Am. J. Trop. Med. Hyg., 61(3), 1999, pp. 457±462 Copyright ᭧ 1999 by The American Society of Tropical Medicine and Hygiene

POINT MUTATIONS IN DIHYDROFOLATE REDUCTASE AND DIHYDROPTEROATE SYNTHASE GENES OF PLASMODIUM FALCIPARUM ISOLATES FROM VENEZUELA

LUDMEL URDANETA, CHRISTOPHER PLOWE, IRA GOLDMAN, AND ALTAF A. LAL Escuela de Malariologia y Saneamiento Ambiental Dr. Arnoldo Gabaldon, Maracay, Estado Aragua, Venezuela; Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Diseases Control and Prevention, Atlanta, Georgia; Molecular Parasitology and Malaria Field Studies Unit, Center for Vaccine Development/Division of Geographic Medicine, University of Maryland School of Medicine, Baltimore, Maryland

Abstract. The present study was designed to characterize mutations in dihydrofolate reductase (DHFR) and dihy- dropteroate synthase (DHPS) genes of Plasmodium falciparum in the Bolivar region of Venezuela, where high levels of clinical resistance to sulfadoxine-pyrimethamine (SP, Fansidar௡; F. Hoffman-La Roche, Basel, Switzerland) has been documented. We used a nested mutation-speci®c polymerase chain reaction and restriction digestion methods to measure 1) the prevalence of DHFR mutations at 16, 50, 51, 59, 108, and 164 codon positions, and 2) the prevalence of mutations in the 436, 437, 581, and 613 codon sites in DHPS gene. In the case of the DHFR gene, of the 54 parasite isolates analyzed, we detected the presence of Asn-108 and Ile-51 in 96% of the isolates and Arg-50 mutation in 64% of the isolates. Each of these mutations has been associated with high level of resistance to pyrimethamine. Only 2 samples (4%) showed the wild type Ser-108 mutation and none showed Thr-108 and Val-16 mutations that are speci®c for resistance to cycloguanil. In the case of DHPS gene, we found a mutation at position 437 (Gly) in 100% of the isolates and Gly-581 in 96% of the isolates. The simultaneous presence of mutations Asn-108 and Ile- 51 in the DHFR gene and Gly-437 and Gly-581 in the DHPS gene in 96% of the samples tested suggested that a cumulative effect of mutations could be the major mechanism conferring high SP resistance in this area.

Morbidity and mortality due to Plasmodium falciparum at position 140 in the DHFR gene was found in the isolate malaria has continued to increase in the past few years. VP8, which also has the characteristic changes at positions While there are several factors that have contributed to the Val-16 and Thr-108, which confer cycloguanil resistance.11 increase in malaria globally, the failure to control the disease In the case of the DHPS gene from P. falciparum lines re- in many parts of the world is partially due to decreased sus- sistant to the most commonly used sulpha drug (sulfadox- ceptibility of the malaria parasites to once effective anti- ine), amino acid changes at the 436, 437, 581, and 613 co- malarial drugs, such as chloroquine. Numerous documented dons have been shown to be associated with resistance.12,13 reports of high-grade chloroquine resistance have prompted A recent study has shown association between mutation at governments in sub-Saharan Africa to reconsider their anti- codon 540 with sulfadoxine resistance.14 malarial treatment guidelines.1 The requirement for alterna- The sulpha and pyrimethamine drugs have been used in tive drugs has led to the extensive use of the sulfadoxine- the treatment of uncomplicated malaria in Venezuela over pyrimethamine (SP) combination drug in the treatment of the past 20 years. Several in vitro and in vivo studies have uncomplicated malaria. documented failures of SP in the treatment of uncomplicated One of the concerns with the use of SP is the rapid de- malaria15 (Caraballo A, unpublished data). Recent drug ef- velopment of resistance. In Thailand, SP replaced chloro- ®cacy studies in the malaria-endemic regions of Bolivar and quine in the early 1970s, but within 5 years, radical cure Amazonas States have revealed 100% resistance to several rates by day 28 had decreased from 83% in 1975 to 22% in drugs such as chloroquine, amodiaquine, and SP (Riggione 1979.2 The long half-life of sulfadoxine and pyrimethamine F, unpublished data). During the period of sample collection, (180 and 95 hr) favor the selection of resistant parasites, and SP was the ®rst-line treatment used by the Malaria Program there is a general concern that in Africa, where the intensity in uncomplicated P. falciparum malaria in Venezuela, where of malaria transmission is considerably higher, the effective chloroquine-resistance predominates. The clinical and para- life of SP may be even shorter. sitologic evidence of a high degree of resistance to SP led Molecular characterization of the DHFR and DHPS genes us to initiate a molecular epidemiologic study to determine has revealed a correlation between pyrimethamine resistance the prevalence of mutations in DHFR and DHPS that have and the following point mutations: an amino acid substitu- been associated with resistance to sulfadoxinepyrimethamine tion at residue 108 from Ser to Asn, mutations at codon 51 in the Bolivar region of Venezuela. that changes Asn to Ile, and a mutation at codon 59 that changes Cys to Arg. A threonine at position 108 paired with MATERIALS AND METHODS an alanine to valine change at position 16 is associated with resistance to cycloguanil. Previous studies have demonstrat- Study area. The sample collection was conducted during ed that a change from Ile to Leu at position 164, coupled the rainy season in 1995 in several communities of the ma- with Asn-108 plus Ile-51 and/or Arg-59 confers high-level larious region of Bolivar State in a tropical, humid forest in resistance to both pyrimethamine and cycloguanil.3±8 New southeastern Venezuela. Sixty-seven fresh isolates of P. fal- mutations in the DHFR gene at position 50 (Cys to Arg) and ciparum were collected from malaria-infected miners and a ®ve amino acid repetitive insert between positions 30 and Amerindians, who had moderate parasitemia detected by mi- 31 have been found to be highly prevalent in Bolivia, where croscopy and had not received drugs during the previous 14 SP resistance is high.9,10 Recently, a novel leucine mutation days. The parasites were cultured for 24±36 hr16 to increase 457 458 URDANETA AND OTHERS the number of parasites, and they were immediately cryo- and 613 was detected using a set of speci®c primers previ- preserved. Clones with known mutations in the DHFR and ously published.20 Genomic DNA (10±20 ng) was ampli®ed DHPS genes were used as controls for polymerase chain following conditions for a nested PCR as described.9 The reaction (PCR) assays and digestion with restriction en- mutations at codon position 436 and 437 in DHPS gene were zymes. studied for enzymatic digestion of the DHPS domain am- The study protocols were approved by the Biomedical pli®ed with the 185 and 218 primers. We used Msp AII, Committee of Malariology of the Ministry of Health of Ven- which exclusively cleaves the Ala-436 mutant, and Ava II, ezuela. which exclusively cleaves the Gly-437 mutant type;9 the di- Extraction of genomic DNA and mutation-speci®c gestions were done in 40-␮l reactions according to manu- PCR assays. The parasite genomic DNA was extracted from facturer's (New England Biolabs, Inc.) speci®cations. All the infected red blood cells using proteinase K, followed by ex- DHPS samples were analyzed by electrophoresis on a 1.5% traction with phenol-chloroform and ethanol precipitation. agarose gel stained with ethidium bromide. Analysis of mutations at the DHFR 16 and 108 sites was done using the PCR method followed by restriction enzyme RESULTS digestion as previously described,17 with the following mod- i®cations: 50±100 ng of genomic DNA, 1 ␮M of each prim- From the 67 P. falciparum isolates collected, we were able er, 200 ␮M of each DNTP, buffer (50 mM KCl, 10 mM Tris, to amplify the DHFR and DHPS genes from 54 parasite pH 8.4, 1.5 mM MgCl2, 0.1 mg/ml of gelatin), and 2.5 units isolates. Allelic frequencies of DHFR and DHPS mutations of Taq polymerase were used in a 100-␮l reaction. The de- were determined for these 54 samples. naturation was performed at 94ЊC for 5 min, the samples The screening of 16 and 108 codon sites in the DHFR were then incubated at 94ЊC for 45 sec, 50ЊC for 1 min, gene was done by enzymatic digestion of the ampli®ed 72ЊC for 2 min for 40 cycles, and a ®nal extension was done DHFR domain with Alu I (for Ser-108), Bsr I (for Asn-108), at 72ЊC for 3 min. The PCR product was puri®ed with Wiz- Scrf I (for Thr-108), and Nla III (for Ala-16 or Val-16). The ard PCR preps (Promega, Madison, WI) and resuspended in digestion of the 708 basepair (bp) of the DHFR domain pro- TE buffer (0.1 M Tris, 0.1 M EDTA, pH 8.0). The restriction duced the characteristic restriction patterns of 322 and 386 endonucleases Alu I, Bsr I, and Scrf I, which exclusively bp for Alu I, Bsr I, and Scrf I. cleave the Ser-108 (wild), the Asn-108 (mutant), and the The 108-Asn mutant type was detected by digestion with Thr-108 (mutant), respectively, were used to digest the am- Bsr I on Venezuelan P. falciparum isolates (Figure 1a; lanes pli®ed DHFR fragment. The enzyme Nla III was used to 3, 7, and 11), and the wild type 108-Ser was revealed by digest Ala-16 (wild) or Val-16 (mutant), showing a pattern digestion with Alu I on a parasite isolate and the 3D7 ref- characteristic for each one as described.17 Digestions were erence clone (Figure 1b; lanes 1 and 5). The 108-Thr and done in 40-␮l reactions containing 10 ␮l of puri®ed PCR 16-Val mutant types present in the FCR3 reference clone fragments according to the manufacturer's (New England were revealed by digestion with Scrf I (Figure 1b; lane 10) Biolabs. Inc., Beverly, MA) speci®cations. The digested and Nla III, respectively (Figure 1b; lane 12 producing the fragments were separated by electrophoresis on a 1.5% aga- characteristic 568- and 140-bp fragments). From 54 DNA rose gel and stained with ethidium bromide for ultraviolet samples of P. falciparum tested, 96% of the isolates con- visualization. The codon sites 50, 51, 59, and 164 in the tained DHFR genes with the Asn-108 mutation and 4% DHFR gene were studied using methods previously de- showed the wild type Ser-108 residue. No sample showed scribed.9±18,19 The DHFR domain was ®rst ampli®ed in a pri- the Thr-108 or Val-16 mutations, which have been linked mary round of PCR using the primers AMPI/AMP2 as de- with cycloguanil resistance. The prevalence of mutations at scribed.18 An aliquot of 0.1±0.5 ␮l of the ®rst ampli®cation 50, 51, 59, and 164 codon sites in the DHFR gene was was used in a mutation-speci®c second round PCR to detect investigated using a nested mutation-speci®c PCR. The Arg- the DHFR mutation sites 51, 59, and 164, as described.9±18 50 mutation (a new mutation recently reported from Bolivian For the analysis of the DHFR 50 codon, we used mutation- malaria-endemic areas with high rates of SP resistance9) was speci®c sense primers FR50w (5Ј-GGAGTATTACCA- present in 64% (34 samples), showing a speci®c band of 570 TGGAAAT-3Ј), which speci®cally ampli®es the sequence bp (Figure 2a). The Ile-51 mutation was found in 96% (52 containing the wild type TGT (Cys) codon, and FR50m (5Ј- samples), giving the characteristic positive signal (574 hp) GGAGTATTACCATGGAAAC-3Ј), which is speci®c for the (Figure 2b). The Arg-59- and Leu-164-speci®c mutations mutant codon CGT (Arg); these primers were paired with were not found in any samples tested. the common antisense primer SP219 to give a ®nal product In the case of the DHPS gene, the prevalence of Phe-436, of 570 basepairs. The cycling parameters for second round Gly-581, Thr-613, and Ser-613 was studied by a nested mu- were initial denaturation at 95ЊC for 3 min, followed by 15 tation-speci®c PCR. We found that 52 (96%) of the 54 sam- cycles of denaturation at 92ЊC for 30 sec, annealing at 55ЊC ples carried Gly-581 mutation; a typical signal for this mu- for 45 sec, and extension at 72ЊC for 45 sec, and a ®nal tant type is shown in Figure 3. None of the other mutations extension at 72ЊC for 3 min. For this assay, the MgCl2 was studied was present. The prevalence of Ala-436 and Gly-437 present in the reaction at a concentration of 1.5 mM; the mutations was tested by speci®c-mutation enzyme analysis; other reaction conditions were as described.18 The PCR prod- the digestion of the ampli®ed DHPS domain (Figure 4a) by ucts were analyzed by electrophoresis on a 2% agarose gel Ava II shows that Gly-437 was present in all isolates, show- and stained with ethidium bromide for ultraviolet visualiza- ing the same pro®le as that of the 3D7 mutant reference tion. clone (Figure 4b, lane 11). The DHFR and DHPS mutations The prevalence of DHPS mutations at residues 436, 581, prevalent in Venezuela, in comparison with previous reports POINT MUTATIONS IN DHFR AND DHPS GENES OF P. FALCIPARUM 459

FIGURE 1. Restriction enzyme digestion of the ampli®ed dihydrofolate reductase domain (708 basepairs [bps]) at codons 108 and 16, using restriction enzymes Alu I (lanes 1, 5, and 9), Scrf I (lanes 2, 6, and 10), Brs I (lanes 3, 7, and 11), and Nla III (lanes 4, 8, and 12). a, Venezuelan samples (lanes 1±12) show the mutant type Asn-108 digested with Brs I; absence of the wild type is revealed by no digestion with Alu I; absence of mutant type Thr-108 is shown by no digestion with Scrf I; and the wild type Ala-16 was digested with Nla III (568- and 93-r [bp] fragments). b, Venezuelan isolate (lanes 1±4) and 3D7 clone (lanes 5±8) were used as controls for the wild type; Alu I digested the DNA in lanes 1 and 5. The FCR3 clone (lanes 9±12) was used as a control for the mutant types Thr-108 and Val-16 (568- and 140-bp fragments), Scrf I digested the DNA in lane 10 and Nla III digested the DNA in lane 12. DNA size markers are shown in lanes M. Values on the left are in basepairs. from areas with a high rate of SP failure in Bolivia, Mali, a single Ala to Gly mutation in codon 437. High levels of Kenya, Malawi,9 Brazil,21 Papua New Guinea,22 and Came- sulfadoxine resistance are associated with double (Ser-436, roon,23,24 are shown in Table 1. Gly-437, Gly-581, Ala-613) or triple (Phe-436, Gly-437, Ala-581, Ser-613) DHPS mutations. Although in most of the

DISCUSSION studies, con®rmatory clinical data are not available, the re- sults point to the association of select point mutations in Plasmodium falciparum resistance to SP is conferred by these two genes with resistance. point mutations in parasite DHFR and DHPS, the enzymes In the present study, we have characterized point muta- targeted by these drugs. This allows the use of standard PCR tions in the DHFR and DHPS genes in P. falciparum isolates techniques for the in vitro monitoring of SP ef®cacy. In these from the Bolivar region of Venezuela, where a high level of assays, either point mutation±speci®c primers are used to clinical resistance to SP has been reported. Studies conduct- amplify the gene or point mutation±speci®c enzymes are ed over the past several years in the Bolivar region have used to speci®cally digest a product of ampli®cation. revealed high (up to 100% in some studies) level of in vitro Previous studies that used reference clones and clinical resistance to SP. At the time parasites were collected for this isolates from different parts of the world have revealed that study, treatment failure of SP was also frequently observed. the in vitro antifolate resistance is associated with point mu- The high levels of clinical failure of SP in the treatment of tations in the DHFR domain of the DHFR±thymidylate syn- malaria in this region provided us an opportunity to char- thetase gene. Among the point mutations in the DHFR gene, acterize mutations in the DHFR and DHPS genes that have a Ser to Asn-108 point mutation is considered the key mu- previously been associated with resistance in areas of less tation that confers resistance to antifolate drugs. Other mu- resistance. tations associated with high levels of pyrimethamine resis- Characterization of the DHFR gene of 54 samples showed tance are codons 50, 51, 59, and 164. In the case of in vitro that 96% had Asn-108 and Ile-51 mutations and 64% had resistance to sulfadoxine, moderate resistance is de®ned by an Arg-50 mutation. The prevalence of mutations in the 460 URDANETA AND OTHERS

FIGURE 2. a, Nested mutation-speci®c polymerase chain reaction (PCR) of the dihydrofolate reductase gene showing the Arg-50 mutation in 3 Venezuelan samples (lanes 1±3). A Plasmodium falciparum isolate containing the wild type Cys-50 (lane 4); the positive control containing Arg-50 (lane 5); and the negative control (without DNA) for the PCR (lane 6) are shown. b, Nested mutation-speci®c PCR showing the Ile- 51 mutation from P. falciparum isolates (lanes 1, 2, and 3). The positive control for the mutation was V/1 S (lane 4), a sample containing the wild type Asn-51 is showed in lane 5, and the negative control (without DNA) for the PCR is in lane 6. DNA size markers are shown lanes M. Ampli®cation primers used in this experiment are described in the Methods and Material section. Values are in basepairs.

DHPS gene from 54 samples studied showed 100% for Gly- 437 and 96% for Gly-581. Similar prevalence rates of these mutations have been recently reported from Bolivia (Table 1), where the prevalences of the new mutation Arg-50 in the DHFR gene and Gly-437 and Gly-581 in the DHPS gene correlate with increasing use of SP use and in vivo resis- tance.9 The prevalence of mutations in the DHFR and DHPS genes detected in this study (Table 1) suggests a strong as- sociation with high levels of in vitro resistance and high clinical resistance present in this area. The simultaneous presence of mutations Asn-108, Ile-51, and Arg-50 in the DHFR gene and Gly-437 and Gly-581 in the DHPS gene suggests that this cumulative effect of mutations is a possible

FIGURE 3. Nested mutation-speci®c polymerase chain reaction FIGURE 4. a, Ampli®cation of the dihydropteroate synthase (PCR) (a 756-basepair [bp] fragment) in the dihydropteroate syn- (DHPS) domain (1,330 basepairs) of Plasmodium falciparum from thase gene showing the Gly-581 mutant from 4 Plasmodium falci- Venezuelan samples (lanes 1±10) and the 3D7 clone (lane 11). b, parum isolates (lanes 1±4) and V/ IS clone, (control for the mutant Restriction enzyme digestion of the DHPS domain at the codon 437 type, lane 8). Two Venezuelan samples with Ala-581 wild type site shows digestion by Ava II of the mutant type Gly-437 (lanes 1± (lanes 5 and 6) and 3D7 clone (control for the wild type, lane 7) 10) and the 3D7 clone (mutant control) (lane 11). The digestion of showed the speci®c band of 441 basepairs. The negative control 1,330 basepairs of the DHPS domain generates 849- and 303-base- (without DNA) for the PCR is shown in lane 9. DNA size markers pair bands. DNA size markers are shown lanes M. Values on the are shown in lanes M. Values on the left are in basepairs. left are in basepairs. POINT MUTATIONS IN DHFR AND DHPS GENES OF P. FALCIPARUM 461

TABLE 1 Prevalence of dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) mutations of Venezuelan Plasmodium falciparum isolates in comparison with previous reports*

DHFR DHPS 16 50 51 59 108 164 436 437 581 613 Country Reference Val Arg Ile Arg Asn Leu Ala Gly Gly Ser/Thr Venezuela This study No. 54 54 54 54 54 54 54 54 54 54 % 0 64 96 0 96 0 0 100 96 0 Bolivia 9 No. 0 41 58 58 59 59 34 34 58 59 % ± 51 100 41 100 56 0 100 100 0 Brazil 21 No. ± ± ± ± 42 ± ± ± ± ± % ± ± ± ± 90 ± ± ± ± ± Mali 9 No. ± 30 56 57 58 58 58 32 59 59 % ± 0 5 13 15 0 60 25 0 12 Kenya 9 No. ± 24 56 56 56 56 40 28 55 55 % ± 0 65 55 80 0 8 18 0 0 Malawi 9 No. ± 32 54 54 54 54 43 42 51 51 % ± 0 100 89 96 0 7 62 0 0 Papua New Guinea 22 No. 23 ± 23 23 23 23 20 20 20 20 % 0 ± 0 30 39 0 0 5 0 5 Cameroon 23, 24 No. ± ± ± ± 127 ± 32 32 32 32 % ± ± ± ± 43 ± 66 22 0 3 * No. ϭ total number of samples tested. major mechanism conferring high SP resistance in this re- Authors' addresses: Ludmel Urdaneta, Escuela de Malariologia y gion. As has been reported in others regions where clinical Saneamiento Ambiental Dr. Arnoldo Gabaldon, Maracay, Estado Ar- agua, Venezuela. Christopher Plowe, Molecular Parasitology and resistance to SP is present, we found the same relationship Malaria Field Studies Unit, Center for Vaccine Development/Divi- between DHFR and DHPS mutations and SP resistance in sion of Geographic Medicine, University of Maryland School of our study site in Venezuela (Table 1). Medicine, Baltimore, MD 21201. Ira Goldman and Altaf A. Lal, While the results of the present and previous studies have Division of Parasitic Diseases, National Center for Infectious Dis- provided a signi®cant body of data on the association be- eases, Centers for Diseases Control and Prevention, Mailstop F-12, Atlanta, GA 30341±3724. tween mutations and resistance, there is a need to undertake longitudinal clinical studies for con®rmatory data using par- asites before and after treatment. These studies will be better REFERENCES done in malaria-endemic areas where historical parasite iso- lates are also available for molecular analysis, which will 1. Bloland PB, Lackritz EM, Kazembe PN, Were JB, Steketee R, allow quantitative assessment of the rates of evolution of Campbell CC, 1993. Beyond chloroquine: implications of these genes under selective drug pressure. drug resistance for evaluating malaria therapy ef®cacy and treatment policy in Africa. J Infect Dis 167: 932±937. The need for clinical studies that combine molecular tools 2. White NJ, 1992. Antimalarial drug resistance: the pace quick- in investigations of drug resistance is most urgent where the ens. J Antimicrob Chemother 30: 571±585. impact of clinical resistance of SP will be a major public 3. Snewin VA, England SM, Sims PF, Hyde JE, 1989. Character- health issue. There is general concern that in Africa, where ization of dihydrofolate reductase-thymidylate synthetase the intensity of malaria transmission is considerably higher, gene from human malaria parasites highly resistant to pyri- methamine. Gene 76: 41±52. the effective life of SP may be even shorter. The molecular 4. Zolg JW, Plitt JR, Chen GX, Palmer S, 1989. Point mutations tools used by us and others in characterization of point mu- in dihydrofolate reductase-thymidylate synthase gene as the tations in the DHFR and DHPS genes are easy to use and molecular basis for pyrimethainine resistance in Plasmodium can be performed faster than the time it takes to conduct in falciparum. Mol Biochem Parasitol 36: 253±262. 5. Sirawarapom W, Sirawarapom R, Cowman AF, Yuthavong Y, vitro drug assays. As countries in Africa and elsewhere Santi DV, 1990. Heterologous expression of active thymidyl- move toward replacing chloroquine with SP as the ®rst-line ate synthase-dihydrofolate reductase from Plasmodium falci- drug for the treatment of uncomplicated P. falciparum ma- parum. Biochemistry 29: 10779±10785. laria, studies of emergence and dispersal of resistant phe- 6. Thaithong S, Chan SW, Songsomboon S, Wilairat P, Seesod N, notypes need to be conducted. The data from these molec- Sueblinwong T, Goman M, Ridley R, Beale G, 1992. Pyri- methamine resistant mutations in Plasmodium falciparum. ular epidemiologic studies, which will reveal information on Mol Biochem Parasitol 52: 149±158. evolutionary trends as parasites are forced through drug 7. Foote S, Galatis D, Cowman A, 1990. Amino acids in dihydro- pressure±induced bottleneck, will provide early signals of folate reductase-TS gene of Plasmodium falciparum involved drug failures. in cycloguanil resistance differ from those involved in pyri- methamine resistance. Proc Natl Acad Sci USA 87: 3014± 3017. Disclaimer: Use of trade names is for identi®cation only and does 8. Basco LK, de Pecoulas PE, Wilson CM, Le Bras J, Mazabraud not imply endorsement by the Public Health Service of the U.S. A, 1995. Point mutations in dihydrofolate reductase-thymi- Department of Health and Human Services. dylate synthase gene and pyrimethamine and cycloguanil re- 462 URDANETA AND OTHERS

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