of one nucleotide at a time. By entering sequence data with repeats coded as variation of a single nucleotide relative to the wild type sequence, the Network software more accurately portrays the relationships between alleles.

RESULTS

Putative resistance-conferring mutations in the P. vivax dhps gene.

We first examined the dhps coding region, specifically focusing on codons linked to putative sulfadoxine resistance. Second, we examined the extended haplotype on which mutant dhps alleles reside to assess whether these putative resistance-conferring alleles have arisen once or many times.

We sequenced both the entire P. vivax pppk-dhps gene and the flanking intergenic region (497 basepairs upstream to 631 basepairs downstream) from this geographically diverse set of isolates. All of the isolates in our dataset carried a valine at P. vivax dhps codon 585. By homology to codon 613 in P. falciparum, this change has been postulated to confer significant sulfa drug resistance to all P. vivax isolates. If this change alone were sufficient to confer clinically significant resistance, then selection of alleles with further mutations might not be observed in response to sulfa drug pressure.

To assess this idea, we focused on P. vivax dhps codons 382, 383, 512, and 553, homologous to P. falciparum dhps codons 436, 437, 540, and 581, 12 because polymorphisms in these P. falciparum dhps codons have been implicated in sulfa drug resistance. Our dataset did not include any polymorphisms in P. vivax dhps codon 512, although a polymorphism in this codon has been observed in Thailand.15

Information about polymorphisms within the dhps coding region, the pppk region, the introns, and the flanking intergenic regions is shown in Table 2. Note that within exons of pppk-dhps, polymorphisms are displayed as amino acid changes. Within introns and flanking intergenic regions, polymorphisms are displayed as nucleotide changes. Because some polymorphisms within the exons of pppk-dhps encoded synonymous changes, some columns in Table 2 list the same amino acid as wild type and mutant. Cells describing these synonymous changes are colored blue, but also contain a one-letter amino acid abbreviation. For simplicity, note that in Table 2 and throughout the remainder of this report dhps alleles are referred to with the single letter amino acid abbreviation of codons 382, 383, and 553. For example, wild-type alleles at these three codons (carrying 382S/383A/553A) are referred to as SAA.

Of the 96 isolates examined, 40 isolates carried a dhps allele wild type at codons 382, 383, and 553. Among these isolates, the pppk-dhps gene contained synonymous and non-synonymous mutations and distinct repeat types within the dhps gene. The repeats are abbreviated as A through H in Table 2 and described in detail in Table 3.

Thirty-one isolates carried the SGA allele, with various polymorphisms seen within the pppk-dhps region in addition to the change at codon 383. Two different double-mutant alleles were identified: the AGA allele was carried by 1 (3.6%) of 28 Thai isolate, and the SGG allele was carried by 12 isolates, 1 (2.9%) of 35 from Indonesia and 11 (39%) of 28 from Thailand. Two triple-mutant alleles were observed, all carried by Thai isolates. The CGG allele was carried by 2 (7.1%) of 28 isolates, and the AGG allele was carried by 6 (2.1%) of 28 isolates.

Arrangement of these alleles according to their geographic origin is shown in Table 4. Significant clustering is obvious. Alleles with mutations in all three codons of interest were found only from at Thailand/Myanmar border, an area under historically high sulfadoxine-pyrimethamine pressure. In contrast, isolates from the Thailand/Cambodia border, Sri Lanka, Indonesia, and Colombia were either wild type at the three codons of interest or carried a single non-synonymous SNP at codon 383. These patterns reflect the sulfadoxine-pyrimethamine use in these regions 14 16 and support the idea that sulfadoxine does exert significant selection on P. vivax dhps and that mutations in these three codons augment resistance to sulfadoxine.

Relatedness of putative sulfa drug resistance-conferring alleles.

Our second goal was to infer the relatedness of these putatively sulfa drug–resistant alleles. The most straightforward approach is to compare the diversity of the extended haplotypes of alleles bearing mutations linked to sulfa drug resistance. Extended haplotypes, including polymorphisms within exons and introns of the pppk-dhps region and within flanking intergenic regions, are shown in Table 2. As expected, wild type alleles at codons of interest are associated with many haplotypes; this is true even for those that have similar geographic origins. For example, the Sri Lankan wild type alleles at all codons of interest are associated with six haplotypes. Additionally, there are three repeat regions within the P. vivax dhps gene represented among the Sri Lanka isolates. Overall, there were 18 extended haplotypes carried by the 40 isolates that were wild type at dhps codons 382, 383, and 553.

Thirty-one isolates carried the single mutant SGA allele. The nine Colombian isolates were all identical, but the five Thai isolates showed two haplotypes and there were seven haplotypes associated with the 17 Indonesian isolates. Eleven Thai isolates carried the double-mutant SGG allele with two haplotypes observed. These two haplotypes were fairly similar, differing only in polymorphisms of the second intron. One Indonesian isolate carried the SGG allele and was associated with a haplotype unlike the Thai SGG haplotypes. One Thai isolate carried the AGA allele (row 33) and was associated with a haplotype similar to that carried by one Thai isolate bearing the triply mutated AGG allele (row 40). These isolates were unique in carrying repeat type G.

All isolates bearing triply mutated dhps alleles were from Mae Sot in northwestern Thailand. Two triple-mutant alleles were identified, the CGG allele and the AGG allele. The two isolates with the CGG allele shared a single haplotype. The remaining six isolates carried the AGG allele, with three haplotypes observed; two of these haplotypes were closely related to each other and to the haplotype associated with the CGG allele. However, one AGG allele was quite divergent (row 40) and carried a novel non-synonymous mutation in dhps codon 619 and silent mutations in codons 622 and 623. Unlike the other isolates, this AGG allele carried no polymorphisms in the introns. As noted above, this unusual haplotype was similar to the haplotype associated with the Thai AGA double mutant (row 33).

Relationships between putative sulfa drug resistance–conferring alleles.

To better visualize relationships among these varied alleles of P. vivax pppk-dhps, we analyzed the data set using the Network program described in detail in the Materials and Methods. The diagram in Figure 2 summarizes this analysis. Inspection of the diagram enables several inferences to be drawn. First, as expected, the wild-type alleles and those that carried few mutations in the positions putatively important for resistance are associated with a wide variety of haplotypes, but most cluster fairly closely. The single mutant SGA allele was found in Colombia, Thailand, and Indonesia, and also is associated with a variety of haplotypes. On the Network diagram, these haplotypes also cluster together for the most part. One isolate from Indonesia (Table 2, row 26) is separated from the other isolates bearing the SGA allele by a large number of mutational steps on the Network diagram. This allele is clearly derived from a different mutational event than the other SGA alleles. However, although most SGA haplotypes cluster together, it is evident from Table 2 that a variety of polymorphisms within the dhps coding region, the pppk region, the introns, and the flanking regions differentiate these haplotypes. The substantial variation distinguishing the haplotypes with the SGA mutation suggests that these have arisen from at least two different origins.

Second, similar reasoning suggests that there are also at least two distinct origins of the double mutant SGG allele. Isolates bearing this allele were found in Indonesia and Thailand. The Thai isolates bearing the SGG allele (Table 2, rows 34 and 35) are clearly closely related, differing only in the second intron, and cluster in the diagram. In contrast, the lone Indonesian isolate bearing the SGG allele (Table 2, row 36) is associated with a haplotype different from that associated with Thai SGG alleles. The one AGA double mutant (Table 2, row 33) is distinct from the other double-mutant haplotypes, and most closely associated with a Thai isolate carrying the AGG triple mutant allele (Table 2, row 40).

From a practical point of view, it is most important to determine how often clinically relevant, highly mutant alleles have arisen. Are the current alleles in this category independent or are they all related, having spread from a single original origin? In this data set, all of the triple-mutant alleles were isolated from a single location (Mae Sot Thailand). Despite this limited geographic origin, there are two distinct AGG alleles. Five of the six AGG alleles carried a polymorphism at nucleotide 262 within the first intron and polymorphisms within the second intron (Table 2, rows 38 and 39), and the CGG triple-mutant shares this haplotype. In contrast, the haplotype of the sixth AGG isolate is clearly distinct (row 40). The unusual isolate also carried repeat type G within dhps, and novel non-synonymous mutations in dhps and pppk not observed in any other isolates. Not surprisingly, this unusual isolate clusters far from the other AGG isolates, highlighting the substantial mutational distances between these two sets of alleles. The haplotype of the AGG isolate in row 40 appears to be most closely related to the lone AGA double mutant isolate, also from Thailand (row 33).

Thus, we have identified numerous alleles bearing putative sulfadoxine resistance–conferring polymorphisms in isolates from Colombia, Thailand, and Indonesia. These mutations have arisen independently on a variety of genetic backgrounds. Most notably, the double-mutant SGG has two distinct origins, one in Thailand and one in Indonesia, and the triple-mutant AGG allele has two origins, both in Thailand.

P. vivax pppk protein.

Despite the bifunctional nature of the PPPK-DHPS protein, previous assessments of P. vivax have focused primarily on the codons within the dhps gene that are implicated in sulfadoxine resistance. 10 15 Because we were interested in complete haplotypes, we sequenced the entire P. vivax pppk-dhps gene from this geographically diverse set of isolates (Table 2). To our surprise, we identified a common polymorphism, M205I, that was present in all of the Thai isolates, and two polymorphisms, E132G and E292D, that were observed in single isolates from Indonesia. There was no association of these polymorphisms with putatively sulfadoxine-resistant domains of dhps, and the significance of these changes on the PPPK enzyme function is unknown.

DISCUSSION

The selection of resistance to sulfadoxine-pyrimethamine has been studied intensively in P. falciparum. In that species, alleles of the dhfr gene that confer high levels of resistance to pyrimethamine have evolved remarkably rarely, but have spread widely from their apparent foci in Southeast Asia and South America. 18 , 20 , 27 32 We have shown previously that pyrimethamine-resistant alleles of P. vivax dhfr appear to have been selected in numerous locations, and spread only locally, a different pattern from that seen in P. falciparum dhfr.24

In this study, we addressed two issues. First, has sulfadoxine exerted significant selection pressure on the P. vivax dhps gene, its presumed target? Second, if these alleles are selected by drug pressure, do resistant alleles arise in many locations, or in just a few locations and then spread as drug use increases?

Because there is no method for testing directly the sensitivity of P. vivax dhps to sulfa drugs or sulfones in vitro, Korsinczky and others used computational models to compare the P. vivax and P. falciparum DHPS enzymes. 10 Based on the structural homology of the two enzymes, they proposed that P. vivax may be less susceptible to sulfadoxine than is P. falciparum because of valine found ubiquitously at P. vivax dhps codon 585. Our data are consistent with this idea, but also demonstrate that mutations in P. vivax dhps codons 382, 383, and 553 augment sulfadoxine resistance. Mutations in these P. vivax dhps codons correspond to positions that have been shown clearly to contribute to sulfa drug resistance in P. falciparum.8 , 33 , 34 Polymorphisms in these key P. vivax dhps codons have arisen repeatedly in a variety of global isolates, an observation that lends credibility to the contention that mutations in P. vivax dhps confer resistance to sulfadoxine in a manner similar to that observed in P. falciparum.

The contribution of these mutations to sulfa drug resistance is further supported by the fact that alleles carrying these P. vivax dhps mutations were more common in parasites from regions under high versus low sulfadoxine-pyrimethamine pressure. For example, all 22 P. vivax dhps alleles from the Thailand/Myanmar border carried mutations in one or more of codons 382, 383, and 553 (Table 4). This region has been subject to significant sulfadoxine-pyrimethamine pressure in the past, and the quadruple-mutant P. vivax dhfr allele is highly prevalent in this region. 14 , 15 In contrast, sulfadoxine-pyrimethamine has not been heavily used in Sri Lanka, and wild-type P. vivax dhfr and dhps predominate.16 In the present study, all Sri Lankan isolates carried dhps wild-type alleles at the codons of interest. Finally, in P. falciparum, mutant alleles of dhps are observed only among isolates that also carry a highly mutant P. falciparum dhfr,6 and this pattern is also observed in P. vivax.10 , 11 , 14 , 15 Taken together, these observations strongly support the idea that, as in P. falciparum, mutations in P. vivax dhps are associated with drug pressure from sulfadoxine-pyrimethamine use, and that these mutant alleles provide a selective advantage to the parasites bearing them.

If these mutations in P. vivax dhps confer resistance to sulfadoxine, then the haplotypes associated with mutant alleles may be compared to infer the relatedness of putative resistance-conferring alleles. The isolates that we studied came principally from Southeast Asia. Within this relatively restricted sample set, we found remarkable variation in the P. vivax pppk-dhps gene and in the flanking intergenic regions, and detected several novel non-synonymous polymorphisms in the pppk and dhps coding regions (Table 2). Most important, we found that double-mutant and triple-mutant alleles of P. vivax dhps have arisen on several distinct haplotype backgrounds. The SGG double-mutant allele has arisen twice, once in Thailand and once in Indonesia. The AGG triple-mutant allele has two distinct origins, both in Thailand. This diversity contrasts markedly with the relatively few haplotypes associated with highly mutant P. falciparum dhps alleles, even those from geographically distant sites in South America and Africa 18 21 and underscores the need for far more extensive examination of the genetic epidemiology of P. vivax.

Further studies will be needed to determine the frequency with which resistance-conferring mutations arise, and to identify the parameters that govern the spread of these mutations through P. vivax populations. Although molecular markers of chloroquine resistance have not been identified in P. vivax, chloroquine resistance has been selected, at least in Indonesia. 35 It is crucial to understand gene flow among P. vivax populations to predict how resistance to chloroquine and to other drugs may spread. Improved understanding of this topic will shed light on the poorly understood biology of P. vivax, and may have important implications for malaria control policy. 17

Table 1

Primers for amplification and sequencing of Plasmodium vivax pyrophosphokinase–dihydropteroate synthase and the flanking intergenic region*

Table 1
Table 2

Plasmodium vivax pyrophosphokinase–dihydropteroate synthase (pppk-dhps) and flanking haplotypes of contemporary global isolates (n = 92) and MR4 samples (n = 4)*

Table 2
Table 3

Plasmodium vivax dihydropteroate synthase repeat motif written as amino acid sequence*

Table 3
Table 4

Polymorphisms in Plasmodium vivax dihydropteroate synthase (Pvdhps) codons 382, 383, and 553 by country and region*

Table 4
Figure 1.
Figure 1.

Diagram of Plasmodium vivax pyrophosphokinase–dihydropteroate synthase (pppk-dhps) region. Boxes represent exons and lines represent introns. Numerals above each box are the exon number, and numerals below each box are the nucleotide coordinates of the exon. In the case of exon 2, pppk occupies nucleotides 272–1241, and dhps occupies nucleotides 1242–2283.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 81, 2; 10.4269/ajtmh.2009.81.348

Figure 2.
Figure 2.

Network diagram of P. vivax dhps and flanking sequences. Note that only haplotypes with more than one isolate are included in the diagram. In the online version, colored circles represent dhps and flanking haplotypes; lines represent mutational steps connecting haplotypes, and black nodes represent hypothetical ancestral haplotypes or haplotypes present in the population but not sampled. Color coding in the online version is as follows: wild type, blue; 383G, purple; 383A/383G, green; 383G/553G, yellow; 382C/383G/553G, orange, 382A/383G/553G, red. The number in parentheses below the country of origin and genotype label refers to the row occupied by the selected haplotype in Table 2. This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 81, 2; 10.4269/ajtmh.2009.81.348

*

Address correspondence to Carol Hopkins Sibley, Department of Genome Science, University of Washington, Seattle, WA 98195-5065. E-mail: sibley@u.washington.edu

Authors’ addresses: Vivian N. Hawkins, Stephanie M. Suzuki, and Carol Hopkins Sibley, Department of Genome Sciences, University of Washington, Seattle, WA 98195-5065. Kanchana Rungsihirunrat, College of Public Health Sciences, Chulalongkorn University, Bangkok Thailand and Faculty of Allied Health Sciences, Thammasat University, Pathumthanee Thailand. Hapuarachchige C. Hapuarachchi, Faculty of Medicine, University of Kelaniya, Ragama Sri Lanka. Amanda Maestre, Grupo Salud y Comunidad, Universidad de Antioquia, Medellín Colombia. Kesara Na-Bangchang, Faculty of Allied Health Sciences, Thammasat University, Pathumthanee Thailand.

Acknowledgments: Vivian H. Hawkins and Carol Hopkins Sibley thank Marnie R. Briceno, Berenger A. A. Ako, Marie-Solange Evehe, and Joseph D. Smith for critical review of the manuscript.

Financial support: This study was supported by grant AI 55604 from the National Institutes of Health to Carol Hopkins Sibley. Kanchana Rungsihirunrat was supported by a Golden Jubilee Scholarship.

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