Drug resistance is an important problem hindering malaria elimination in tropical areas. Point mutations in Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes confer resistance to antifolate drug, sulfadoxine-pyrimethamine (SP) while P. falciparum chloroquine-resistant transporter (Pfcrt) genes caused resistance to chloroquine (CQ). Decline in Pfdhfr/Pfdhps and Pfcrt mutations after withdrawal of SP and CQ has been reported. The aim of present study was to investigate the prevalence of Pfdhfr, Pfdhps, and Pfcrt mutation from 2 endemic areas of Thailand. All of 200 blood samples collected from western area (Thai-Myanmar) and southern area (Thai-Malaysian) contained multiple mutations in Pfdhfr and Pfdhps genes. The most prevalent haplotypes for Pfdhfr and Pfdhps were quadruple and double mutations, respectively. The quadruple and triple mutations of Pfdhfr and Pfdhps were common in western samples, whereas low frequency of triple and double mutations was found in southern samples, respectively. The Pfcrt 76T mutation was present in all samples examined. Malaria isolated from 2 different endemic regions of Thailand had high mutation rates in the Pfdhfr, Pfdhps, and Pfcrt genes. These findings highlighted the fixation of mutant alleles causing resistance of SP and CQ in this area. It is necessary to monitor the re-emergence of SP and CQ sensitive parasites in this area.
Although malaria is an ancient disease caused by Plasmodium parasite, it remains important to public health to present era. Plasmodium falciparum infection causes variable clinical symptoms ranging from asymptomatic to severe manifestations. The emergence of resistance of P. falciparum to the available antimalarial drugs is an important factor for malaria control [1]. In Thailand, resistance to many antimalarial drugs, including chloroquine (CQ), sulfadoxine-pyrimethamine (SP), mefloquine, and artemisinin has been reported [2,3]. CQ resistant P. falciparum was reported in the early 1960s [4,5]. In 1973, SP replaced CQ as the first-line treatment for uncomplicated falciparum malaria due to widespread resistance [1,6], but after 10 years, SP was ineffective [1,7]. Then, mefloquine was introduced in 1985 and resistance emerged in the same decade [8]. Artemisinin-based combination therapy was introduced as first-line treatment in 1995 [9].
Molecular epidemiological investigation provides information for detecting the emergence and spread of antimalarial drug resistance. Mutations in the P. falciparum dihydrofolate reductase (Pfdhfr) and P. falciparum dihydropteroate synthase (Pfdhps) genes (at codons 51, 59, 108, and 164 of Pfdhfr and 437, 540, and 581 of Pfdhps) are associated with SP treatment failures [10,11]. The mutations in Pfdhfr and Pfdhps genes were staged, resulting in increased levels of SP drug resistance [12]. The P. falciparum CQ resistance transporter gene (Pfcrt) K76T mutation has been linked to P. falciparum CQ resistance [13].
In some countries, the withdrawal of CQ for P. falciparum treatment, Pfcrt mutation (K76T) gently decreased and disappeared completely [14–17]. Similar to withdrawal of SP for P. falciparum treatment, Pfdhfr and Pfdhps gene mutations also decreased in some countries [18–21]. Conversely, alleles conferring CQ and SP resistance still occur at high frequency after discontinuation of these drugs [22,23]. However, declining of Pfdhfr, Pfdhps, and Pfcrt mutations might be associated with duration of drug withdrawal and geographical differences.
The objective of the present study was to investigate the prevalence of 5 Pfdhfr (A16V, N51I, C59R, S108N/T, and I164L), 5 Pfdhps (S436A, A437G, K540E, A581G, and A613S/T) and 1 Pfcrt (K76T) mutation from 2 different endemic areas of Thailand.
MATERIALS AND METHODS
Ethics approval
The study protocol was reviewed and approved by the Ethics Committee of Thammasat University (COA No. 134/2561). All patients were informed about the study objectives, sampling technique, and the benefits of the study. Informed consents were obtained according to the ethical standards from all patients.
Sample collection
A total of 200 dried blood spot samples were collected during 2007–2017 from patients with P. falciparum infection who attended malaria clinics in the western (Tak Province) and southern (Yala Province) regions along the Thai-Myanmar and Thai-Malaysian border, respectively.
Extraction of parasite genomic DNA
Genomic DNA of all blood samples was prepared using a QIAamp DNA extraction mini-kit (QIAGEN, Valencia, California, USA) according to manufacturer’s instruction and used as a template for polymerase chain reaction (PCR) amplification.
Amplification and detection of the Pfdhfr and Pfdhps
Pfdhfr and Pfdhps genes were amplified by nested PCR using Pfdhfr and Pfdhps specific primers (Table 1) according to the previously described methods with some modification [24]. Briefly, the PCR was carried out with the following reaction mixture including 0.25 μM of each primer, 1.5 mM MgCl2 (Thermo scientific, Waltham, Massachusetts, USA), 1×Taq buffered with KCl (Thermo scientific), 200 μM deoxynucleotides (dNTPs) (Bioline, London, UK), 2 μl of genomic DNA in the primary PCR, and 1 μl of primary PCR product in nested PCR and 1 unit of Taq DNA polymerase (Thermo scientific). All of the PCR products were then analyzed on 1% agarose gel and visualized under UV illuminator. PCR products were digested with restriction enzymes (Table 1) [24] then the restriction fragments were analyzed on 1.2% agarose gel and visualized under UV illuminator.
Amplification and detection of the Pfcrt
Amplification of K76T was performed by nested PCR using Pfcrt specific primers (Table 1) according to the previously described methods with some modification [25–27]. Briefly, the PCR was carried out with the following reaction mixture including 0.1 μM of each primer, 2.5 mM MgCl2 (Thermo scientific), 1×Taq buffered with KCl (Thermo scientific), 100 μM deoxynucleotides (dNTPs) (Bioline), 0.5 μl of genomic DNA in the primary PCR, and 0.5 μl of primary PCR product in nested PCR and 0.5 unit of Taq DNA polymerase (Thermo scientific). All of the PCR products were then analyzed on 1.5% agarose gel and visualized under UV illuminator. PCR products were digested with restriction enzymes ApoI (New England Biolabs Inc., Hertfordshire, UK) (Table 1), as described by the manufacturer. Then the restriction fragments were analyzed on 2.0% agarose gel and visualized under UV illuminator.
Statistical Analysis
Data analysis was performed by SPSS software version 21.0 (IBM Corporation, Armonk, New York, USA). The chi-square test was used to compare the frequencies and correlations of all data. The level of significance was set at P<0.05.
RESULTS
Analysis of Pfdhfr and Pfdhps mutations
A total of 200 P. falciparum samples were successfully amplified and analyzed for both the Pfdhfr and Pfdhps genes. The polymorphisms at each codon were demonstrated by restriction fragments (Fig. 1A, B). The frequencies of Pfdhfr and Pfdhps mutations was summarised in Table 2. All samples had at least 1 codon mutation in the Pfdhfr (A16V, N51I, C59R, S108N/T, and I164L) and Pfdhps (S436A, A437G, K540E, A581G, and A613S/T). Four codon mutations were detected in Pfdhfr (51I, 59R, 108N, and 164L) in samples from 2 areas. All isolates carried mutations at codon 59 and 108 in Pfdhfr. Four codon mutations were detected in Pfdhps (436A, 437G, 540E, and 581G) in samples from Tak Province, whereas 3 codon mutations were detected from Yala Province. All isolates carried wild-type alleles at codon 613 in Pfdhps. Mixed genotypes were detected in thirty isolates by codon 51 and 164 of Pfdhfr, and 436 and 540 of Pfdhps that were no processed further. There was no wildtype allele ANCSI, but 3 alleles (AIRNI, AIRNL, and ANRNL) of Pfdhfr were identified in this study (Table 3). AIRNL was the most prevalent allele (52.4%) in all P. falciparum isolates collected from Tak Province (88.9%) (Fig. 2). AIRNI was the most prevalent allele in P. falciparum isolates from Yala Province (80.9%). Statistical significance was found between 2 study areas (P<0.001). For Pfdhps, 6 alleles were identified (Table 3). SGKGA was the most prevalent allele (50.0%) found in P. falciparum isolates from Yala Province (83.1%). SGEGA was the most prevalent allele in P. falciparum isolates from Tak Province (70.4%). Statistical significance was found between 2 study areas (P<0.001). Eleven allele combination of Pfdhfr-Pfdhps were found in this study (Table 4). The quintuple mutation (AIRNI-SGKGA), which comprise triple mutations in Pfdhfr and 2 mutations in Pfdhps were found to be most prevalent in study population (35.3%) and in isolates from Yala Province (66.3%). The septuple mutation (AIRNL-SGEGA), which comprised quadruple mutations in Pfdhfr and 3 mutations in Pfdhps were most frequent in isolates from Tak Province (64.2%).
Analysis of Pfcrt mutations
A total of 187 samples (93.5%) were analyzed by nested PCR for the Pfcrt K76T gene. The Pfcrt mutation resulting in substitution of threonine (T) for lysine (K) at position 76 was present in all studied samples from 2 endemic areas (Figs. 1C, 2).
DISCUSSION
Mutations on Pfdhfr and Pfdhps genes associated with SP resistance have been reported in several malaria endemic areas such as Guinea [28], Indonesia [29], Malaysia [30], Myanmar [31], and Thailand [32,33]. In Thailand, a previous study revealed that the change of Pfdhfr point mutations from double mutations to triple and quadruple mutations in some areas [34,35] which the number of mutations is correlated with increased level of SP resistance [12]. In the present study, all P. falciparum isolates had at least three mutation point in Pfdhfr genes, indicated persistence of highly mutations on SP resistant markers. The Pfdhps mutation studies conducted between 2001 and 2007 in Thailand indicated that there has been fluctuation of the Pfdhps mutations between triple and quadruple mutations. In this study, predominant frequency of double mutation was found especially in isolates from southern endemic area. The predominance of triple mutations was also found in isolates from western area. This result indicated that high mutation in Pfdhfr and Pfdhps genes with different frequency existed in these 2 different localities. In Thailand, SP was withdrawn from P. falciparum treatment for many years. Although decreased of Pfdhfr and Pfdhps mutations were reported from some countries after the withdrawal of these drugs, high frequency of Pfdhfr and Pfdhps mutations still present in Thailand. This existence of mutations on Pfdhfr and Pfdhps genes may be associated with using of other antifolate drugs that can also induce pressure on Pfdhfr and Pfdhps.
A previous study has demonstrated that high prevalence of Pfcrt K76T mutation in study isolates might contribute to CQ resistance to P. falciparum [36]. A high prevalence rate of Pfcrt K76T mutation was previously observed in several countries such as Mali [25], Kenya [37], Indonesia [26], Philippines [38] and Thailand [39–41]. Declining of Pfcrt mutations after withdrawal of CQ has been reported in Malawi [14], Tanzania [15], Kenya [16], and China [17]. However, even CQ was withdrawn from Thailand for long period, the CQ resistance allele still remains with high frequency. This complete fixation of CQ resistance in P. falciparum is might due to the co-existence of P. falciparum and P. vivax infections in this country while CQ is a standard regimen for P. vivax malaria treatment, leading to the phenomenon of continuous exposure to drug pressure in P. falciparum.
Our study demonstrates a high prevalence of Pfdhfr, Pfdhps, and Pfcrt mutations of P. falciparum isolates from 2 endemic areas in Thailand, emphasizing the fixation of mutant Pfdhfr, Pfdhps and Pfcrt alleles that confer consistent resistance of SP and CQ. SP and CQ drugs are still not appropriate for P. falciparum treatment in Thailand and other antimalarial groups should be considered.
ACKNOWLEDGMENT
This research was supported by the Program Management Unit for Human Resources and Institutional Development, Research and Innovation, NXPO [grant number B05F630043].
The authors have no conflict of interest.
REFERENCES
Na-BangchangKCongpuongKCurrent malaria status and distribution of drug resistance in East and Southeast Asia with special focus to ThailandTohoku J Exp Med200721199113https://doi.org/10.1620/tjem.211.99WongsrichanalaiCPickardALWernsdorferWHMeshnickSREpidemiology of drug-resistant malariaLancet Infect Dis20022209218https://doi.org/10.1016/s1473-3099(02)00239-6NoedlHSocheatDSatimaiWArtemisinin-resistant malaria in AsiaN Engl J Med2009361540541https://doi.org/10.1056/NEJMc0900231HarinasutaTSuntharasamaiPViravanCChloroquine resistant falciparum malaria in ThailandLancet1965286657660https://doi.org/10.1016/s0140-6736(65)90395-8YoungMDContacosPGStitcherJEMillarJWDrug resistance in Plasmodium falciparum from ThailandAm J Trop Med Hyg196312305314https://doi.org/10.4269/ajtmh.1963.12.305ChinWBearDMColwellEJKosakalSA comparative evaluation of sulfalene-trimethoprim and sulphormethoxine-pyrimethamine against falciparum malaria in ThailandAm J Trop Med Hyg197322308312https://doi.org/10.4269/ajtmh.1973.22.308JohnsonDERoendejPWilliamsRGFalciparum malaria acquired in the area of the Thai-Khmer border resistant to treatment with FansidarAm J Trop Med Hyg198231907912https://doi.org/10.4269/ajtmh.1982.31.907WongsrichanalaiCSirichaisinthopJKarwackiJJCongpuongKMillerRSPangLThimasarnKDrug resistant malaria on the Thai-Myanmar and Thai-Cambodian bordersSoutheast Asian J Trop Med Public Health2001324149WHOMekong Malaria Programme Malaria in the Greater Mekong Subregion: Regional and Country ProfilesWorld Health OrganizationGeneva, Switzerland2008http://www.whothailand.org/EN/Section3/Section113.htmReederJCRieckmannKHGentonBLorryKWinesBCowmanAFPoint mutations in the dihydrofolate reductase and dihydropteroate synthetase genes and in vitro susceptibility to pyrimethamine and cycloguanil of Plasmodium falciparum isolates from Papua New GuineaAm J Trop Med Hyg199655209213https://doi.org/10.4269/ajtmh.1996.55.209BascoLKEldin de PecoulasPWilsonCMLe BrasJMazabraudAPoint mutations in the dihydrofolate reductase-thymidylate synthase gene and pyrimethamine and cycloguanil resistance in Plasmodium falciparumMolBiochem Parasitol199569135138https://doi.org/10.1016/0166-6851(94)00207-4PloweCVCorteseJFDjimdeANwanyanwuOCWatkinsWMWinstanleyPAEstrada-FrancoJGMollinedoREAvilaJCCespedesJLCarterDDoumboOKMutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase and epidemiologic patterns of pyrimethamine–sulfadoxine use and resistanceJ Infect Dis199717615901596https://doi.org/10.1086/514159BabikerHAPringleSJAbdel-MuhsinAMackinnonMHuntPWallikerDHigh-level chloroquine resistance in Sudanese isolates of Plasmodium falciparum is associated with mutations in the chloroquine resistance transporter gene pfcrt and the multidrug resistance gene pfmdr1J Infect Dis200118315351538https://doi.org/10.1086/320195KublinJGCorteseJFNjunjuEMMukadamRAWirimaJJKazembePNDjimdéAAKouribaBTaylorTEPloweCVReemergence of chloroquine-sensitive Plasmodium falciparum malaria after cessation of chloroquine use in MalawiJ Infect Dis200318718701875https://doi.org/10.1086/375419MohammedANdaroAKalingaAManjuranoAMoshaJFMoshaDFvan ZwetselaarMKoenderinkJBMoshaFWAlifrangisMReyburnHRoperCKavisheRATrends in chloroquine resistance marker, Pfcrt-K76T mutation ten years after chloroquine withdrawal in TanzaniaMalar J201312415https://doi.org/10.1186/1475-2875-12-415MwaiLOchongEAbdirahmanAKiaraSMWardSKokwaroGSasiPMarshKBorrmannSMackinnonMNzilaAChloroquine resistance before and after its withdrawal in KenyaMalaria J20098106https://doi.org/10.1186/1475-2875-8-106WangXMuJLiGChenPGuoXFuLChenLSuXWellemsTEDecreased prevalence of the Plasmodium falciparum chloroquine resistance transporter 76T marker associated with cessation of chloroquine use against P. falciparum malaria in Hainan, People’s Republic of ChinaAm J Trop Med Hyg200572410414McCollumAMMuellerKVillegasLUdhayakumarVEscalanteAACommon origin and fixation of Plasmodium falciparum dhfr and dhps mutations associated with sulfadoxine-pyrimethamine resistance in a low-transmission area in South AmericaAntimicrob Agents Chemother20075120852091https://doi.org/10.1128/AAC.01228-06HailemeskelEKassaMTaddesseGMohammedHWoyessaATasewGSleshiMKebedeAPetrosBPrevalence of sulfadoxine-pyrimethamine resistance-associated mutations in dhfr and dhps genes of Plasmodium falciparum three years after SP withdrawal in Bahir Dar, Northwest EthiopiaActa Trop2013128636641https://doi.org/10.1016/j.actatropica.2013.09.010PearceRJOrdRKaurHLupalaCSchellenbergJShirimaKManziFAlonsoPTannerMMshindaHRoperCSchellenbergDA community-randomized evaluation of the effect of intermittent preventive treatment in infants on antimalarial drug resistance in southern TanzaniaJ Infect Dis2013207848859https://doi.org/10.1093/infdis/jis742RamanJSharpBKleinschmidtIRoperCStreatEKellyVBarnesKIDifferential effect of regional drug pressure on dihydrofolate reductase and dihydropteroate synthetase mutations in southern MozambiqueAm J Trop Med Hyg200878256261https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3748784KhimNBouchierCEkalaMTIncardonaSLimPLegrandEJambouRDoungSPuijalonOMFandeurTCountrywide survey shows very high prevalence of Plasmodium falciparum multilocus resistance genotypes in CambodiaAntimicrob Agents Chemother20054931473152https://doi.org/10.1128/AAC.49.8.3147-3152.2005McCollumAMMuellerKVillegasLUdhayakumarVEscalanteAACommon origin and fixation of Plasmodium falciparum dhfr and dhps mutations associated with sulfadoxine-pyrimethamine resistance in a low-transmission area in South AmericaAntimicrob Agents Chemother20075120852091https://doi.org/10.1128/AAC.01228-06DuraisinghMTCurtisJWarhurstDCPlasmodium falciparum: detection of polymorphisms in the dihydrofolate reductase and dihydropteroate synthetase genes by PCR and restriction digestionExp Parasitol19988918https://doi.org/10.1006/expr.1998.4274DjimdéADoumboOKCorteseJFKayentaoKDoumboSDiourtéYCoulibalyDDickoASuXZNomuraTFidockDAWellemsTEPloweCVA molecular marker for chloroquine-resistant falciparum malariaN Engl J Med2001344257263https://doi.org/10.1056/NEJM200101253440403MaguireJDAIKrisinSusantiSismadiPFryauffDJBairdJKThe T76 mutation in the pfcrt gene of Plasmodium falciparum and clinical chloroquine resistance phenotypes in Papua, IndonesiaAnn Trop Med Parasitol200195559572https://doi.org/10.1080/00034980120092516GolassaLEnwejiNErkoBAseffaASwedbergGDetection of a substantial number of submicroscopic Plasmodium falciparum infections by polymerase chain reaction: a potential threat to malaria control and diagnosis in EthiopiaMalar J201312352https://doi.org/10.1186/1475-2875-12-352JiangTChenJFuHWuKYaoYEyiJUMMatesaRAObonoMMODuWTanHLinMLiJHigh prevalence of Pfdhfr–Pfdhps quadruple mutations associated with sulfadoxine–pyrimethamine resistance in Plasmodium falciparum isolates from Bioko Island, Equatorial GuineaMalar J201918101https://doi.org/10.1186/s12936-019-2734-xBasukiSFitriahRiyantoSBudionoDachlanYPUemuraHTwo novel mutations of pfdhps K540T and I588F, affecting sulphadoxine-pyrimethamine-resistant response in uncomplicated falciparum malaria at Banjar district, South Kalimantan Province, IndonesiaMalar J201413135https://doi.org/10.1186/1475-2875-13-135LauTYSylviMWilliamTMutational analysis of Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase genes in the interior division of Sabah, MalaysiaMalar J201312445https://doi.org/10.1186/1475-2875-12-445ZhaoYLiuZSoeMTWangLSoeTNWeiHThanAAungPLLiYZhangXHuYWeiHZhangYBurgessJSiddiquiFAMenezesLWangQKyawMPCaoYCuiLGenetic variations associated with drug resistance markers in asymptomatic Plasmodium falciparum infections in MyanmarGenes201910692https://doi.org/10.3390/genes10090692AlamMTVinayakSCongpuongKWongsrichanalaiCSatimaiWSlutskerLEscalanteAABarnwellJWUdhayakumarVTracking origins and spread of sulfadoxine-resistant Plasmodium falciparum dhps alleles in ThailandAntimicrob Agents Chemother2011155164https://doi.org/10.1128/AAC.00691-10SugaramRSuwannasinKKunasolCMathemaVBDayNPJSudathipPPrempreePDondorpAMImwongMMolecular characterization of Plasmodium falciparum antifolate resistance markers in Thailand between 2008 and 2016Malar J202019107https://doi.org/10.1186/s12936-020-03176-xImwongMJindakhadTKunasolCSutawongKVejakamaPDondorpAMAn outbreak of artemisinin resistant falciparum malaria in Eastern ThailandSci Rep2015517412https://doi.org/10.1038/srep17412KrudsoodSImwongMWilairatanaPPukrittayakameeSNonprasertASnounouGWhiteNJLooareesuwanSArtesunate–dapsone–proguanil treatment of falciparum malaria: genotypic determinants of therapeutic responseTrans R Soc Trop Med Hyg200599142149https://doi.org/10.1016/j.trstmh.2004.07.001SetthaudomCTan-ariyaPSitthichotNKhositnithikulRSuwandittakulNLeelayoovaSMungthinMRole of Plasmodium falciparum chloroquine resistance transporter and multidrug resistance 1 genes on in vitro chloroquine resistance in isolates of Plasmodium falciparum from ThailandAm J Trop Med Hyg201185606611https://doi.org/10.4269/ajtmh.2011.11-0108HolmgrenGGilJPFerreiraPMVeigaMIObonyoCOBjörkmanAAmodiaquine resistant Plasmodium falciparum malaria in vivo is associated with selection of pfcrt 76T and pfmdr1 86YInfect Genet Evol20066309314https://doi.org/10.1016/j.meegid.2005.09.001ChenNKyleDEPasayCFowlerEVBakerJPetersJMChengQpfcrt allelic types with two novel amino acid mutations in chloroquine-resistant Plasmodium falciparum isolates from the PhilippinesAntimicrob Agents Chemother20034735003505https://doi.org/10.1128/AAC.47.11.3500-3505.2003LopesDRungsihirunratKNogueiraFSeugornAGilJPdo RosárioVECravoPMolecular characterisation of drug-resistant Plasmodium falciparum from ThailandMalaria J2002112https://doi.org/10.1186/1475-2875-1-12CongpuongKNa BangchangKMungthinMBualombaiPWernsdorferWHMolecular epidemiology of drug resistance markers of Plasmodium falciparum malaria in ThailandTrop Med Int Health20058717722https://doi.org/10.1111/j.1365-3156.2005.01450.xRungsihirunratKChaijareonkulWSeugornANa-BangchangKThaithongSAssociation between chloroquine resistance phenotypes and point mutations in pfcrt and pfmdr1 in Plasmodium falciparum isolates from ThailandActa Trop20091093740https://doi.org/10.1016/j.actatropica.2008.09.011
Figures and Tables
The polymorphism of pfdhfr (A), pfdhps (B), and pfcrt (C) gene by gel electrophoresis. W-wildtype, M-mutant, S/N-serine/threonine, Mi-mixed, K1-P. falciparum K1 strain, 3D7- P. falciparum 3D7 strain, U-undigested fragment. Fragment sizes in base pair (bp) are shown.
The proportions of mutations in 3 resistance genes (pfdhfr, pfdhps, and pfcrt) observed in P. falciparum isolates in this study.
The primers and enzymes for genotyping of Pfdhfr, Pfdhps and Pfcrt genes
Gene
PCR
Primer
Primer sequence (5′ to 3′)
RFLP position
Restrictionenzyme
PCR size (bp)
Restriction product size (bp)
Wild type
Mutation
Pfdhfr
Primary
M1
TTTATGATGGAACAAGTCTGC
M5
AGTATATACATCGCTAACAGA
Secondary
M3
TTTATGATGGAACAAGTCTGCGACGTT
A16V
Nlalll
522
376, 93, 53
376, 146
(16, 51, 108, 164)
F/
AAATTCTTGATAAACAACGGAACCTTTTA
N51I
MluCI
154, 120, 65, 55
218, 120, 65, 55
S108T
BstNI
522
181, 145
S108N
Bsrl
522
332, 190
I164L
DraI
245, 171, 107
245, 143, 107, 27
Secondary
F
GAAATGTAATTCCCTAGATATGGAATATT
C59R
Xmnl
326
189, 137
163, 137, 26
(59)
M4
TTAATTTCCCAAGTAAAACTATTAGAGCTTC
Pfdhps
Primary
R2
AACCTAAACGTGCTGTTCAA
R/
AATTGTGTGATTTGTCCACAA
Secondary
K
TGCTAGTGTTATAGATATAGGATGAGCATC
S436A
MnlI
438
317, 121
278, 121, 39
(436, 437, 540)
K/
CTATAACGAGGTATTGCATTTAATGCAAGAA
A437G
AvaII
438
404, 34
K540E
FokI
405, 33
320, 85, 33
Secondary
L
ATAGGATACTATTTGATATTGGACCAGGATTCG
A581G
BslI
161
161
128, 33
(581, 613)
L/
TATTACAACATTTTGATCATTCGCGCAACCGG
A613S
BsaWI
161
131, 30
A613T
AgeI
161
128, 33
Pfcrt
Primary
CRTP1
CCGTTAATAATAAATACACGCAG
CRTP2
CGGATGTTACAAAACTATAGTTACC
Secondary
CRTD1
TGTGCTCATGTGTTTAAACTT
K76T
ApoI
134
100, 34
134
CRTD2
CAAAACTATAGTTACCAATTTTG
Prevalence of Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) single nucleotide polymorphisms (SNPs) in 200 P. falciparum isolates from 2 endemic areas of Thailand
Gene
Amino acid position
SNPs
Prevalence (%)
P-value
Total n=200
Tak Province n=100
Yala Province n=100
Pfdhfr
16
A (wild-type)
200 (100.0)
100 (100.0)
100 (100.0)
-
V (mutant)
0 (0.0)
0 (0.0)
0 (0.0)
51
N (wild-type)
3 (1.5)
3 (3.0)
0 (0.0)
0.001a
I (mutant)
187 (93.5)
87 (87.0)
100 (100.0)
M (mix)
10 (5.0)
10 (10.0)
0 (0.0)
59
C (wild-type)
0 (0.0)
0 (0.0)
0 (0.0)
-
R (mutant)
200 (100.0)
100 (100.0)
100 (100.0)
108
S (wild-type)
0 (0.0)
0 (0.0)
0 (0.0)
-
T (mutant)
0 (0.0)
0 (0.0)
0 (0.0)
N (mutant)
200 (100.0)
100 (100.0)
100 (100.0)
164
I (wild-type)
84 (42.0)
6 (6.0)
78 (78.0)
<0.001a
L (mutant)
103 (51.5)
86 (86.0)
17 (17.0)
M (mix)
13 (6.5)
8 (8.0)
5 (5.0)
Pfdhps
436
S (wild-type)
158 (79.0)
79 (79.0)
79 (79.0)
0.946
A (mutant)
29 (14.5)
14 (14.0)
15 (15.0)
M (mix)
13 (6.5)
7 (7.0)
6 (6.0)
437
A (wild-type)
0 (0.0)
0 (0.0)
0 (0.0)
-
G (mutant)
200 (100.0)
100 (100.0)
100 (100.0)
540
K (wild-type)
116 (58.0)
16 (16.0)
100 (100.0)
<0.001a
E (mutant)
83 (41.5)
83 (83.0)
0 (0.0)
M (mix)
1 (0.5)
1 (1.0)
0 (0.0)
581
A (wild-type)
7 (3.5)
7 (7.0)
0 (0.0)
0.007a
G (mutant)
193 (96.5)
93 (93.0)
100 (100.0)
613
A (wild-type)
100 (100.0)
100 (100.0)
100 (100.0)
-
S/T (mutant)
0 (0.0)
0 (0.0)
0 (0.0)
aP-value were statistically significant between 2 areas.
Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) alleles in 170 P. falciparum isolates from 2 endemic areas of Thailand
Pfdhfr haplotypesa
Amino acid position
Prevalence (%)
16
51
59
108
164
Total n=170
Tak Province n=81
Yala Province n=89
Triple mutation
A
I
R
N
I
78 (45.9)
6 (7.4)
72 (80.9)
Triple mutation
A
N
R
N
L
3 (1.8)
3 (3.7)
0 (0.0)
Quadruple mutation
A
I
R
N
L
89 (52.4)
72 (88.9)
17 (19.1)
Pfdhps haplotypesa
Amino acid position
Prevalence (%)
436
437
540
581
613
Total n=170
Tak Province n=81
Yala Province n=89
Double mutation
S
G
E
A
A
2 (1.2)
2 (2.5)
0 (0.0)
Double mutation
S
G
K
G
A
85 (50.0)
11 (13.6)
74 (83.1)
Triple mutation
A
G
E
A
A
2 (1.2)
2 (2.5)
0 (0.0)
Triple mutation
A
G
K
G
A
16 (9.4)
1 (1.2)
15 (16.9)
Triple mutation
S
G
E
G
A
57 (33.5)
57 (70.4)
0 (0.0)
Quadruple mutation
A
G
E
G
A
8 (4.7)
8 (9.9)
0 (0.0)
aP-value were statistically significant between 2 areas.
Allele combinations of Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) in 170 P. falciparum isolates from 2 endemic areas of Thailand
