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Am. J. Trop. Med. Hyg., 76(6), 2007, pp. 1057-1065
Copyright © 2007 by The American Society of Tropical Medicine and Hygiene

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SENSITIVITY TO ANTIFOLATES AND GENETIC ANALYSIS OF PLASMODIUM VIVAX ISOLATES FROM THAILAND

KANCHANA RUNGSIHIRUNRAT, KESARA NA-BANGCHANG, VIVIAN N. HAWKINS, MATHIRUT MUNGTHIN, AND CAROL HOPKINS SIBLEY*
Faculty of Allied Health Sciences, Thammasat University, Pathumtani, Thailand; Department of Parasitology, Phramongkutklao College of Medicine, Bangkok, Thailand; Department of Genome Sciences, University of Washington, Seattle, Washington


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We investigated the association between the Plasmodium vivax dihydrofolate reductase (Pvdhfrtas) and the P. vivax dihydropteroate synthase (Pvdhps) genotype and in vitro sensitivity to the antifolates pyrimethamine, WR99210, chlorcycloguanil, sulfadoxine, and dapsone. Drug responses of 32 P. vivax isolates were assessed in two in vitro systems: schizont maturation inhibition and a yeast expression system. The geometric mean of 50% inhibition concentration (IC50) values for pyrimethamine, chlorcycloguanil, WR99210, sulfadoxine, and dapsone were 85 ± 88, 784 ± 662, 95 ± 87, 2,424 ± 2,784, and 1,625 ± 1,801 nM, respectively, for the schizont maturation assay. Five different Pvdhfr alleles and four Pvdhps alleles were observed: 26 of 32 quadruple mutant alleles of Pvdhfr (F57I,L/S58R/T61M/S117T), four triple mutants (S58R/T61M/S117T, K49C/S58R/S117N), and two double mutant isolates (S58R/S117N). All isolates carried Pvdhps 585V. Twenty four isolates carried double mutant Pvdhps (A383G/A553G), six an additional mutation, S382A,C/A383G/A553G, and two a single mutation, A383G. Increasing geometric mean IC50 values were observed with increased number of Pvdhfr mutations from double to quadruple. Results suggest that quadruple mutant alleles confer decreased sensitivity to pyrimethamine but retain sensitivity to WR99210.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Malaria affects more than 300 million people around the world and kills 2–3 million annually. More than 40% of the global population is at risk. Plasmodium falciparum and Plasmodium vivax are responsible for most cases of malaria. Plasmodium vivax is responsible for approximately 70–80 million cases of malaria worldwide and causes extensive morbidity in Central and South America and Asia.1

The spread of chloroquine resistance in P. falciparum has led to the use of sulfadoxine-pyrimethamine (SP) as the first-line drug for malaria treatment in several countries including Thailand, where P. vivax and P. falciparum often co-exist and occur at approximately equal frequencies, although the relative proportions differ greatly in different areas.2,3 This antifolate combination, known as Fansidar® (F. Hoffmann LaRoche, Basel, Switzerland), acts synergistically to inhibit the folate biosynthesis pathway in the malaria parasite. Treatment of chloroquine-resistant P. falciparum malaria with SP also led to inadvertent drug treatment against P. vivax. The result was antifolate resistance in P. vivax.4 Pyrimethamine acts against malaria parasites by selectively inhibiting their dihydrofolate reductase–thymidylate synthase (DHFR-TS) and sulfadoxine inhibits dihydropteroate synthase (DHPS); both are key enzymes in the biosynthesis and recycling of tetrahydrofolate.5–9 Resistance seems to occur by a series of sequential mutations at the homologous positions in the parasite dhfr and dhps genes that alter the drug binding sites of the encoded enzymes. Correlations of sequence alterations with resistance to SP were refined by expression and purification of the various polymorphic recombinant enzymes and by transfection in laboratory studies.7,10–12 A major question is how mutations in dhfr and dhps predict drug resistance measured in vitro. Cloning of the dhfr gene from P. vivax has allowed molecular comparisons of alleles from different regions.13 Detection of these mutations in field isolates has provided valuable information for monitoring the emergence of drug resistance.

Monitoring of in vitro drug susceptibility of P. vivax to antimalarial drugs has been limited by the difficulties in culturing this malaria species. Plasmodium vivax requires immature red blood cells for reinvasion. To maintain this species in vitro, a large supply of reticulocytes is needed. The concentration of reticulocytes in peripheral blood is only 0.5–1.5%, a level that is insufficient for the sustenance of P. vivax in culture. As a result, assessment of P. vivax sensitivity to antimalarial drugs has been performed only in short-term culture.14–17 Therefore, the association between the Pvdhfr mutations and the response of P. vivax to antifolates is still unclear.

Recently, assays based on expression of P. vivax dhfr in the budding yeast system, Saccharomyces cerevisiae, have been developed to rapidly screen drugs directed against parasite target molecules.18–20 Limited clinical assessments support the hypothesis that mutations within the Pvdhfr gene affect the clinical response to SP treatment.2 A small clinical study has suggested that SP is effective against P. vivax isolates carrying alleles of Pvdhfr with three or fewer mutations.21 The objective of the present study was to investigate in P. vivax isolates from Thailand the association between Pvdhfr mutations and in vitro sensitivity to three inhibitors of DHFR: pyrimethamine, chlorcycloguanil (the active metabolite of chlorproguanil), and the experimental inhibitor, WR99210, as well as two inhibitors of DHPS, sulfadoxine and dapsone. Two in vitro systems for assessment of sensitivity of P. vivax isolates were applied, the sensitivity assay based on schizont maturation inhibition14 and the yeast expression system.18


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Sample collection. Samples were collected from April to August 2005 at Mae Sot General Hospital and Section 4, Vector Borne Disease Control, Mae Sot district, Tak Province, Thailand. This malaria- endemic area is located along the Thailand-Myanmar border and has been well-documented as a region in which P. falciparum is multidrug resistant.22,23 Patients with mono-infection of P. vivax with asexual parasitemias between 1,000 and 50,000 ring-stage parasites per microliter were included in the study. Written informed consent for study participation was obtained from all patients, and the study was reviewed and approved by the Ethics Committee of the Ministry of Public Health, Thailand. Prior to treatment, a blood sample (5 mL) was collected by venipuncture into lithium–heparin collecting tubes; blood was also spotted onto filter paper for genotyping. All patients were treated by clinic personnel with a standard regimen of 3 days of chloroquine and 14 days of primaquine. All patients recovered and had no relapse or recrudescence during the 28-day follow-up period.

Assessment of sensitivity of P. vivax isolates to drugs in short-term culture. The schizont maturation assay was performed with P. vivax field isolates using a modified method of Russell and others.14 Briefly, a 2-mL blood sample was mixed with phosphate-buffered saline at the ratio of 1:1 and added to the CF11 column (a 10-mL syringe tipped with glass wool and filled with CF11 cellulose powder (Whatman, Florham Park, NJ). The supernatant was then removed and the pellet was resuspended in RPMI 1640 medium. The blood mixture was centrifuged and the supernatant was removed. The pellet was then resuspended in human AB serum to obtain a hematocrit of 40%. The blood–serum suspension was mixed with McCoy’s 5A medium at the ratio of 1:10. The concentrations of folic acid and p-aminobenzoic acid in McCoy’s medium are 10 mg/L and 1 mg/L, respectively. Fifty microliters of this mixture were added to each well of a 96-well microtiter plates pre-dosed with drug.

Plasmodium vivax field isolates were tested for their sensitivities against pyrimethamine, WR99210, chlorcycloguanil, sulfadoxine, and dapsone. All antifolate drugs were obtained from Jacobus Pharmaceutical, Inc. (Princeton, NJ). Drug plates were prepared fresh to avoid possible degradation. A stock solution of each drug was prepared in 1% dimethyl sulfoxide (DMSO) and was subsequently diluted in RPMI 1640 medium to obtain the desired drug concentrations. Fifty microliters of the final drug solution was added to each well of a 96-well microtiter plate. This plate contained varying concentrations of drug in each column and well A was free of drug and served as control. Wells B–H contained ascending concentrations of drug, each concentration of which was tested in triplicate. The concentration ranges for each drug used were 0–2,500 nM for pyrimethamine, 0–2,560 nM for WR99210, and 0–200,000 nM for chlorcycloguanil, sulfadoxine, and dapsone. The tested plate was incubated at 37.5°C in a gas chamber containing 5% CO2 for 24–36 hours depending on the stage of the parasite before culturing. After incubation, a thick blood film was prepared from each well and the number of normal schizonts (containing > 8 nuclei) per 200 asexual stage parasites was counted. The number of schizonts in each well that contained drug was compared with that in the control well and expressed as a percentage of the control. The dose–response curve was analyzed by nonlinear regression analysis to obtain the IC50 value, the concentration that inhibits schizont maturation by 50% compared with the no drug control.

Detection of mutations in the Pvdhfr and Pvdhps genes. Parasite DNA was extracted from dried blood spots on filter paper using a QIAamp DNA extraction mini-kit (Qiagen, Valencia, CA) and used as template for amplification by polymerase chain reaction (PCR). 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 with a pair of primers (forward: 5'-ATG GAG GAC CTT TCA GAT GTA TTT GAC ATT-3' and reverse: 5'-CCA CCT TGC TGT AAA CCA AAA AGT CCA GAG -3'). The 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 deoxynucleotides (dNTPs), 15–20 µL of genomic DNA, and 0.5 unit of Taq DNA polymerase (Promega, Madison, WI). The PCR cycling parameters were as follows: initial denaturation at 94°C for 3 minutes, followed by 5 cycles at 94°C for 1 minute, 60°C for 1 minute, and 72°C for 1 minute, then followed by 25 cycles at 94°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute. 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 a 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'). The 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 dNTPs, 15–20 µL of genomic DNA, and 0.5 unit of Taq DNA polymerase (Promega). The PCR was performed using 40 cycles at 94°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute. The PCR products were fractioned by electrophoresis on a 1.5% agarose gel, purified with the QIAquick PCR purification kit (Qiagen), and sequenced using the fluorescent dye chemical system (MegaBACE; Amersham Pharmacia Biotech, Piscataway, NJ).

Multiclonal detection in field isolates. Detection of multiclonal infection of the PVMSP-3{alpha} gene was performed using the PCR-restriction fragment length polymorphism method described by Bruce and others.24 The PVMSP-3{alpha} was amplified by nested PCR and the sizes of the PCR products were estimated after electrophoresis on a 1.8% agarose gel. Ten microliters of the PCR product was digested with Hha I (New England BioLabs, Ipswich, MA) and analyzed by electrophoresis on a 1.8% agarose gel. Multiple infections were distinguished when the summed size of the DNA fragments resulting from Hha I digestion exceeded the size of the uncut PCR product.24,25

Cloning and expression of Pvdhfr alleles in yeast. The TH5 S. cerevisiae strain, which lacks endogenous DHFR activity and requires supplemental dTMP for growth,26 was used for expression of Pvdhfr as previously described.18,20,27 The shuttle plasmid that can be propagated in both Escherichia coli and S. cerevisiae was used as vector for expression of the Pvdhfr coding region.28 The PCR primers for amplification of the Pvdhfr plus 19 downstream nucleotides were designed for homologous recombination in yeast.20 The PCR cycling parameters used were the same as for Pvdhfr amplification. Yeast were transformed using a high-efficiency lithium acetate protocol29 and plated onto medium lacking tryptophan and dTMP to select for the plasmid and functional DHFR activity. This produced a series of genetically matched yeast strains dependent upon different alleles of Pvdhfr.

Determination of drug sensitivity in the yeast expression system. Sensitivity assays in the yeast expression system were performed in 96-well microtiter plates.19,20 Each strain of transformed yeast was grown for 18–24 hours in complete medium lacking dTMP and from 0 to 5 x 10–4 M pyrimethamine, chlorcycloguanil, or WR99210. The growth of the yeast in each well was measured by reading the optical density at 660 nm. The growth of each yeast strain at each drug concentration was used to plot the percent growth relative to the yeast in the control well that contained only DMSO. The IC50 value was calculated from the slope and intercept of the line defined by the two data points that bracket 50% relative growth. Comparisons of the IC50 values of yeast dependent upon the mutant alleles to yeast dependent upon the wild-type allele were used to assess the relative resistance level of each Pvdhfr allele to the drug.

Statistical analysis. Statistical analysis to investigate the association between number of point mutations in Pvdhfr and in vitro sensitivity of P. vivax isolates was performed using a two-sided Student’s t-test of IC50 values. Statistical significance level was set at {alpha} = 0.05 for all tests.


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Assessment of sensitivity of P. vivax isolates in vitro. Our first goal was to assess in short-term culture the response of 32 P. vivax field isolates to three inhibitors of DHFR: pyrimethamine, chlorcycloguanil (the active metabolite of chlorproguanil), and the experimental antifolate WR99210, and to two inhibitors of DHPS, sulfadoxine and dapsone. Thirty-two P. vivax isolates were assessed by comparing the maturation of rings to schizonts in the absence and presence of a range of drug concentrations; not all isolates had sufficient parasites to assay their response to all five drugs. All stages of asexual development (rings, trophozoites, and schizonts) were present in the peripheral blood and the initial parasitemia in the isolates varied between 1,280 and 18,000/µL of blood. For each of the drugs, a wide range of IC50 values was obtained. The IC50 values are shown in Figure 1Go according to their log10 values and the geometric means, and median values and ranges are shown in Table 1Go. The range of IC50 values was most pronounced for pyrimethamine where the IC50 values covered almost four orders of magnitude. Despite the wide range, the mean values showed consistent differences in the response to the five drugs. First, considering all of the isolates, WR99210 inhibited growth most effectively; not even the highest IC50 values exceeded 600 nM. Second, chlorcycloguanil was substantially less effective than either pyrimethamine or WR99210, even among the most sensitive isolates. Finally, the IC50 values measured for the two sulfa drugs were extremely high, but dapsone was somewhat more effective than sulfadoxine, as has been observed when similar measurements were made in P. falciparum.30


Figure 1
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  		    FIGURE 1. Box plot of the log10 of the 50% inhibitory concentration (IC50) values measured in the short-term culture system in Plasmodium vivax field isolates from Mae Sot, Thailand. The ranges of values for pyrimethamine (PYR), chlorcycloguanil (CCG) WR99210 (WR), sulfadoxine (SD), and dapsone (DDS) are shown. The box indicates the central 50% of values and the line shows the median of all values, with the upper and lower range of all the values shown by the bars.

 

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TABLE 1
Geometric means, arithmetic median and range of the IC50 values for each of the drugs in the short term culture system*
 
Detection of mutations in the Pvdhfr and Pvdhps genes. The sensitivity of P. falciparum isolates to antifolates in vitro depends principally upon the genotypes of the dhfr and dhps genes in the parasite.6,8,9,30,31 To test whether this correlation is valid for P. vivax, we sequenced dhfr and dhps from all isolates. We identified five different Pvdhfr alleles and four Pvdhps alleles. The frequencies of the genotypes are shown in Table 2Go. Twenty-six of the 32 isolates carried quadruple mutant alleles of Pvdhfr; one allele has been observed in isolates from several sites previously (F57L/S58R/T61M/S117T)2,19,32–34, and the other is an allele that differs at amino acid 57 (F57I/S58R/T61M/S117T) and has been observed previously only in Thailand.32 In addition, there were four triple mutants (S58R/T61M/S117T, K49C/S58R/S117N) and two isolates with the double mutant genotype commonly seen (S58R/S117N).17,34–36 All isolates carried a valine at DHPS residue 585; this residue has been hypothesized to account for the low susceptibility P. vivax to sulfadoxine.37 In addition to 585V, two isolates were mutant solely at dhps residue 383. Twenty four isolates carried double mutant alleles of Pvdhps at residues 383 and 553 (A383G/A553G) and six isolates carried an additional mutation at residue 382 (S382A/A383G/A553G, S382C/A383G/A553G). No wild type alleles for either gene were identified.


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TABLE 2
Allele frequencies of Pvdhfr and Pvdhps in Plasmodium vivax isolates from Thailand*
 
Association between sensitivity in the short-term culture system and Pvdhfr genotype. Our primary goal was to correlate the genotype of each isolate with the response to each drug in the short-term culture system. Table 3Go shows this information for each of the 32 field isolates tested against all or some of the drugs. Each group of dhfr alleles was analyzed for its association with sensitivity of the parasites in that isolate, as measured in the short-term culture system. The most surprising result is that isolates carrying the same dhfr and dhps alleles still showed wide variance in their IC50 values for each of the drugs. For example, among isolates carrying quadruple mutant allele (F57L/S58R/T61M/S117T), the IC50 values for pyrimethamine varied from 4 to more than 5,000 nM, and for the F57I/S58R/T61M/S117T allele from 3 to more than 3000 nM. However, a trend of increasing mean IC50 values was observed as we compared isolates with increased number of Pvdhfr mutations from double to quadruple. The variance was wide, but we compared the mean IC50 values for both pyrimethamine and chlorcycloguanil of the isolates with ≤ 3 mutations with that of the isolates that carried a quadruple mutant dhfr allele. These two groups were significantly different (pyrimethamine, P = 0.02, and chlorcycloguanil, P = 0.002, by two-sided Student’s t-test). In contrast, these groups differed only slightly in their response to WR99210 (P = 0.1).


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TABLE 3
Relationship between Pvdhfr and Pvdhps genotypes in Plasmodium vivax field isolates and susceptibility to pyrimethamine, chlorcycloguanil, WR99210, sulfadoxine, and dapsone, as determined by short-term culture*
 
Table 3Go is arranged in ascending order of the IC50 value for pyrimethamine. The range of values for response to the other drugs is also wide, but no isolate that had the lowest value for pyrimethamine showed the lowest value in response to chlorcycloguanil or WR99210. Overall, the trends suggest strongly that chlorcycloguanil is less effective than pyrimethamine, but these trends did not reach statistical significance. Moreover, WR99210 retained its effectiveness even against parasites that carry the quadruple mutant alleles of Pvdhfr.

As expected, the IC50 values for both sulfadoxine and dapsone were high. This reflects both the relative ineffectiveness of drugs of this class when used alone against Plasmodia38 and the fact that all of the isolates carried dhps alleles with 585V as well as at least one other mutation, as defined by Korsinczky and others.37 Sulfa drugs inhibit DHPS, not DHFR, so one would not expect that the dhfr allele in an isolate would affect the response to sulfa drugs, and that is what we observed. When we stratified the isolates according to their dhps genotypes and compared the responses to sulfadoxine and dapsone, there was also no difference in the IC50 values in the groups.

Clonality of the isolates. One possible explanation for the wide variance in the IC50 values among isolates with the same Pvdhfr genotype might be that the isolate contains more than one parasite clone. None of the sequences of the Pvdhfr or Pvdhps genes showed evidence for polymorphism within these loci. However, we did assess the clonality of the infections by assaying the size of the insert in the PvMSP-3{alpha} gene.24 Three size classes were observed, type A (1.9 Kb), B (1.5 Kb), and C (1.1 Kb). All isolates carried the class A repeat except for one isolate that carried size class B and three isolates that carried size class C.

After digestion with Hha I, the PCR-restriction fragment length polymorphism pattern suggested that some isolates showed patterns indicative of polyclonal infection. Polyclonality was distinguished when the summed size of the fragments digested with Hha I exceeded the size of the uncut PCR product. We assumed that if the sum of the fragments exceeded the size of the uncut fragment, the isolate was polyclonal. By that criterion, nine isolates were polyclonal and none of these isolates showed a minority allele at Pvdhfr or Pvdhps. Moreover, the IC50 values that were far from the expected median for that Pvdhfr genotype were observed in apparently monoclonal isolates.

Determination of drug sensitivity in the yeast expression system. Because there is no long-term culture system for P. vivax, we also used a heterologous yeast system to compare the efficacy of DHFR inhibitors against various alleles of Pvdhfr.19,20 To compare directly the short-term culture response of these field isolates with the yeast system, the P. vivax DHFR domain from each of the five alleles, including the wild-type control allele,13 was amplified, cloned into the yeast shuttle vector by homologous recombination, and transformed into yeast that lacked endogenous DHFR activity. The IC50 values for each strain against pyrimethamine, WR99210, and chlorcycloguanil were measured by the relative growth of the yeast in vitro. The yeast were grown in a range of drug concentrations up to 5 x 10–4 M and the IC50 value for each allele was calculated. Figure 2Go shows representative data, but the values for 4 replicate experiments were within a factor of 2 for each determination. These IC50 values were then used to assess the relative resistance level compared with the wild-type allele (Table 4Go). All mutant alleles showed increased resistance to pyrimethamine and chlorcycloguanil by more than 50 fold when compared with the wild type allele. In contrast, all mutant alleles were relatively sensitive to WR99210 (relative resistance = 1.3–83), particularly the S58R/S117N alleles. As expected, the values are consistent with those measured previously for the same Pvdhfr alleles.19,20 The current data also extend the usefulness of the yeast expression system, demonstrating that chlorcycloguanil was particularly ineffective against all but the wild-type allele.


Figure 2
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  		    FIGURE 2. Sensitivity of yeast dependent on various Plasmodium vivax dihydrofolate reductase (Pvdhfr) alleles to pyrimethamine, chlorcycloguanil, and WR99210. Yeast strains dependent upon the indicated Pvdhfr alleles were expressed in yeast deficient in DHFR activity and the sensitivity to pyrimethamine, chlorcycloguanil, and WR99210 was tested as described in the Materials and Methods.

 

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TABLE 4
IC50 values for yeast dependent upon each Pvdhfr allele and the resistance relative to wild type (RR)*
 

DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It has only recently been possible to determine whether the mutations observed in the Pvdhfr and Pvdhps genes account for antifolate resistance seen in vitro and in vivo. Because of the early assertion that P. vivax was intrinsically resistant to antifolates, most studies have been focused only on P. falciparum and information on P. vivax is extremely limited. The lack of a continuous culture system for P. vivax has limited in vitro studies to the assay of progression in a single cycle.14,15,17 The initial parasitemia in the blood sample is usually less than 30,000/µL of blood, which is below the threshold of sensitivity for the assay that uses radiolabeled hypoxanthine incorporation. The morphologic method is time-consuming and requires considerable practical skill, and it has therefore been applied for determination of drug sensitivity relatively rarely. A yeast system has been used as a surrogate, but its relationship to sensitivity of the parasites to drug is not known. In this work, our goal was to correlate sensitivity to drug as determined in short-term culture with sensitivity as determined in the yeast expression system. Furthermore, we sought to correlate drug sensitivity with Pvdhfr and Pvdhps genotype.

The poor correlation between dhfr genotype and sensitivity to DHFR inhibitors as measured in short-term culture was unexpected. We have examined the data in detail to identify technical difficulties that might explain the wide variance. In general, the IC50 value measured for some of the quadruple mutant strains was far lower than expected. This is not the result of poor growth of a particular isolate because those at the low end for pyrimethamine were not consistently lowest for other drugs. It is also not likely to be a result of differences in actual drug concentration in the plates because these drugs are soluble and stable in DMSO, the plates were prepared fresh on the day of assay, and the low values are not all observed in the same plate nor on the same day. It is possible that some of the isolates were polyclonal, but that effect seems far more likely to yield a resistant IC50 value in a culture with a minority population of highly mutant alleles, and that is the opposite of this anomaly. In any case, the isolates that were polyclonal were not responsible for the most extreme IC50 values. All of the assays were done using a common source of media and serum. However, the patient red blood cells differ and could contribute to differences in outcome, particularly if the internal level of folate or p-aminobenzoic acid varies widely.

The standard approach to wide variation of in vitro assays in P. falciparum depends on continuous culture. These standard reference strains whose sensitivity is known can be included in assays of fresh field isolates. However, the lack of cultured P. vivax reference strains makes it impossible to use this approach. Even for P. falciparum, variances of 10–100 fold in IC50 values between parasites of the same apparent genotype are not uncommon. In our data, the variance is approximately 1,000-fold for the quadruple mutant genotypes that have the largest numbers of isolates. The inherent variability of an assay where only small numbers can be counted seems the most likely explanation. The microscopic method is extremely laborious; for each isolate, a thick blood film was prepared from each well and the number of normal schizonts (containing > 8 nuclei) per 200 asexual stage parasites was counted, and each determination was done in triplicate. The tyranny of small numbers is unavoidable. Recently, a comparison of this microscopic method with the isotopic and fluorescence-based methods was published.16 These new approaches are likely to greatly reduce the difficulty and the variance inherent in the present assay.

The IC50 values measured in yeast were well correlated with the Pvdhfr genotype. This is the expected result because the yeast host strain is the same for all of the lines compared, and Pvdhfr is the single variable. These values also correlate with the determination of the Ki values for pyrimethamine and WR99210 measured in purified DHFR-TS in vitro.39 The IC50 value measured in the yeast system depends not only on the interaction between the drug and the DHFR enzyme, but also on the level of expression of the P. vivax genes in the yeast host. Even more problematic, the growth of the P. vivax measured in short-term culture requires media with high levels of folate and p-aminobenzoic acid, and both are known to increase the IC50 value for pyrimethamine measured in P. falciparum in vitro.40 For that reason, the sensitivity of P. falciparum to antifolates is usually evaluated in medium deficient in folic acid. Because of the differences in assay conditions and culture medium, the IC50 values obtained from the present study could not be compared with the susceptibility of P. falciparum as measured in culture. Therefore, neither the short term culture assay nor the yeast assay allows us to draw conclusions on the relation between the IC50 absolute value measured in vitro and the drug concentration that might be predicted to be effective in the human host.

In contrast to the lack of correlation of the genotypes with the short-term culture results for individual isolates, both the average values of the short-term culture assays and the yeast results did reflect the expected trends. The relative effectiveness of the various drugs is faithfully reflected in the output and enables us to draw some valuable conclusions. For example, both assays show that for all genotypes, chlorcycloguanil is less effective than pyrimethamine. This is important because chlorcycloguanil is the active DHFR inhibitor in chlorcycloguanil/dapsone (LapDap®; GlaxoSmithKline, Research Triangle Park, NC). This drug has been shown to be more effective in P. falciparum against strains that carry a triple mutant Pfdhfr allele,41,42 and LapDap® and LapDap® plus artesunate are currently proposed for use in Africa. Our results suggest that neither drug would be a wise choice for treatment of P. vivax malaria.

A second conclusion that can be drawn from both datasets is that WR99210 continues to show promise as a possible treatment of P. vivax malaria. This idea is based on the limited data that show that SP is effective in vivo even against parasites that carry Pvdhfr alleles with fewer than three mutations.21 The demonstration that pyrimethamine and chlorcycloguanil were significantly more effective in vitro against isolates that carried triple or double mutations compared with those that had quadruple mutant alleles is consistent with those data. Furthermore, the relative effectiveness of the WR99210 in both the short-term culture and yeast expression systems reported here, and against the purified enzyme in vitro39, suggests that this class of drug may be effective even against parasites that carry the quadruple mutant alleles.

Four Pvdhps alleles were detected in our samples. Twenty-four isolates (75%) carried the A383G/A553G allele, six isolates (19%) carried the S382A,C/A383G/A553G allele, and two isolates (6%) carried the single mutation A383G. In addition, all isolates carried DHPS 585V; this residue has been implicated in the presumed intrinsic refractoriness of P. vivax to sulfa drugs. Notably, all P. vivax isolates assessed to date have carried 585V.37,43 Mutations at residues 382, 383, and 553 have been identified previously in parasite isolates from Thailand.37,43 By analogy with the equivalent changes in P. falciparum dhps, it has been suggested that these changes are associated with reduced sensitivity to both sulfa drugs and sulfones. In one clinical trial, parasites harboring six or more combined mutations of Pvdhfr and Pvdhps genes were cleared more slowly from the blood after treatment with SP than parasites with fewer mutations in these genes.43 Because we did not identify any parasites with the proposed wild type Pvdhps sequence,37 we are unable to determine whether that is the reason for the high IC50 values measured in our short-term culture assays.

The clinical use of SP as first-line treatment for P. falciparum malaria in Thailand was discontinued in 1996 and the drug has never been recommended for treatment of P. vivax malaria. However, infections with both P. vivax and P. falciparum are common,44–46 and drug pressure would be expected to have been progressively continued from the use of SP as presumptive treatment of P. falciparum malaria and its use in combination with mefloquine (Fansimef®; F. Hoffmann La Roche) as first-line treatment of P. falciparum malaria until the termination of these drugs in 2001.47 In our study of isolates collected in 2005, 26 of 32 isolates carried a quadruple mutant allele of Pvdhfr and only 2 of 32 carried the double mutant 58R/117N allele. In contrast, Imwong and others identified 18 of 44 double mutant and 13 of 44 quadruple mutant alleles in isolates from Thai patients between 1992 and 1996.2 Although the source of drug pressure is not clear, it appears that progressive development of resistance still was ongoing between 2000 and 2005.

Currently, the major strategy to delay the emergence of antimalarial drug resistance is combination therapy using two or more drugs that target different pathways. If drugs continue to be the main therapeutic weapon against malaria, thorough understanding of the interaction between drug and parasite is essential. The development of new antifolates like WR99210 that are effective against SP-resistant parasites, and their combination with appropriate partners, can play an important role in a rational drug treatment strategy. Understanding the resistance mechanism of P. vivax to antifolate drugs may help in formulating a better antifolate combination that is effective against both P. falciparum and P. vivax.

Received December 6, 2006. Accepted for publication February 11, 2007.

Acknowledgments: We thank the patients who participated in this study and the staff of the Mae Sot General Hospital and Section 4, Vector Borne Disease Control, Mae Sot District, Tak Province for their kind assistance during blood sample collection. We also thank Dr. David Jacobus (Jacobus Pharmaceutical, Inc.) for the gift of the drugs used in the studies and for his thoughtful comments on the manuscript.

Financial support: This work was supported by Thailand Research Fund for the scholarship of The Royal Golden Jubilee Ph.D. program and National Institutes of Health grant AI 55604 to Carol Hopkins Sibley.

* Address correspondence to Carol Hopkins Sibley, Department of Genome Sciences, University of Washington, Seattle, WA 98195-5065. E-mail: sibley{at}u.washington.edu Back

Authors’ addresses: Kanchana Rungsihirunrat and Kesara Na-Bangchang, Faculty of Allied Health Sciences, Thammasat University, Pathumtani, Thailand. Mathirut Mungthin, Department of Parasitology, Phramongkutklao College of Medicine, Bangkok, Thailand. Vivian N. Hawkins and Carol Hopkins Sibley, Department of Genome Sciences, University of Washington, Seattle, WA 98195-5065.


REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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