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Plasmodium vivax:Rapid Detection by Polymerase Chain Reaction and Restriction Fragment Length Polymorphism of the Key Mutation in Dihydrofolate Reductase-Thymidylate Synthase Gene Associated with Pyrimethamine Resistance
EXPERIMENTAL PARASITOLOGY ARTICLE NO.
89, 343–346 (1998)
PR974281
RESEARCH BRIEF Plasmodium vivax: Rapid Detection by Polymerase Chain Reaction and Restriction Fragment Length Polymorphism of the Key Mutation in Dihydrofolate Reductase–Thymidylate Synthase Gene Associated with Pyrimethamine Resistance
Rachida Tahar,1 Philippe Eldin de Pecoulas, Andre Mazabraud, Leonardo K. Basco2 ´ ´ ´ Centre de Genetique Moleculaire, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette, France
Tahar, R., Eldin de Pecoulas, P., Mazabraud, A., and Basco, L. K. 1998. Plasmodium vivax: Rapid detection by polymerase chain reaction and restriction fragment length polymorphism of the key mutation in dihydrofolate reductase–thymidylate synthase gene associated with pyrimethamine resistance. Experimental Parasitology 89, 343– 346. q 1998 Academic Press Index Descriptors and Abbreviations: malaria; Plasmodium falciparum; drug resistance; point mutation; chloroquine; sulfadoxine; ACD, acid citrate-dextrose; EDTA, ethylene diaminetetraacetic acid; DHFR, dihydrofolate reductase; DHFR-TS dihydrofolate reductase thmidylate synthase; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphisme.
Clinical studies conducted in Asia and South America, essentially in the 1950s and 1960s, suggested that Plasmodium vivax developed resistance to pyrimethamine, alone or in combination with sulfadoxine, rapidly (Peters 1987). Because P. vivax cannot be propagated in vitro easily, there are no experimental data on its in vitro sensitivity to pyrimethamine (Brockelman et al. 1989; Basco and Le Bras 1994). Furthermore, limited clinical data on pyrimethamine sensitivity of P. vivax are available from recent studies because chloroquine has been the only recommended blood schizontocidal drug for the treatment and 1
To whom correspondence should be addressed. Fax: 33 1 69 82 43 86. E-mail: [email protected]. 2 Present address: Institut Franc¸ais de Recherche Scientifique pour ´ ´ le Developpement en Cooperation (ORSTOM) - Organisation de la ´ Lutte contre les Endemies en Afrique Centrale (OCEAC), B. P. 288, ´ Yaounde, Cameroon.
0014-4894/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
prophylaxis against P. vivax infections. In view of several reports from Asia and South America on the development of chloroquine resistance in P. vivax (Rieckmann et al. 1989; Baird et al. 1995), there are rising concerns that treatment and chemoprophylaxis against P. vivax infections may require alternative antimalarial drugs. The mechanism of action of pyrimethamine is based on the specific inhibition of dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3), leading to the disruption of folate and pyrimidine biosynthesis in malaria parasites (Walter 1991). Several in vivo and in vitro studies have strongly suggested that there is a direct correlation between the presence of specific point mutations in the DHFR domain of the bifunctional dihydrofolate reductase–thymidylate synthase (DHFR-TS) gene and pyrimethamine resistance in P. falciparum, P. berghei, and P. chabaudi (Cowman et al. 1988; Cowman and Lew 1989; Foote et al. 1990; Peterson et al. 1990; Cheng and Saul 1994; Van Dijk et al. 1994; Basco et al. 1995). Furthermore, an analysis based on the X-ray crystallographic structure of prokaryotic and eukaryotic DHFR enzymes has shown that these point mutations result in amino acid substitutions within the active site of the enzyme, which is consistent with the considerable decrease in the enzyme affinity for pyrimethamine in the presence of these mutations (Sirawaraporn et al. 1997). Despite various obstacles that render the comprehension of the molecular basis of pyrimethamine resistance in P. vivax difficult, experience based on P. falciparum and rodent malaria parasites leads us to expect pyrimethamine-resistant P. vivax to possess an equivalent key mutation in the DHFR-TS gene. Recently, we isolated, cloned, and sequenced the entire P. vivax DHFR-TS gene. Alignment with DHFRTS genes of other malaria parasites revealed that the DHFR domain of the P. vivax DHFR-TS gene undergoes a nonsynonymous point mutation at position 117, which is homologous to the key codon 108 in P. falciparum and codon 110 in P. berghei (Fig. 1). In P. vivax, there are two alternative codons at this position: serine (AGC) and asparagine
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FIG. 1. Alignment of DNA sequences of the DHFR-TS gene (334–366 nucleotides, P. vivax numbering; 307–339 nucleotides, P. falciparum numbering) showing key point mutations that confer pyrimethamine resistance in malaria parasites. The deduced amino acid sequences are given below the nucleotide sequences. The DHFR-TS sequences were obtained from the following sources: pyrimethamineresistant (R) P. falciparum HB3/Honduras clone (Cowman et al. 1988; Genbank accession number J03772), pyrimethamine-sensitive P. chabaudi adami DS line (Cowman and Lew 1989; accession number M30834), pyrimethamine-sensitive P. berghei ANKA clone (Van Dijk et al. 1994; accession number U12275), pyrimethamine-sensitive P. yoelii 173 strain (Cheng and Saul 1994; accession number L28122), and P. vinckei (Cheng and Saul 1994; accession number L28121). For comparison, the homologous sequence of a wild-type, pyrimethaminesensitive (S) P. falciparum strain is given. The complete nucleotide sequence of the P. vivax DHFR-TS gene was determined in our laboratory (accession number X98123). Because incomplete sequences of the DHFR-TS gene of P. vinckei and P. yoelii have been determined, their numbering is not known. Amino acid residue 117 deduced from the P. vivax DHFR-TS gene sequence corresponds to amino acid residues 108, 106, and 110 in the P. falciparum, P. chabaudi, and P. berghei homologues, respectively. The direct association between the Ser-toAsn point mutation at these homologous codons and in vitro or in vivo pyrimethamine resistance has been demonstrated in P. falciparum and rodent malaria parasites in previous studies. The restriction site of Pvu II is indicated by a vertical bar.
(AAC). Because pyrimethamine-sensitive P. falciparum, P. berghei, P. chabaudi, and P. vinckei are characterized by serine at the homologous position and serine-to-asparagine substitution confers resistance to pyrimethamine in these malaria parasites, we hypothesize that pyrimethamine sensitivity in P. vivax is also associated with Ser-117, whereas pyrimethamine resistance is associated with Asn-117. On the basis of these findings, we designed a simple, rapid, and reproducible protocol to detect the presence of either Ser- or Asn-117 in the P. vivax DHFRTS gene with the ultimate aim of facilitating future studies to establish a direct correlation between phenotype and genotype in P. vivax field isolates. With the use of our PCR protocol and primer pairs that are specific for the P. vivax DHFR-TS gene, negative controls, with or without human DNA, and controls with parasite DNA from P. falciparum, P. ovale, and P. malariae yielded no visible bands. All samples containing
TAHAR ET AL.
P. vivax DNA samples yielded a single band of 715 bp. No false negative results were obtained from samples containing P. vivax DNA. After PCR amplification of the DHFR domain of the P. vivax DHFRTS gene, PCR products were treated with the restriction enzyme Pvu II. The restriction site (CAG|CTG) is present at only one site within the amplified fragment if a given parasite is a wild type (Ser-117). A mutant isolate does not possess the restriction site for Pvu II. A typical example of the PCR-RFLP results is presented in Fig. 2. Eleven Burmese field isolates and two other clinical isolates (DUF/French Guiana, LFT/Cambodia) had Asn-117. Six isolates (ARI/Pakistan, CRV/Yemen, PHI/Djibouti, HRT/Comoros, PIT/Madagascar, one Burmese isolate) displayed the wild-type codon Ser-117. None of the isolates studied showed mixed genotype. DNA sequencing confirmed the results derived from the PCR-RFLP technique. To determine the capability of the method to distinguish the two genotypes in a mixed isolate, we mixed small amounts of the two types of genes ARI (Ser 117) and a Burma (Asn 117). As little as 1 pg of DNA can be used to drive the Burmese isolate Asn 117 PCR. However, starting with 1 ng of a mixture of the two DNAs, 1/10 of either type was easily detectable, and 1/100 was at the limit of the assay sensitivity. The PCR-RFLP method described in this paper allows a rapid, reliable, and reproducible detection of the key codon of the P. vivax DHFR-TS associated with pyrimethamine resistance. Analysis of the DNA sequence of the P. vivax DHFR-TS gene and restriction sites showed that only two commonly available endonucleases, Alu I and Pvu II, cut within codon Asn-117. Of these two enzymes, Alu I possesses numerous other restriction sites within the DHFR domain, rendering its use impractical for the interpretation of results after agarose gel electrophoresis. The restriction enzyme Pvu II has one other restriction site within the TS domain of the gene. In our protocol, PCR amplification was designed to limit the number of restriction sites of Pvu II to a single site. The specificity of the technique is ensured at two levels. First, PCR amplification using species-specific oligonucleotide primers yielded positive results only from samples containing P. vivax DNA. Even though P. ovale and P. malariae DHFR-TS genes have not been sequenced yet, our PCR results indicate that the 58 or 38 end or both ends of the DHFR domain of the latter two human malaria species are different and, under stringent conditions, our primers do not amplify other homologous genes. Second, DNA sequence alignment suggests that nucleotide sequences that are immediately upstream of the key codon 117 in the P. vivax DHFR-TS gene or its corresponding codons in other malaria species so far studied are not conserved; so the same restriction sites do not exist in different malaria species. The rapid survey described in this paper was based essentially on imported malaria cases in France. Despite the limited number of P. vivax isolates studied in this present work, the aim of which was to develop a simple PCR-RFLP protocol, an interesting trend was observed. Thirteen P. vivax isolates with mutant-type Asn-117 originated from endemic areas (French Guiana, Cambodia, and Burma) where pyrimethamine, alone or in combination with sulfadoxine, was deployed massively in the past (Peters 1987). The other five P. vivax isolates (plus one isolate from Burma) that we studied originated from areas (Djibouti, Madagascar, Comoros Islands, Yemen, and Pakistan) where antifolate drugs have not been used extensively. These preliminary results are in agreement with the hypothesis that the epidemiology of drug-resistant malaria parasites is associated with a massive distribution of an antimalarial drug in an endemic area, probably due to the selection of resistant mutant parasites (Peters 1987). Although the limited number of isolates does not allow us to draw any conclusion
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PYRIMETHAMINE-RESISTANT P. vivax AND MUTATION
FIG. 2. Ser-to-Asn substitution at codon 117 revealed by PCRRFLP on a 1.5% agarose gel. Genomic DNA of 7 P. vivax clinical isolates from various geographic origins and 12 field isolates from Dawae, Myanmar, was extracted by phenol–chloroform mixture. On the basis of our nucleotide sequence of the P. vivax DHFR-TS gene, two oligonucleotide primers were designed to amplify the entire DHFR domain (711 base pairs plus four nucleotides downstream) of the gene: 58 -ATGGAGGACCTTTCAGATGTATT-38 (forward primer), corresponding to nucleotides 1–23, and 58-CCACCTTGCTGTAAACCAAAAAGTCCAGAG-38 (reverse primer), corresponding to nucleotides 715–686. PCR was performed with the following reaction mixture: 15 pmol of each primer, approximately 0.5–1.0 mg of total DNA (contaminated with human leukocyte DNA), 1.5 mM MgCl2, 50mM KCl, 10mM Tris-HCl buffer (pH 8.4), 200 mM deoxynucleotides, and 1 unit of Taq DNA polymerase in a total volume of 50 ml. The thermocycler was programmed to run 30 cycles, with each cycle consisting of denaturation (958C for 1 min), annealing (508C for 1 min), and elongation (728C for 2 min), with an additional 15 min of elongation phase after the last cycle. One-fourth of the total volume (12.5 ml) of the PCR product was digested with Pvu II restriction enzyme (20 units; New England Biolabs, Beverly, MA) at 378C for 3 h in a total volume of 50 ml. Digestion products (7 ml) were loaded in 1.5% agarose gel and resolved by electrophoresis. The gel was stained with ethidium bromide, and the bands were revealed by ultraviolet transillumination. A wild-type isolate with Ser-117 is indicated by digestion of the PCR product into two fragments (350 and 365 bp; indicated by “B” in the figure). Because a mutant isolate does not possess a restriction site for Pvu II enzyme, the original fragment (715 bp) remains intact (indicated by “A” in the figure). PCR products were cloned directly into pMOS Blue T-vector (Amersham International, Buckinghamshire, UK) and transformed into competent Escherichia coli XL-1 Blue strain. Recombinant plasmid was purified by alkaline lysis method for primer-directed DNA sequencing by dideoxy chain termination method. Lane 1, PHI/ Djibouti; lane 2, Burma-9; lane 3, DUF/French Guiana; lane 4, Burma7; lane 5, Burma-5; lane 6, Burma-6; lane 7, Burma-4; lane 8, COU/ Yemen; lane 9, ARI/Pakistan; lane 10, Burma-2; lane 11, Burma-1; L, 1-kb ladder. The size difference of the digestion products of PHI/ Djibouti (lane 1) is 33 bp, instead of 15 bp in Burma-6 (lane 6), COU/ Yemen (lane 8), and ARI/Pakistan (lane 9), because of an 18-bp deletion in the repetitive sequence within the DHFR domain, as determined by DNA sequencing.
on the epidemiology of drug-resistant P. vivax, our results may stimulate malaria researchers to conduct a more thorough epidemiological survey using simple techniques to detect pyrimethamine-resistant genotype. The PCR-RFLP technique requires basic equipment and reagents that are commonly found in even a modest molecular biology laboratory in malaria endemic countries. The method is relatively rapid, with results available within 24–48 h, and applies basic molecular biology techniques that do not require highly specialized skills. These advantages may favor the application of the PCR-RFLP technique in reference laboratories in endemic areas. The method developed in this paper is similar to the PCR-RFLP technique that we developed to determine codons 108 and 16 of the P. falciparum DHFR-TS gene (Eldin de ´ Pecoulas et al. 1995). The robustness of our technique was shown by successfully applying it to a large number of P. falciparum field isolates ´ (Eldin de Pecoulas et al. 1996; Basco and Ringwald 1998). The reliability of our technique was subsequently confirmed by other authors using the same technique to detect codon 108 of the P. falciparum DHFRTS gene (Curtis et al. 1996; Zindrou et al. 1996). The same approach was developed to detect the key point mutation in the pfmdr 1 gene (Frean et al. 1992). The PCR-RFLP technique described in this report has numerous advantages compared with other molecular techniques: robustness, rapidity, simplicity, high reproducibility, relatively low cost, and use of nonradioactive reagents. This technique may thus be suitable for pursuing molecular studies in reference laboratories in endemic countries with the aim of establishing a correlation between the presence of the putative key point mutation at codon 117 of the P. vivax DHFR-TS gene and in vitro or in vivo pyrimethamine sensitivity in field isolates of P. vivax or both.
ACKNOWLEDGMENTS
We thank Professor Jean-Pierre Coulaud (Department of Infectious ˆ and Tropical Diseases, Hopital Bichat-Claude Bernard, Paris), for providing P. vivax, P. ovale, and P. malariae isolates, and Professor Philippe ˆ Brasseur (Laboratoire de Parasitologie, Hopital Charles Nicolle, ´ ´ Rouen) and Dr. Pierre Druilhe (Unite de Parasitologie Experimentale, Institut Pasteur, Paris), for facilitating collection of blood samples in Burma. Rachida Tahar was supported by the Fondation pour la Recher´ che Medicale. Leonardo Basco was supported by UNDP/World Bank/ WHO Special Programme for Research and Training in Tropical Diseases (TDR). This work was supported in part by grants from AUPELF´ UREF (Action Concertee pour la Recherche).
REFERENCES
Baird, J. K., Basri, H., Subianto, B., Fryauff, D. J., McElroy, P. D., Leksana, B., Richie, T. L., Masbar, S., Wignall, F. S., and Hoffman, S. L. 1995. Treatment of chloroquine-resistant Plasmodium vivax with chloroquine and primaquine or halofantrine. Journal of Infectious Diseases 171, 1678–1682. Basco L. K., and Le Bras J. 1994. Short-term in vitro culture of
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Plasmodium vivax and P. ovale for drug susceptibility testing. Parasitology Research 80, 262–264.
point mutation in Plasmodium falciparum isolates. Transactions of the Royal Society of Tropical Medicine and Hygiene 90, 181–182.
Basco, L. K., and Ringwald, P. (1998). Molecular epidemiology of ´ malaria in Yaounde, Cameroon. I. Analysis of point mutations in the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum. American Journal of Tropical Medicine and Hygiene 58, 369–373.
Foote, S. J., Galatas, D., and Cowman, A. F. 1990. Amino acids in the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum involved in cycloguanil resistance differ from those involved in pyrimethamine resistance. Proceedings of the National Academy of Science USA 87, 3014–3017.
Basco, L. K., Eldin de Pecoulas, P., Wilson, C. M., Le Bras, J., and Mazabraud, A. 1995. Point mutations in the dihydrofolate reductasethymidylate synthase gene and pyrimethamine and cycloguanil resistance in Plasmodium falciparum. Molecular and Biochemical Parasitology 69, 135–138.
Frean, J. A., Awad El Kariem, F. M., Warhurst, D. C., and Miles, M. A. 1992. Rapid detection of pfmdr 1 mutations in chloroquineresistant Plasmodium falciparum malaria by polymerase chain reaction analysis of blood spots. Transactions of the Royal Society of Tropical Medicine and Hygiene 86, 29–30.
Brockelman, C. R., Tan-Ariya, P., and Bunnag, D. (1989). Development of in vitro microtest for the assessment of Plasmodium vivax sensitivity to chloroquine. Southeast Asian Journal of Tropical Medicine and Public Health 20, 41–47.
Peters, W. 1987. “Chemotherapy and Drug Resistance in Malaria.” 2nd ed. Academic Press, London.
Cheng, Q., and Saul, A. 1994. The dihydrofolate reductase domain of rodent malarias: Point mutations and pyrimethamine resistance. Molecular and Biochemical Parasitology 65, 361–363. Cowman, A. F., and Lew, A. M. 1989. Antifolate drug selection results in duplication and rearrangement of chromosome 7 in Plasmodium chabaudi. Molecular and Cellular Biology 9, 5182–5188. Cowman, A. F., Morry, M. J., Biggs, B. A., Cross, G. A., and Foote, S. J. 1988. Amino acid changes linked to pyrimethamine resistance in the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum. Proceedings of the National Academy of Science USA 85, 9109–9113. Curtis, J., Duraisingh, M. T., Trigg, J. K., Mbwana, H., Warhurst, D. C., and Curtis, C. F. 1996. Direct evidence that asparagine at position 108 of the Plasmodium falciparum dihydrofolate reductase is involved in resistance to antifolate drugs in Tanzania. Transactions of the Royal Society of Tropical Medicine and Hygiene 90, 678–680.
Peterson, D. S., Milhous, W. K., and Wellems, T. E. 1990. Molecular basis of differential resistance to cycloguanil and pyrimethamine in Plasmodium falciparum malaria. Proceedings of the National Academy of Science USA 87, 3018–3022. Rieckmann, K. H., Davis, D. R., and Hutton, D. C. 1989. Plasmodium vivax resistance to chloroquine? Lancet 2, 1183–1184. Sirawaraporn, W., Sathitkul, T., Sirawaraporn, R., Yuthavong, Y., and Santi, D. V. 1997. Antifolate-resistant mutants of Plasmodium falciparum dihydrofolate reductase. Proceedings of the National Academy of Sciences USA 94, 1124–1129. Van Dijk, M. R., McConkey, G. A., Vinkenoog, R., Waters, A. P., and Janse, C. J. 1994. Mechanisms of pyrimethamine resistance in two different strains of Plasmodium berghei. Molecular and Biochemical Parasitology 68, 167–171. Walter, R. D. 1991. Folate metabolism as a target for chemotherapy of malaria. In “Biochemical Protozoology” (G. H. Coombs and M. J. North, Eds.), pp. 560–568. Taylor & Francis, London.
Eldin de Pecoulas, P., Basco, L. K., Abdallah, B., Dje, M. K., Le Bras, J., and Mazabraud, A. 1995. Plasmodium falciparum: Detection of antifolate resistance by mutation-specific restriction enzyme digestion. Experimental Parasitology 80, 483–487.
Zindrou, S., Le, D. D. Pham, T. X,. Nguyen, P. D., Nguyen, D. S., Skold, O., and Swedberg, G. 1996. Rapid detection of pyrimethamine susceptibility of Plasmodium falciparum by restriction endonuclease digestion of the dihydrofolate reductase gene. American Journal of Tropical Medicine and Hygiene 54, 185–188.
Eldin de Pecoulas, P., Basco, L. K., Le Bras, J., and Mazabraud, A. 1996. Association between antifol resistance in vitro and DHFR
Received 28 August 1997; accepted with revision 30 December 1997
89, 343–346 (1998)
PR974281
RESEARCH BRIEF Plasmodium vivax: Rapid Detection by Polymerase Chain Reaction and Restriction Fragment Length Polymorphism of the Key Mutation in Dihydrofolate Reductase–Thymidylate Synthase Gene Associated with Pyrimethamine Resistance
Rachida Tahar,1 Philippe Eldin de Pecoulas, Andre Mazabraud, Leonardo K. Basco2 ´ ´ ´ Centre de Genetique Moleculaire, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette, France
Tahar, R., Eldin de Pecoulas, P., Mazabraud, A., and Basco, L. K. 1998. Plasmodium vivax: Rapid detection by polymerase chain reaction and restriction fragment length polymorphism of the key mutation in dihydrofolate reductase–thymidylate synthase gene associated with pyrimethamine resistance. Experimental Parasitology 89, 343– 346. q 1998 Academic Press Index Descriptors and Abbreviations: malaria; Plasmodium falciparum; drug resistance; point mutation; chloroquine; sulfadoxine; ACD, acid citrate-dextrose; EDTA, ethylene diaminetetraacetic acid; DHFR, dihydrofolate reductase; DHFR-TS dihydrofolate reductase thmidylate synthase; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphisme.
Clinical studies conducted in Asia and South America, essentially in the 1950s and 1960s, suggested that Plasmodium vivax developed resistance to pyrimethamine, alone or in combination with sulfadoxine, rapidly (Peters 1987). Because P. vivax cannot be propagated in vitro easily, there are no experimental data on its in vitro sensitivity to pyrimethamine (Brockelman et al. 1989; Basco and Le Bras 1994). Furthermore, limited clinical data on pyrimethamine sensitivity of P. vivax are available from recent studies because chloroquine has been the only recommended blood schizontocidal drug for the treatment and 1
To whom correspondence should be addressed. Fax: 33 1 69 82 43 86. E-mail: [email protected]. 2 Present address: Institut Franc¸ais de Recherche Scientifique pour ´ ´ le Developpement en Cooperation (ORSTOM) - Organisation de la ´ Lutte contre les Endemies en Afrique Centrale (OCEAC), B. P. 288, ´ Yaounde, Cameroon.
0014-4894/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
prophylaxis against P. vivax infections. In view of several reports from Asia and South America on the development of chloroquine resistance in P. vivax (Rieckmann et al. 1989; Baird et al. 1995), there are rising concerns that treatment and chemoprophylaxis against P. vivax infections may require alternative antimalarial drugs. The mechanism of action of pyrimethamine is based on the specific inhibition of dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3), leading to the disruption of folate and pyrimidine biosynthesis in malaria parasites (Walter 1991). Several in vivo and in vitro studies have strongly suggested that there is a direct correlation between the presence of specific point mutations in the DHFR domain of the bifunctional dihydrofolate reductase–thymidylate synthase (DHFR-TS) gene and pyrimethamine resistance in P. falciparum, P. berghei, and P. chabaudi (Cowman et al. 1988; Cowman and Lew 1989; Foote et al. 1990; Peterson et al. 1990; Cheng and Saul 1994; Van Dijk et al. 1994; Basco et al. 1995). Furthermore, an analysis based on the X-ray crystallographic structure of prokaryotic and eukaryotic DHFR enzymes has shown that these point mutations result in amino acid substitutions within the active site of the enzyme, which is consistent with the considerable decrease in the enzyme affinity for pyrimethamine in the presence of these mutations (Sirawaraporn et al. 1997). Despite various obstacles that render the comprehension of the molecular basis of pyrimethamine resistance in P. vivax difficult, experience based on P. falciparum and rodent malaria parasites leads us to expect pyrimethamine-resistant P. vivax to possess an equivalent key mutation in the DHFR-TS gene. Recently, we isolated, cloned, and sequenced the entire P. vivax DHFR-TS gene. Alignment with DHFRTS genes of other malaria parasites revealed that the DHFR domain of the P. vivax DHFR-TS gene undergoes a nonsynonymous point mutation at position 117, which is homologous to the key codon 108 in P. falciparum and codon 110 in P. berghei (Fig. 1). In P. vivax, there are two alternative codons at this position: serine (AGC) and asparagine
343
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FIG. 1. Alignment of DNA sequences of the DHFR-TS gene (334–366 nucleotides, P. vivax numbering; 307–339 nucleotides, P. falciparum numbering) showing key point mutations that confer pyrimethamine resistance in malaria parasites. The deduced amino acid sequences are given below the nucleotide sequences. The DHFR-TS sequences were obtained from the following sources: pyrimethamineresistant (R) P. falciparum HB3/Honduras clone (Cowman et al. 1988; Genbank accession number J03772), pyrimethamine-sensitive P. chabaudi adami DS line (Cowman and Lew 1989; accession number M30834), pyrimethamine-sensitive P. berghei ANKA clone (Van Dijk et al. 1994; accession number U12275), pyrimethamine-sensitive P. yoelii 173 strain (Cheng and Saul 1994; accession number L28122), and P. vinckei (Cheng and Saul 1994; accession number L28121). For comparison, the homologous sequence of a wild-type, pyrimethaminesensitive (S) P. falciparum strain is given. The complete nucleotide sequence of the P. vivax DHFR-TS gene was determined in our laboratory (accession number X98123). Because incomplete sequences of the DHFR-TS gene of P. vinckei and P. yoelii have been determined, their numbering is not known. Amino acid residue 117 deduced from the P. vivax DHFR-TS gene sequence corresponds to amino acid residues 108, 106, and 110 in the P. falciparum, P. chabaudi, and P. berghei homologues, respectively. The direct association between the Ser-toAsn point mutation at these homologous codons and in vitro or in vivo pyrimethamine resistance has been demonstrated in P. falciparum and rodent malaria parasites in previous studies. The restriction site of Pvu II is indicated by a vertical bar.
(AAC). Because pyrimethamine-sensitive P. falciparum, P. berghei, P. chabaudi, and P. vinckei are characterized by serine at the homologous position and serine-to-asparagine substitution confers resistance to pyrimethamine in these malaria parasites, we hypothesize that pyrimethamine sensitivity in P. vivax is also associated with Ser-117, whereas pyrimethamine resistance is associated with Asn-117. On the basis of these findings, we designed a simple, rapid, and reproducible protocol to detect the presence of either Ser- or Asn-117 in the P. vivax DHFRTS gene with the ultimate aim of facilitating future studies to establish a direct correlation between phenotype and genotype in P. vivax field isolates. With the use of our PCR protocol and primer pairs that are specific for the P. vivax DHFR-TS gene, negative controls, with or without human DNA, and controls with parasite DNA from P. falciparum, P. ovale, and P. malariae yielded no visible bands. All samples containing
TAHAR ET AL.
P. vivax DNA samples yielded a single band of 715 bp. No false negative results were obtained from samples containing P. vivax DNA. After PCR amplification of the DHFR domain of the P. vivax DHFRTS gene, PCR products were treated with the restriction enzyme Pvu II. The restriction site (CAG|CTG) is present at only one site within the amplified fragment if a given parasite is a wild type (Ser-117). A mutant isolate does not possess the restriction site for Pvu II. A typical example of the PCR-RFLP results is presented in Fig. 2. Eleven Burmese field isolates and two other clinical isolates (DUF/French Guiana, LFT/Cambodia) had Asn-117. Six isolates (ARI/Pakistan, CRV/Yemen, PHI/Djibouti, HRT/Comoros, PIT/Madagascar, one Burmese isolate) displayed the wild-type codon Ser-117. None of the isolates studied showed mixed genotype. DNA sequencing confirmed the results derived from the PCR-RFLP technique. To determine the capability of the method to distinguish the two genotypes in a mixed isolate, we mixed small amounts of the two types of genes ARI (Ser 117) and a Burma (Asn 117). As little as 1 pg of DNA can be used to drive the Burmese isolate Asn 117 PCR. However, starting with 1 ng of a mixture of the two DNAs, 1/10 of either type was easily detectable, and 1/100 was at the limit of the assay sensitivity. The PCR-RFLP method described in this paper allows a rapid, reliable, and reproducible detection of the key codon of the P. vivax DHFR-TS associated with pyrimethamine resistance. Analysis of the DNA sequence of the P. vivax DHFR-TS gene and restriction sites showed that only two commonly available endonucleases, Alu I and Pvu II, cut within codon Asn-117. Of these two enzymes, Alu I possesses numerous other restriction sites within the DHFR domain, rendering its use impractical for the interpretation of results after agarose gel electrophoresis. The restriction enzyme Pvu II has one other restriction site within the TS domain of the gene. In our protocol, PCR amplification was designed to limit the number of restriction sites of Pvu II to a single site. The specificity of the technique is ensured at two levels. First, PCR amplification using species-specific oligonucleotide primers yielded positive results only from samples containing P. vivax DNA. Even though P. ovale and P. malariae DHFR-TS genes have not been sequenced yet, our PCR results indicate that the 58 or 38 end or both ends of the DHFR domain of the latter two human malaria species are different and, under stringent conditions, our primers do not amplify other homologous genes. Second, DNA sequence alignment suggests that nucleotide sequences that are immediately upstream of the key codon 117 in the P. vivax DHFR-TS gene or its corresponding codons in other malaria species so far studied are not conserved; so the same restriction sites do not exist in different malaria species. The rapid survey described in this paper was based essentially on imported malaria cases in France. Despite the limited number of P. vivax isolates studied in this present work, the aim of which was to develop a simple PCR-RFLP protocol, an interesting trend was observed. Thirteen P. vivax isolates with mutant-type Asn-117 originated from endemic areas (French Guiana, Cambodia, and Burma) where pyrimethamine, alone or in combination with sulfadoxine, was deployed massively in the past (Peters 1987). The other five P. vivax isolates (plus one isolate from Burma) that we studied originated from areas (Djibouti, Madagascar, Comoros Islands, Yemen, and Pakistan) where antifolate drugs have not been used extensively. These preliminary results are in agreement with the hypothesis that the epidemiology of drug-resistant malaria parasites is associated with a massive distribution of an antimalarial drug in an endemic area, probably due to the selection of resistant mutant parasites (Peters 1987). Although the limited number of isolates does not allow us to draw any conclusion
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PYRIMETHAMINE-RESISTANT P. vivax AND MUTATION
FIG. 2. Ser-to-Asn substitution at codon 117 revealed by PCRRFLP on a 1.5% agarose gel. Genomic DNA of 7 P. vivax clinical isolates from various geographic origins and 12 field isolates from Dawae, Myanmar, was extracted by phenol–chloroform mixture. On the basis of our nucleotide sequence of the P. vivax DHFR-TS gene, two oligonucleotide primers were designed to amplify the entire DHFR domain (711 base pairs plus four nucleotides downstream) of the gene: 58 -ATGGAGGACCTTTCAGATGTATT-38 (forward primer), corresponding to nucleotides 1–23, and 58-CCACCTTGCTGTAAACCAAAAAGTCCAGAG-38 (reverse primer), corresponding to nucleotides 715–686. PCR was performed with the following reaction mixture: 15 pmol of each primer, approximately 0.5–1.0 mg of total DNA (contaminated with human leukocyte DNA), 1.5 mM MgCl2, 50mM KCl, 10mM Tris-HCl buffer (pH 8.4), 200 mM deoxynucleotides, and 1 unit of Taq DNA polymerase in a total volume of 50 ml. The thermocycler was programmed to run 30 cycles, with each cycle consisting of denaturation (958C for 1 min), annealing (508C for 1 min), and elongation (728C for 2 min), with an additional 15 min of elongation phase after the last cycle. One-fourth of the total volume (12.5 ml) of the PCR product was digested with Pvu II restriction enzyme (20 units; New England Biolabs, Beverly, MA) at 378C for 3 h in a total volume of 50 ml. Digestion products (7 ml) were loaded in 1.5% agarose gel and resolved by electrophoresis. The gel was stained with ethidium bromide, and the bands were revealed by ultraviolet transillumination. A wild-type isolate with Ser-117 is indicated by digestion of the PCR product into two fragments (350 and 365 bp; indicated by “B” in the figure). Because a mutant isolate does not possess a restriction site for Pvu II enzyme, the original fragment (715 bp) remains intact (indicated by “A” in the figure). PCR products were cloned directly into pMOS Blue T-vector (Amersham International, Buckinghamshire, UK) and transformed into competent Escherichia coli XL-1 Blue strain. Recombinant plasmid was purified by alkaline lysis method for primer-directed DNA sequencing by dideoxy chain termination method. Lane 1, PHI/ Djibouti; lane 2, Burma-9; lane 3, DUF/French Guiana; lane 4, Burma7; lane 5, Burma-5; lane 6, Burma-6; lane 7, Burma-4; lane 8, COU/ Yemen; lane 9, ARI/Pakistan; lane 10, Burma-2; lane 11, Burma-1; L, 1-kb ladder. The size difference of the digestion products of PHI/ Djibouti (lane 1) is 33 bp, instead of 15 bp in Burma-6 (lane 6), COU/ Yemen (lane 8), and ARI/Pakistan (lane 9), because of an 18-bp deletion in the repetitive sequence within the DHFR domain, as determined by DNA sequencing.
on the epidemiology of drug-resistant P. vivax, our results may stimulate malaria researchers to conduct a more thorough epidemiological survey using simple techniques to detect pyrimethamine-resistant genotype. The PCR-RFLP technique requires basic equipment and reagents that are commonly found in even a modest molecular biology laboratory in malaria endemic countries. The method is relatively rapid, with results available within 24–48 h, and applies basic molecular biology techniques that do not require highly specialized skills. These advantages may favor the application of the PCR-RFLP technique in reference laboratories in endemic areas. The method developed in this paper is similar to the PCR-RFLP technique that we developed to determine codons 108 and 16 of the P. falciparum DHFR-TS gene (Eldin de ´ Pecoulas et al. 1995). The robustness of our technique was shown by successfully applying it to a large number of P. falciparum field isolates ´ (Eldin de Pecoulas et al. 1996; Basco and Ringwald 1998). The reliability of our technique was subsequently confirmed by other authors using the same technique to detect codon 108 of the P. falciparum DHFRTS gene (Curtis et al. 1996; Zindrou et al. 1996). The same approach was developed to detect the key point mutation in the pfmdr 1 gene (Frean et al. 1992). The PCR-RFLP technique described in this report has numerous advantages compared with other molecular techniques: robustness, rapidity, simplicity, high reproducibility, relatively low cost, and use of nonradioactive reagents. This technique may thus be suitable for pursuing molecular studies in reference laboratories in endemic countries with the aim of establishing a correlation between the presence of the putative key point mutation at codon 117 of the P. vivax DHFR-TS gene and in vitro or in vivo pyrimethamine sensitivity in field isolates of P. vivax or both.
ACKNOWLEDGMENTS
We thank Professor Jean-Pierre Coulaud (Department of Infectious ˆ and Tropical Diseases, Hopital Bichat-Claude Bernard, Paris), for providing P. vivax, P. ovale, and P. malariae isolates, and Professor Philippe ˆ Brasseur (Laboratoire de Parasitologie, Hopital Charles Nicolle, ´ ´ Rouen) and Dr. Pierre Druilhe (Unite de Parasitologie Experimentale, Institut Pasteur, Paris), for facilitating collection of blood samples in Burma. Rachida Tahar was supported by the Fondation pour la Recher´ che Medicale. Leonardo Basco was supported by UNDP/World Bank/ WHO Special Programme for Research and Training in Tropical Diseases (TDR). This work was supported in part by grants from AUPELF´ UREF (Action Concertee pour la Recherche).
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Received 28 August 1997; accepted with revision 30 December 1997