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    Map of distribution of mutant pvdhfr alleles in Thailand. The pie charts show the proportions of wild-type and mutant pvdhfr alleles.

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    Map of distribution of mutant pvdhps alleles in Thailand. The pie charts show the proportions of wild-type and mutant pvdhps alleles.

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Geographical Distribution of Amino Acid Mutations in Plasmodium vivax DHFR and DHPS from Malaria Endemic Areas of Thailand

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  • 1 Faculty of Allied Health Sciences, Thammasat University, Pathumtani, Thailand; Department of Parasitology, Phramongkutklao College of Medicine, Thailand; Department of Genome Sciences, University of Washington, Seattle, Washington

Both malaria treatment and prophylaxis target the parasite dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) enzymes. Specific point mutations in these genes confer resistance to sulfadoxine-pyrimethamine (SP) in both Plasmodium falciparum and P. vivax. We used direct sequencing to examine the prevalence of point mutations in pvdhps and pvdhfr in 160 P. vivax isolates collected from areas along the international borders of Thailand. Results show that the majority of the isolates harbored a quadruple mutant allele of pvdhfr and a double mutant allele of pvdhps, but the distribution was not uniform. The highly mutant allele combination was especially prevalent along the Thai-Myanmar border, whereas the majority of the isolates from areas along the Thai-Cambodian and Thai-Malaysian borders carried double mutant alleles of pvdhfr and single mutant alleles of pvdhps. Novel mutations that have not been identified previously at codon 512 of pvdhps (K512M, K512E, K512T) were also found.

INTRODUCTION

Widespread resistance to antimalarial drugs is a major public health problem in the Southeast Asia Region including Thailand.1 Although annual reported malaria incidence in Thailand shows a downward trend, people in rural areas, especially along the Thai-Myanmar and Thai-Cambodian borders where multi-drug resistant P. falciparum malaria is highly prevalent, remain at great risk.2,3 Migration of foreign workers across the borders has been implicated in the spread of malaria transmission.4 The antimalarial combination sulfadoxine-pyrimethamine (SP, Fansidar®) was introduced to Thailand in 1972 as the first-line treatment of chloroquine-resistant Plasmodium falciparum malaria. Soon after its introduction for clinical use, resistance of the parasite to this drug was reported along the Thai-Cambodian border, and by 1982, resistance was widespread throughout the country.5,6

The two components of SP target dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) and point mutations within the genes that encode these enzymes are the primary cause of resistance to the drug.7 SP has not been the recommended treatment of P. vivax, but in Thailand, P. falciparum and P. vivax often co-exist with relative equal frequencies.8 As a result, P. vivax has often been exposed inadvertently to SP during treatment of P. falciparum, and this has caused a progressive selection of SP-resistant alleles in P. vivax as well.9 However, the extent of SP resistance in the P. vivax population has not been well defined.

Because different drug regiments are used for treatment of these two species, correct diagnosis is essential. Chloroquine plus primaquine remains the standard treatment of P. vivax infection in Thailand. Nevertheless, chloroquine-resistant P. vivax has recently been reported in some countries in Southeast Asia.10 Such resistance has not yet been reported in Thailand,11 although a trend of declining of in vitro susceptibility of the parasite to the drug has been noted during the past ten years. This emphasizes the urgent need to search for alternative treatments for P. vivax infection, possibly including drugs of the antifolate class.

Molecular studies have clearly demonstrated that resistance to sulfadoxine-pyrimethamine results from a series of sequential mutations in the parasite dihydropteroate synthase (dhps) and dihydrofolate reductase (dhfr) genes.9,1215 Mutations in dhfr and dhps have been intensively studied in P. falciparum whereas data on P. vivax dhfr and dhps are limited. Distribution of these alleles varies among different geographical regions and is related to the intensity of SP use for treatment of P. falciparum in the past. Highly mutant alleles are common in areas where SP has been intensively used, especially in Southeast Asia. The prevalence of pvdhfr and pvdhps mutations has been studied in many regions in Southeast Asia, including Indonesia, Papua New Guinea, India, and Sri Lanka but this information is very limited in some parts of the world (especially in Central and South America).16 In Thailand, most data have been drawn from Mae Sot, the highest malaria endemic area on the Thai-Myanmar border. It is essential to monitor the prevalence and diversity of pvdhfr and pvdhps mutant alleles in field isolates to understand the emergence of drug resistance and provide effective treatment in both P. falciparum and P. vivax.

Very early in the deployment of SP, Young and Burgess reported that P. vivax was “intrinsically resistant” to the drug, and that idea has persisted in the literature.17 However, recent work has demonstrated that SP is effective in patients infected with P. vivax that carry a wild-type genotype or a 58R/117N double mutant allele.18 Data from a randomized controlled trial demonstrated that SP and chlorproguanil-dapsone are still effective against P. vivax infection compared with chloroquine in Pakistan.19 These antifolates therefore may have a much greater potential use in areas of mixed infection than previously thought. Moreover, other antifolates such as drugs of the WR99210 class may be a novel strategy for treatment of P. vivax as parasites bearing highly pyrimethamine-resistant alleles of pvdhfr were still susceptible to WR99210.20 Because P. vivax cannot be maintained in culture, the assessment of the association between pvdhfr and pvdhps genotypes and its resistance to antifolates is still limited. However, the prevalence of alleles in pvdhfr and pvdhps associated with drug resistance can be used as surrogate markers to estimate the level of antifolate resistance in a P. vivax population. In the present study, we determined the prevalence and diversity of dhfr and dhps of P. vivax isolates collected from various endemic areas of Thailand along the international borders.

MATERIALS AND METHODS

Study areas and sample collection.

A total of 160 blood samples infected with P. vivax were collected during May–August 2005 from patients attending the malaria clinics in different geographical locations in Thailand along the Thai-Myanmar (10, 6, 44, 13, 13, and 31 isolates from Chiang Mai, Mae Hong Son, Tak, Kanchanaburi, Ratchaburi, and Ranong Province, respectively), Thai-Cambodian (9 and 4 isolates from Chanthaburi, and Trad Province, respectively) and Thai-Malaysian (30 isolates from Yala Province) borders. Two-hundred to 300 μL finger prick blood samples were collected and dotted onto filter paper (Whatman No. 3). The dried filter paper samples were stored in small plastic zip lock bags prior to the extraction of parasite DNA and analysis of parasite genotypes by the polymerase chain reaction and DNA sequencing. Giemsa-stained thin and thick blood smears were prepared and examined microscopically for the presence of P. vivax parasites. Approval of the study protocol was obtained from the Ethics Committees of the Ministry of Public Health, Thailand.

Parasite DNA extraction and pvdhfr and pvdhps amplification.

Parasite genomic DNA was extracted from individual dried blood spots on filter paper using a QIAamp DNA extraction mini-kit (QIAGEN) and used as template for PCR amplification. Primers were designed according to the published sequence of dhfr-ts (GenBank accession no. X98123) and dhps gene (GenBank accession no. AY186730) of P. vivax. pvdhfr was amplified by semi-nested PCR. The primary amplification was performed using a pair of outer primers (forward: 5′-atg gag gac ctt tca gat gta tt -3′ and reverse: 5′-cca cct tgc tgt aaa cca aaa agt cca gag-3′). This primary amplification product was then used for a second round of PCR using semi nested primers (forward: 5′-tac gcc atc tgc gca tg and and reverse: 5′-cca cct tgc tgt aaa cca aaa agt cca gag-3′). PCR was carried out in a total volume of 50 μL with the following reaction mixture: 0.1 μM of each primer, 2.5 mM MgCl2, 100 mM KCl, 20 mM Tris-HCl pH 8.0, 100 μM deoxy-nucleotides (dNTPs), 10–15 μL of genomic DNA in the primary PCR, and 1–2 μL of primary PCR product in semi-nested PCR and 0.5 unit of Taq DNA polymerase (Promega). PCR cycling parameters were as follows: initial denaturation at 94°C for 3 min, followed by 5 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min, then followed by 5 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min, and finally with 20 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min. pvdhps was amplified by nested PCR with a first round pair of PCR primers (forward: 5′-aaa gcg tag cga cag aag aac-3′ and reverse: 5′-ttg aaa cac gca tta tgg tat cg-3′) and in the second round pair of PCR primers (forward: 5′-ctc gcc atg ctc gta att tt-3′ and reverse: 5′-gag att acc cta agg ttg atg tat c-3′). PCR was carried out in a total volume of 50 μL with the following reaction mixture: 0.1 μM of each primers, 2.5 mM MgCl2, 100 mM KCl, 20 mM Tris-HCl pH 8.0, 100 μM deoxy-nucleotides (dNTPs), 10–15 μL of genomic DNA in primary PCR and 1–2 μL of primary PCR product in semi-nested PCR and 0.5 unit of Taq DNA polymerase (Promega). PCR was performed using 40 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min. The amplified PCR products were then analyzed on 1.5% agarose gel. The amplified products were purified using QIAquick PCR purification kit (QIAGEN) and sequenced by an automated DNA sequencer (ABI system). Sequence alignments and analysis was carried out using DNASTAR software (Lasergene).

RESULTS

Detection of mutations in the pvdhfr gene.

Sequence comparison revealed that 3 isolates carried the wild-type allele for pvdhfr; the remaining parasites carried mutant pvdhfr genotypes. Among these mutant isolates, the most common pvdhfr alleles were quadruple (59.4%) and double mutants (35.6%). Only one isolate carried a single mutation at codon 117, and 4 isolates carried triple mutations. The frequency of pvdhfr genotypes of the identified alleles are listed in Table 1. Double mutant alleles (58R/117N) were observed frequently in isolates collected from the Thai-Cambodian (Chanthaburi and Trad) and Thai-Malaysian (Yala) borders. Two quadruple mutant alleles of 57L/58R/61M/117T and 57I/58R/61M/117T were found commonly in isolates collected along the Thai-Myanmar border. A single mutant allele at codon 117 (S117T) was observed in only one isolate collected from Mae Hong Son and 2 mutant alleles (58R/61M/117T and 49G/61M/117N) were found in Tak. Figure 1 shows the location of each sampling areas and the pie charts indicate the proportions of wild-type and mutant pvdhfr alleles present in each location.

Detection of mutations in the pvdhps gene.

All identified pvdhps genotypes are listed in Table 2. The most prevalent pvdhps allele was an allele that differed from the reference sequence at two positions (383G/553G; 60%), followed by an allele with a single change (383G; 26.9%). Only 2 isolates (1.3%) presented a gene sequence identical to the reference sequence for pvdhfr. Fifteen isolates carried 3 changes and 3 isolates carried alleles with 4 differences. Novel mutations that have not been previously identified at codon 512 (K512M, K512E, K512T) were found in 7 isolates collected from Chiang Mai, Mae Hong Son, and Kanchanaburi Provinces. The proportions of pvdhps alleles distributed in different geographical areas of Thailand are shown in Figure 2. The pattern of resistance was similar to that observed for pvdhfr; isolates with highly mutant alleles were more prevalent from the western areas than from the eastern borders.

Distribution of pvdhfr and pvdhps alleles from different geographical areas of Thailand.

Our samples were taken from various geographical areas and showed allelic diversity of pvdhfr and pvdhps genes. There were 7 and 11 alleles identified for pvdhfr and pvdhps, respectively, with a total of 20 different combinations of dhfr and dhps alleles. Distribution of these alleles from different geographical malaria endemic areas is shown in Table 3. The most prevalent combination (36.3%) carried a quadruple mutant allele (57I/58R/61M/117T) of dhfr combined with a double mutant allele (383G/553G) of dhps, followed by the double mutant allele (58R/117N) of dhfr combined with a single mutant allele (383G) of dhps (24.2%). No isolates carried wild type alleles of both genes. Three isolates contained single or double mutant dhps coupled with dhfr wild-type, whereas 2 isolates contained double mutant dhfr coupled with wild-type dhps. Ninety-seven isolates (60.6%) contained at least the “quintuple” combination of 5 mutations among the dhfr-dhps allele. There was no evidence for preferential association of the dhfr and dhps alleles within isolates; the observed proportions were those predicted based on the prevalence of the individual alleles in the P. vivax population.

DISCUSSION

Currently, approximately half of the malaria cases in Thailand are caused by a monoinfection with P. vivax or mixed infection of P. vivax and P. falciparum.1 P. vivax infections have become more prevalent than P. falciparum infections in some regions of Thailand.21,22 Co-infections of these two species are also common in many other areas of Asia.8,23,24 In both species, mutations in dhfr and dhps are associated with pyrimethamine and sulfadoxine resistance.7,16,25

In this study, we determined the prevalence of mutations in the pvdhps and pvdhfr genes from a total of 160 P. vivax field isolates collected from different geographic areas of Thailand. In worldwide surveys, the diversity of pvdhfr alleles is strikingly greater than that observed for P. falciparum, but we only observed a subset of these alleles in our geographic area.9,13,16,18,20,2628 The vast majority of isolates contained mutant genotypes for both dhfr and dhps; only 3 (1.9%) and 2 isolates (1.3%) showed wild-type sequence for dhfr and dhps, respectively. Double mutant alleles of dhfr at residues 58 and 117 (S58R/S117N) were found frequently in isolates obtained from Thai-Cambodian and Thai-Malaysian borders, and similar observations have been reported from Cambodia and India.28,29 However, quadruple mutant alleles of dhfr at residues 57, 58, 61, and 117 (F57L,I/S58R/T61M/S117T), predominated in field isolates collected from the Thai-Myanmar border, where the highest malaria incidence rates and multi-drug resistant P. falciparum exist. These highly mutant alleles have also been observed in Myanmar, Indonesia, the Car Nicobar Islands of India and previously in isolates from Thailand.9,13,18,20,30,31 A similar distribution pattern of dhps alleles was also observed, where the alleles with higher numbers of mutations were more common in isolates collected from Thai-Myanmar border, and alleles with only single mutations were observed in isolates obtained from the Eastern international border areas.

Sulfadoxine-pyrimethamine was used as presumptive treatment of malaria in Thailand in all malaria endemic areas and phased out by the end of 2001.1 The resistance patterns we observed are thus likely to reflect the past use of SP and the current drug use in these neighboring countries. Genetic studies on the genes encoding both chloroquine resistance and antifolate resistance show that gene flow of antimalarial drug resistance in malaria parasites is most often a consequence of human migration rather than the emergence of new mutations.32,33 In the past, areas along the Thai-Myanmar and Thai-Cambodian borders were considered as highly multi-drug resistant in falciparum malaria due to population movement across each border for gem mining. However, after the closure of the Thai-Cambodian border in 1992, the number of malaria cases for both falciparum and vivax malaria declined markedly. Reduction of population migration and movement may be responsible for this observation.

Data based on yeast expression system and some clinical studies indicate that parasites harboring 3 or more pvdhfr mutation were highly resistant to sulfadoxine-pyrimethamine and even to chlorcycloguanil but not to WR99210.13,18,34 Recently, a randomized clinical efficacy trial of two antifolate drugs, sulfadoxine-pyrimethamine and chlorproguanil-dapsone, compared with chloroquine, indicated that antifolates are effective against P. vivax in Afghanistan and Pakistan. However, in this study the dhfr/dhps genotype was unknown.19 By analogy with P. falciparum, the prevalence of mutant alleles of P. vivax dhfr and dhps should also be valuable information for prediction of the clinical efficacy of antifolate drugs. Using different methods, several in vitro methods allow the assessment the correlation of dhfr genotype with sensitivity to antifolates.20,3537 Quadruple and double mutant allele of dhfr predominated in field isolates from Thailand but in vitro sensitivity data showed that WR99210 was still highly active against these parasites whereas chlorcycloguanil was less effective than pyrimethamine in both in vitro assays as previously reported.38 This suggests that new antifolates that are structurally related to the WR99210 class may be valuable against P. vivax.

The molecular basis of sulfadoxine resistance in P. vivax is not clearly understood, and there is no in vitro method for testing the sensitivity to sulfas of the parasites that carry various pvdhps alleles. Thus, we cannot determine directly whether some of the novel amino acid replacements that we observed increase sulfa resistance. Earlier studies have shown that sulfa drugs are less active against P. vivax than P. falciparum.39,40 Table 2 shows the equivalent codons in P. falciparum and P. vivax dhps, based on alignment of the amino acid sequences. All pvdhps alleles identified thus far carry the V585 residue that is orthologous to the V613 codon, and it has been suggested that this amino acid may be a key determinant of “innate” sulfadoxine resistance of P. falciparum to sulfadoxine.34,41 The S436A/F, A437G, and A581G changes in pfdhps are also associated with increased sulfa resistance and these correspond to codons S382A/C, A383G, and A553G changes that we observed in P. vivax. Therefore, it seems likely that these changes also affect sulfa sensitivity. In this study, we identified novel mutations at residue 512 (K512M, K512E, K512T) that have not previously been reported in Thai isolates. This codon corresponds to position 540 in P. falciparum, and the K540E bearing allele is commonly associated with sulfa resistance in P. falciparum in African isolates, so even these novel changes in pvdhps are likely to impact sulfa sensitivity.42 Finally, the prevalence of the highly mutant pvdhps alleles on the western border in isolates that also carry highly pyrimethamine-resistant alleles of pvdhfr supports the idea that alleles with mutations at these positions do confer sulfa resistance.

Among these mutant alleles, more than 60% of parasites carried at least 5 combined dhfr-dhps mutations. These polymorphic allelic variants might be useful molecular markers for predicting sulfadoxine-pyrimethamine treatment failure in P. vivax malaria. Patients with early treatment failures are likely to be infected with parasites harboring 6 or more combined mutations of pvdhfr and pvdhps.34 Monitoring mutations in P. vivax DHFR and DHPS by molecular analysis is valuable for understanding the antifolate resistance and designing optimal strategies for their therapeutic use.

Table 1

Prevalence of pvdhfr mutant alleles in P. vivax isolates from Thailand

Amino acid position, pvdhfr
Allele49575861117Number (%)
Bold letters indicate mutant amino acids.
Wild typeCFSTS3 (1.9%)
SingleCFSTT1 (0.6%)
DoubleCFRTN57 (35.6%)
Triple 1CFRMT3 (1.9%)
Triple 2GFRTN1 (0.6%)
Quadruple 1CLRMT18 (11.3%)
Quadruple 2CIRMT77 (48.1%)
Table 2

Prevalence of pvdhps mutant alleles in P. vivax isolates from Thailand, and the corresponding codon in P. falciparum dhps

Codon with non-synonymous change
AlleleSpecies pfdhps pvdhpsS436F/A 382A437G 383K540E 512A581G 553A613S 585Number (%)
Bold letters indicate mutant amino acids. The # allele had non-synonymous change at codon 554 K=aaa, K* = aag.
Wild typeSAKAV2 (1.3%)
#SAKAV1 (0.6%)
SingleSGKAV42 (26.3%)
DoubleSGKGV96 (60.0%)
SGKCV1 (0.6%)
TripleAGKGV8 (5.0%)
CGKGV3 (1.9%)
SGMGV2 (1.3%)
SGTGV2 (1.3%)
QuadrupleCGEGV2 (1.3%)
AGMGV1 (0.6%)
Table 3

Distribution of pvdhfr and pvdhps allelic combinations in P. vivax isolates from Thailand K*=aag

Areas
Thai-MyanmarThai-CambodiaThai-MalaysiaFrequency (%)
Genotype of dhfr alleleGenotype of dhps alleleCMMHTKRBRNCHBRYL
Wild type383G11 (0.6)
383G/553G112 (1.3)
117T383G/553G11 (0.6)
58R/117NWild type22 (1.3)
383G11742639 (24.4)
383G/553G139215 (9.4)
383G/553C11 (0.6)
58R/61M/117T383G/553G33 (1.9)
49G/58R/117N383G11 (0.6)
57L/58R/61M/117T57L/58R/61M/117T16117 (10.6)
382A/383G/553G11 (0.6)
57I/58R/61M/117T383G11 (0.6)
554K*11 (0.6)
383G/553G51168819158 (36.6)
382A/383G/553G13217 (4.4)
382C/383G/553G123 (1.9)
383G/512T/553G112 (1.3)
383G/512M/553G22 (1.3)
382A/383G/512M/553G11 (0.6)
382C/383G/512E/553G112 (1.3)
Total106441313319430160
Figure 1.
Figure 1.

Map of distribution of mutant pvdhfr alleles in Thailand. The pie charts show the proportions of wild-type and mutant pvdhfr alleles.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 3; 10.4269/ajtmh.2008.78.462

Figure 2.
Figure 2.

Map of distribution of mutant pvdhps alleles in Thailand. The pie charts show the proportions of wild-type and mutant pvdhps alleles.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 3; 10.4269/ajtmh.2008.78.462

*

Address correspondence to Kesara Na-Bangchang, Faculty of Allied Health Sciences, Thammasat University, Pathumtani, Thailand, 12121. E-mail: kesaratmu@yahoo.com

Authors’ addresses: Kanchana Rungsihirunrat and Kesara Na-Bangchang, Faculty of Allied Health Sciences, Thammasat University, Pathumtani 12121, Thailand, E-mail: kesaratmu@yahoo.com. Carol Hopkins Sibley, Department of Genome Sciences, University of Washington, Seattle, WA 98195-5065. Mathirut Mungthin, Department of Parasitology, Phramongkutklao College of Medicine, Bangkok, Thailand.

Acknowledgments: The authors thank the patients who participated in this study and the staff of Malaria Clinic for their kind assistance during blood sample collection.

Financial support: This work was supported by Thailand Research Fund through The Royal Golden Jubilee PhD program.

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