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    (A) Annual numbers of Plasmodium falciparum–infected cases in Thailand during 1990 and 2016. Vertical broken demarcation lines indicate the period of nationwide implementation of antimalarial treatment regimens: MSP, mefloquine combined with sulfadoxine and pyrimethamine; M, mefloquine monotherapy; 2-day artemisinin combination therapy (ACT), artesunate plus mefloquine for 2 days; 3-day ACT, 2-day ACT plus artesunate on day 3. (B) Biannual frequencies of mutant Plasmodium falciparum chloroquine resistance transporter (Pfcrt) haplotypes (spots) relative to the Dd2 sequence (GenBank accession no. AF030694). Correlation between frequencies of mutant haplotypes and years of sample collection (during 1991 and 201 represented in broken line: Pearson’s correlation coefficient, r = 0.382; P = 0.350, and during 2003 and 2016 shown in solid line: r = 0.780; P = 0.038).

  • 1.

    Wellems TE, Plowe CV, 2001. Chloroquine-resistant malaria. J Infect Dis 184: 770776.

  • 2.

    Wongsrichanalai C, Pickard AL, Wernsdorfer WH, Meshnick SR, 2002. Epidemiology of drug-resistant malaria. Lancet Infect Dis 2: 209218.

  • 3.

    Fidock DA 2000. Mutations in the P. falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Mol Cell 6: 861871.

    • Search Google Scholar
    • Export Citation
  • 4.

    Ecker A, Lehane AM, Clain J, Fidock DA, 2012. PfCRT and its role in antimalarial drug resistance. Trends Parasitol 28: 504514.

  • 5.

    Petersen I 2015. Balancing drug resistance and growth rates via compensatory mutations in the Plasmodium falciparum chloroquine resistance transporter. Mol Microbiol 97: 381395.

    • Search Google Scholar
    • Export Citation
  • 6.

    Gabryszewski SJ 2016. Evolution of fitness cost-neutral mutant PfCRT conferring P. falciparum 4-aminoquinoline drug resistance is accompanied by altered parasite metabolism and digestive vacuole physiology. PLoS Pathog 12: e1005976.

    • Search Google Scholar
    • Export Citation
  • 7.

    Callaghan PS, Hassett MR, Roepe PD, 2015. Functional comparison of 45 naturally occurring isoforms of the Plasmodium falciparum chloroquine resistance transporter (PfCRT). Biochemistry 54: 50835094.

    • Search Google Scholar
    • Export Citation
  • 8.

    Agrawal S 2017. Association of a novel mutation in the Plasmodium falciparum chloroquine resistance transporter with decreased piperaquine sensitivity. J Infect Dis 216: 468476.

    • Search Google Scholar
    • Export Citation
  • 9.

    Parker D, Lerdprom R, Srisatjarak W, Yan G, Sattabongkot J, Wood J, Sirichaisinthop J, Cui L, 1994. Longitudinal in vitro surveillance of Plasmodium falciparum sensitivity to common anti-malarials in Thailand between 1994 and 2010. Malar J 11: 290.

    • Search Google Scholar
    • Export Citation
  • 10.

    Chaijaroenkul W, Ward SA, Mungthin M, Johnson D, Owen A, Bray PG, Na-Bangchang K, 2011. Sequence and gene expression of chloroquine resistance transporter (Pfcrt) in the association of in vitro drugs resistance of Plasmodium falciparum. Malar J 10: 42.

    • Search Google Scholar
    • Export Citation
  • 11.

    Takahashi N 2012. Large-scale survey for novel genotypes of Plasmodium falciparum chloroquine-resistance gene Pfcrt. Malar J 11: 92.

  • 12.

    Harinasuta T, Suntharasamai P, Viravan C, 1965. Chloroquine-resistant falciparum malaria in Thailand. Lancet 2: 657660.

  • 13.

    Malikul S, 2000. Malariology 1999 in Commemoration of 50 Years of Malaria Control in Thailand. Bangkok, Thailand: The Agricultural Co-operative Federation of Thailand Press (in Thai).

  • 14.

    Thimasarn K, Sirichaisinthop J, Vijaykadga S, Tansophalaks S, Yamokgul P, Laomiphol A, Palananth C, Thamewat U, Tháithong S, Rooney W, 1995. In vivo study of the response of Plasmodium falciparum to standard mefloquine/sulfadoxine/pyrimethamine (MSP) treatment among gem miners returning from Cambodia. Southeast Asian J Trop Med Public Health 26: 204212.

    • Search Google Scholar
    • Export Citation
  • 15.

    Wongsrichanalai C, Prajakwong S, Meshnick SR, Shanks GD, Thimasarn K, 2004. Mefloquine-its 20 years in the Thai malaria control program. Southeast Asian J Trop Med Public Health 35: 300308.

    • Search Google Scholar
    • Export Citation
  • 16.

    Zhou G, Sirichaisinthop J, Sattabongkot J, Jones J, Bjørnstad ON, Yan G, Cui L, 2005. Spatio-temporal distribution of Plasmodium falciparum and P. vivax malaria in Thailand. Am J Trop Med Hyg 72: 256262.

    • Search Google Scholar
    • Export Citation
  • 17.

    World Health Organisation, 2014. Status Report on Artemisinin Resistance: September 2014. Geneva, Switzerland: WHO.

  • 18.

    Putaporntip C, Kuamsab N, Kosuwin R, Tantiwattanasub W, Vejakama P, Sueblinvong T, Seethamchai S, Jongwutiwes S, Hughes AL, 2016. Natural selection of K13 mutants of Plasmodium falciparum in response to artemisinin combination therapies in Thailand. Clin Microbiol Infect 285: e1e8.

    • Search Google Scholar
    • Export Citation
  • 19.

    Jongwutiwes S, Buppan P, Kosuvin R, Seethamchai S, Pattanawong U, Sirichaisinthop J, Putaporntip C, 2011. Plasmodium knowlesi malaria in humans and macaques, Thailand. Emerg Infect Dis 17: 17991806.

    • Search Google Scholar
    • Export Citation
  • 20.

    Sidhu AB, Verdier-Pinard D, Fidock DA, 2002. Chloroquine resistance in Plasmodium falciparum malaria parasites conferred by Pfcrt mutations. Science 298: 210213.

    • Search Google Scholar
    • Export Citation
  • 21.

    Johnson DJ, Fidock DA, Mungthin M, Lakshmanan V, Sidhu AB, Bray PG, Ward SA, 2004. Evidence for a central role for PfCRT in conferring Plasmodium falciparum resistance to diverse antimalarial agents. Mol Cell 15: 867877.

    • Search Google Scholar
    • Export Citation
  • 22.

    Dhingra SK 2017. A variant PfCRT isoform can contribute to Plasmodium falciparum resistance to the first-line partner drug piperaquine. MBio 8: e00303e00317.

    • Search Google Scholar
    • Export Citation
  • 23.

    Conrad MD 2014. Comparative impacts over 5 years of artemisinin-based combination therapies on Plasmodium falciparum polymorphisms that modulate drug sensitivity in Ugandan children. J Infect Dis 210: 344353.

    • Search Google Scholar
    • Export Citation
  • 24.

    Hemming-Schroeder E 2018. Impacts of antimalarial drugs on Plasmodium falciparum drug resistance markers, western Kenya, 2003–2015. Am J Trop Med Hyg 98: 692699.

    • Search Google Scholar
    • Export Citation
  • 25.

    Tumwebaze P 2017. Changing antimalarial drug resistance patterns identified by surveillance at three sites in Uganda. J Infect Dis 215: 631635.

    • Search Google Scholar
    • Export Citation
  • 26.

    Putaporntip C, Buppan P, Jongwutiwes S, 2011. Improved performance with saliva and urine as alternative DNA sources for malaria diagnosis by mitochondrial DNA-based PCR assays. Clin Microbiol Infect 17: 14841491.

    • Search Google Scholar
    • Export Citation
  • 27.

    Librado P, Rozas J, 2009. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25: 14511452.

  • 28.

    Tamura K, Stecher G, Peterson D, Filipski A, Kumar S, 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30: 27252729.

    • Search Google Scholar
    • Export Citation
  • 29.

    Martin DP, Murrell B, Khoosal A, Muhire B, 2017. Detecting and analyzing genetic recombination using RDP4. Methods Mol Biol 1525: 433460.

  • 30.

    Tajima F, 1983. Evolutionary relationship of DNA sequences in finite populations. Genetics 105: 437460.

  • 31.

    Excoffier L, Lischer HE, 2010. Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows. Mol Ecol Resour 10: 564567.

    • Search Google Scholar
    • Export Citation
  • 32.

    Looareesuwan S, Viravan C, Webster HK, Kyle DE, Hutchinson DB, Canfield CJ, 1996. Clinical studies of atovaquone, alone or in combination with other antimalarial drugs, for treatment of acute uncomplicated malaria in Thailand. Am J Trop Med Hyg 54: 6266.

    • Search Google Scholar
    • Export Citation
  • 33.

    Thaithong S, Beale GH, 1981. Resistance of ten Thai isolates of Plasmodium falciparum to chloroquine and pyrimethamine by in vitro tests. Trans R Soc Trop Med Hyg 75: 271273.

    • Search Google Scholar
    • Export Citation
  • 34.

    Foote SJ, Kyle DE, Martin RK, Oduola AM, Forsyth K, Kemp DJ, Cowman AF, 1990. Several alleles of the multidrug-resistance gene are closely linked to chloroquine resistance in Plasmodium falciparum. Nature 345: 255258.

    • Search Google Scholar
    • Export Citation
  • 35.

    Durrand V, Berry A, Sem R, Glaziou P, Beaudou J, Fandeur T, 2004. Variations in the sequence and expression of the Plasmodium falciparum chloroquine resistance transporter (Pfcrt) and their relationship to chloroquine resistance in vitro. Mol Biochem Parasitol 136: 273285.

    • Search Google Scholar
    • Export Citation
  • 36.

    Jongwutiwes S, Putaporntip C, Hughes AL, 2010. Bottleneck effects on vaccine-candidate antigen diversity of malaria parasites in Thailand. Vaccine 28: 31123117.

    • Search Google Scholar
    • Export Citation
  • 37.

    Kublin JG, Cortese JF, Njunju EM, Mukadam RA, Wirima JJ, Kazembe PN, Djimdé AA, Kouriba B, Taylor TE, Plowe CV, 2003. Reemergence of chloroquine-sensitive Plasmodium falciparum malaria after cessation of chloroquine use in Malawi. J Infect Dis 187: 18701875.

    • Search Google Scholar
    • Export Citation
  • 38.

    Hughes AL, 2008. Near neutrality: leading edge of the neutral theory of molecular evolution. Ann N Y Acad Sci 1133: 162179.

  • 39.

    Nash D, Nair S, Mayxay M, Newton PN, Guthmann JP, Nosten F, Anderson TJ, 2005. Selection strength and hitchhiking around two anti-malarial resistance genes. Proc Biol Sci 272: 11531161.

    • Search Google Scholar
    • Export Citation
  • 40.

    Hughes AL, Packer B, Welch R, Bergen AW, Chanock SJ, Yeager M, 2003. Widespread purifying selection at polymorphic sites in human protein-coding loci. Proc Natl Acad Sci USA 100: 1575415757.

    • Search Google Scholar
    • Export Citation
  • 41.

    Price MN, Arkin AP, 2015. Weakly deleterious mutations and low rates of recombination limit the impact of natural selection on bacterial genomes. MBio 6: e01302e01315.

    • Search Google Scholar
    • Export Citation

 

 

 

 

Multiple Novel Mutations in Plasmodium falciparum Chloroquine Resistance Transporter Gene during Implementation of Artemisinin Combination Therapy in Thailand

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  • 1 Molecular Biology of Malaria and Opportunistic Parasites Research Unit, Department of Parasitology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand;
  • 2 Inter-Department Program of Biomedical Sciences, Faculty of Graduate School, Chulalongkorn University, Bangkok, Thailand;
  • 3 Department of Biology, Faculty of Science, Naresuan University, Pitsanulok Province, Thailand;
  • 4 Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand

Mutations in the chloroquine resistance transporter gene of Plasmodium falciparum (Pfcrt) are associated with drug susceptibility status of chloroquine and other antimalarials that interfere with heme detoxification process including artemisinin. We aim to investigate whether an increase in duration of artemisinin combination therapy (ACT) in Thailand could affect mutations in Pfcrt. The complete coding sequences of Pfcrt and dihydrofolate reductase (Pfdhfr), and size polymorphisms of the merozoite surface proteins-1 and 2 (Pfmsp-1 and Pfmsp-2) of 189 P. falciparum isolates collected during 1991 and 2016 were analyzed. In total, 12 novel amino acid substitutions and 13 novel PfCRT haplotypes were identified. The most prevalent haplotype belonged to the Dd2 sequence and no wild type was found. A significant positive correlation between the frequency of Pfcrt mutants and the year of sample collection was observed during nationwide ACT implementation (r = 0.780; P = 0.038). The number of haplotypes and nucleotide diversity of isolates collected during 3-day ACT (2009–2016) significantly outnumbered those collected before this treatment regimen. Positive Darwinian selection occurred in the transmembrane domains only among isolates collected during 3-day ACT but not among those collected before this period. No remarkable change was observed in the molecular indices for other loci analyzed when similar comparisons were performed. An increase in the duration of artesunate in combination therapy in Thailand could exert selective pressure on the Pfcrt locus, resulting in emergence of novel variants. The impact of these novel haplotypes on antimalarial susceptibilities requires further study.

INTRODUCTION

A worldwide effort to control malaria has been highly reliant on antimalarial chemotherapy; therefore, drug resistance in malaria parasites can substantially preclude effective therapeutic and preventive interventions. One of the most devastating impacts of this phenomenon has been widely concerned since the emergence and spread of chloroquine resistance in Plasmodium falciparum, the most prevalent and malignant malaria species in the tropics. To date, the global dispersal of chloroquine-resistant P. falciparum has rendered the drug ineffective in almost all disease-endemic countries.1,2

Plasmodium falciparum chloroquine resistance transporter or chloroquine resistance transporter of P. falciparum is a 48.6-kDa transmembrane protein on the digestive vacuolar membrane that confers chloroquine resistance.3 It is encoded by a single copy 13-exon gene, Pfcrt, comprising 424 codons. A point mutation at codon 76 (K76T) is a key determinant of chloroquine resistance phenotype.35 However, the hitherto mutation per se may not be the sole predictor of drug resistance, whereas specific substitutions beyond codon 76 could modulate drug susceptibility status or compensate for the fitness costs of chloroquine-resistant parasites.3,5,6 To date, at least 30 amino acid substitutions and ∼51 haplotypes were identified in PfCRT of natural isolates.7,8 In Thailand, all P. falciparum isolates collected during the past two decades exhibited chloroquine-resistant phenotypes, and the key mutations at codons 72–76 of the Pfcrt gene revealed CVIET as the most predominant haplotype.911

Antimalarial treatment in Thailand has been relied on parasite-based diagnosis by microscopy, whereas antimalarial drug policies have been implemented and changed in response to therapeutic efficacy based on national surveillance system. Chloroquine was introduced to treat malaria caused by all Plasmodium species in 1945 and was implemented nationwide in 1965 despite the first report on drug resistance in falciparum malaria patients in 1957.12,13 Because of the widespread occurrence of chloroquine resistance in P. falciparum, national treatment policy was changed to the combination of sulfadoxine and pyrimethamine (SP) in 1974 and was implemented until 1981 when antifolate resistance deteriorated in most endemic areas of the country.14 During 1982 and 1985, quinine plus tetracycline were used as a standard treatment regimen and was replaced by the combination of mefloquine and SP because of the problem of drug compliance. Because the lack of evidence for delaying mefloquine resistance in combination therapy and the adverse effects from SP, mefloquine monotherapy was initiated in 1990, fully implemented in 1992, and used until 1994 when severe deterioration of mefloquine efficacy occurred.1416 During 1995 and 2008, artemisinin combination therapy (ACT) with mefloquine as a partner drug was deployed as a 2-day treatment regimen (Figure 1). Because of emergence and increasing tendency in the treatment failure rate of 2-day ACT, treatment with 3-day ACT regimen was initiated in 2008 and completely implemented nationwide in 2009.17 However, a recent increase in treatment failure of 3-day ACT has prompted the change in treatment regimen to dihydroartemisinin plus piperaquine in 2017 and fully implemented in 2018. Nevertheless, chloroquine has been used as the first-line drug to treat vivax and other non-falciparum malaria since its first implementation in 1965. Importantly, about 8–10% of vivax malaria patients had submicroscopic P. falciparum infection and were treated with chloroquine.18,19 Therefore, it is likely that a small proportion of P. falciparum populations in Thailand could have been exposed to chloroquine.

Figure 1.
Figure 1.

(A) Annual numbers of Plasmodium falciparum–infected cases in Thailand during 1990 and 2016. Vertical broken demarcation lines indicate the period of nationwide implementation of antimalarial treatment regimens: MSP, mefloquine combined with sulfadoxine and pyrimethamine; M, mefloquine monotherapy; 2-day artemisinin combination therapy (ACT), artesunate plus mefloquine for 2 days; 3-day ACT, 2-day ACT plus artesunate on day 3. (B) Biannual frequencies of mutant Plasmodium falciparum chloroquine resistance transporter (Pfcrt) haplotypes (spots) relative to the Dd2 sequence (GenBank accession no. AF030694). Correlation between frequencies of mutant haplotypes and years of sample collection (during 1991 and 201 represented in broken line: Pearson’s correlation coefficient, r = 0.382; P = 0.350, and during 2003 and 2016 shown in solid line: r = 0.780; P = 0.038).

Citation: The American Journal of Tropical Medicine and Hygiene 99, 4; 10.4269/ajtmh.18-0401

Although mutations in Pfcrt are not considered to be the key molecular markers for resistance of P. falciparum to other antimalarial drugs, transfection-based approaches have shown that certain mutations at this locus conferred alteration in susceptibilities to artemisinin and some partner drugs in ACT, particularly those that interfere with heme detoxification process in the parasite’s digestive vacuole such as mefloquine and piperaquine.6,2022 Importantly, changing antimalarial treatment regimens to ACT have remarkable impacts on P. falciparum drug resistance markers, including Pfcrt of parasite populations in Uganda and Kenya where chloroquine resistance deteriorated around the turn of the century.2325 To determine whether nationwide implementation of ACT and its modification could influence mutations in Pfcrt, we analyzed the complete coding sequences of Pfcrt and the dihydrofolate reductase gene (Pfdhfr) along with length polymorphisms in the merozoite surface proteins-1 and 2 (Pfmsp-1 and Pfmsp-2) genes of P. falciparum isolates in Thailand. Results revealed multiple novel Pfcrt haplotypes with greater molecular diversity indices among isolates collected during implementation of the 3-day ACT regimen than those collected before this period, consistent with ongoing selective pressure on this locus.

Methods

Parasite populations.

Venous blood samples were obtained from 383 P. falciparum–infected patients initially diagnosed by microscopy and confirmed by species-specific polymerase chain reaction (PCR) assay.26 Blood samples were collected during 1991, 2003–2006, 2008–2011, and 2013–2016 from seven provinces of Thailand bordering Myanmar, Cambodia, and Malaysia (Supplemental Figure 1). The parasite density determined from thick blood films ranged from 35 to 217,800 parasites/μL (geometric mean = 2,761 parasites/μL). Each blood sample was aliquoted; one preserved in EDTA anticoagulant and the other in RNA preservative for DNA and RNA extractions, respectively. The annual P. falciparum cases and the national treatment regimens for falciparum malaria in Thailand during sample collection period are depicted in Figure 1.

Detection of multiple-clone infections.

Clonality of samples was determined by PCR analysis of size polymorphisms in block 2 of the Pfmsp-1 and the central repeats of the Pfmsp-2 of P. falciparum (Supplemental Material 1).

Sequencing of Pfcrt.

The complete coding region of Pfcrt was obtained from direct sequencing of the PCR products amplified from cDNA and genomic DNA of each isolate (Supplemental Material 2). All singleton substitutions were re-determined using PCR products from independent amplifications of newly synthesized cDNA template from the same isolate.

Sequencing of Pfdhfr.

The complete Pfdhfr sequence was amplified by PCR using genomic DNA of each isolate (Supplemental Material 3). Sequences were determined directly from purified PCR products.

Ethics.

Written informed consent was obtained from all participants or from their parents or guardians before blood sample collection. The ethical aspects of this study were reviewed and approved by the Institutional Review Board on Human Research of Faculty of Medicine, Chulalongkorn University (IRB No. 257/57).

Statistical analysis.

The sequences were aligned against the corresponding gene of the 3D7 clone. Haplotype diversity (h) and its sampling variance were calculated using the DnaSP program.27 Nucleotide diversity (π), the rates of synonymous substitutions per synonymous site (dS), and nonsynonymous substitutions per nonsynonymous site (dN) and phylogenetic tree were analyzed by using the MEGA 6.0 program.28 Evidence of genetic recombination was determined by various recombination tests implemented in the RDP4 package.29 Tajima’s D statistics was used for computing deviation from neutrality.30 Tajima’s D test determines the differences between the average number of nucleotide differences and an estimate of θ from the number of segregating sites where θ = 4Neμ, in which Ne and μ are the effective population size and the mutation rate, respectively.30 Under an equilibrium model or no influence from population history, significant positive Tajima’s D values imply balancing or positive selection, whereas significant negative values suggest purifying or negative selection. Analysis of population genetic structure was performed by using molecular variance approach implemented in the Arlequin 3.5 software.31

Results

Novel Pfcrt coding sequences.

Analysis of size polymorphisms in Pfmsp-1 and Pfmsp-2 revealed that 242 of 383 isolates harbored single alleles of both loci. The complete coding sequences of Pfcrt could be obtained from 189 of these 242 isolates (78.1%). Compared with the 3D7 sequence, 26 nucleotide substitutions were detected among 189 Pfcrt sequences, resulting in 21 amino acid changes. Most nucleotide substitutions (84.6%) occurred in the putative transmembrane domain-encoding regions. Newly identified amino acid substitutions included 76I, 78L, 97R, 147D, 166L, 172M, 218F, 220P, 320T, 333I, 337N, and 351V (Table 1). In total, 21 genotypes were identified, yielding 18 different amino acid haplotypes. Haplotype I was most prevalent (87.83%) that shared the same sequence with the Dd2 clone (GenBank accession no. AF030694). Single synonymous substitutions were detected in three isolates at 411T>C, 543A>G, and 567T>C, all belonged to haplotype I. Isolates in haplotype I were heterogeneous based on analysis of the Pfmsp-1 and Pfmsp-2 loci. Haplotype II was found in four isolates (2.1%) that were identical with a Thai isolate TM93 collected two decades ago.32 Haplotype III possessed the same sequence as the K1 strain first isolated from Kanchanaburi in 197933 and the Colombian FCB strain.34 Haplotypes IV and V shared perfect sequence identity with the Cambodian isolate 734 and the Ghanaian isolate GB4, respectively.7,35 The remaining haplotypes VI-XVIII were novel and occurred as single isolates (Table 1). The wild-type Pfcrt was not found. Although the number of isolates collected from each endemic area and from different period in this study was uneven, the overall frequencies of mutant codons relative to the wild-type (3D7) and the most common Dd2 haplotypes were very low, ranging from 0.5% to 2.1% (Table 2).

Table 1

Distribution of Plasmodium falciparum chloroquine resistance transporter haplotypes among 189 clinical isolates in Thailand

Positions found in experimental clones/lines induced by selective drug pressure.21 Italicized amino acids denote induced mutations by drug pressures. Dots are identical residues with the 3D7 sequence (GenBank accession no. KM28867). Known isolates/strains carrying haplotypes I–V are shown in bold. Novel residues are highlighted. Representative isolates are in parentheses. Demarcations of exons are indicated by dashes.

Table 2

Frequencies of amino acid variants in Pfcrt of Thai isolates

Province-year (n)N75DK76IF78LH97RH97LA144FG147DL148IF172LI174MI194TI218FA220PA320TT333ST333II337NI351V
Trat-1991 (N = 19)15.8%
Trat-2003 (N = 13)
Ranong-2003 (N = 1)
Trat-2004 (N = 2)
Ranong-2004 (N = 10)
Trat-2005 (N = 2)50%50%50%50%
Tak-2006 (N = 14)
Ranong-2006 (N = 2)
Tak-2008 (N = 14)7.1%
Ubon-2008 (5)
Yala-2008 (N = 3)
Narathiwat-2008 (N = 7)14.3%
Tak-2009 (N = 3)
Chantaburi-2009 (N = 6)16.7%16.7%
Tak-2010 (N = 21)4.8%4.8%
Tak-2011 (N = 22)4.5%4.5%4.5%
Ubon-2012 (N = 1)
Tak-2013 (N = 10)
Ubon-2014 (N-10)20%10%20%20%10%20%10%20%
Ubon-2015 (N = 10)
Yala-2015 (N = 8)12.5%
Yala-2016 (N = 5)20%20%20%20%20%20%20%20%
Ubon-2016 (N = 1)100%
Total (N = 189)2.1%0.5%0.5%1.1%0.5%2.1%0.5%2.1%1.6%0.5%2.1%1.1%0.5%0.5%1.6%0.5%0.5%0.5%

Pfcrt = Plasmodium falciparum chloroquine resistance transporter; Ubon = Ubon Ratchathani. Amino acid variants are those differed from the Pfcrt sequences of either the Dd2 or the 3D7 clones (GenBank accession nos. AF030694 and KM288867, respectively).

The complete Pfdhfr coding sequences.

The complete Pfdhfr sequences from the same 189 isolates contained four nonsynonymous nucleotide substitutions, resulting in amino acid changes at N51I, C59R, S108N, and I164L. In total, four amino acid haplotypes were identified, characterized by IRNL (64.02%), IRNI (31.75%), NRNL (2.64%), and NRNI (1.59%).

Population genetics inferred from Pfmsp-1 and Pfmsp-2.

We observed 13 Pfmsp-1, 9 Pfmsp-2, and 73 combined Pfmsp-1 and Pfmsp-2 alleles among these 189 isolates. The number of haplotypes and haplotype diversity of these loci were almost comparable for populations bordering Myanmar and Cambodia, whereas lower levels of these parameters occurred for parasites near Malaysia (Supplemental Table 1). Genetic structuring was observed because all pairwise Fst values deviated significantly from zero, suggesting limited gene flow across these distant endemic areas and consistent with previous analyses using other loci (Supplemental Table 2).36

Variations in Pfcrt and other loci relative to change in ACT regimens.

There was a significant positive correlation between frequency of Pfcrt mutants and the year of sample collection during implementation of ACT (during 2003 and 2016) (r = 0.780; P = 0.038) but not when all sample collection period was considered (during 1991 and 2016) (r = 0.382; P = 0.350) (Figure 1). For further analyses, these samples were, therefore, assigned in the “pre-3-day ACT” and “3-day ACT” groups for those collected during 1991 and 2008, and 2009 and 2016, respectively. The number of Pfcrt haplotypes was significantly increased in the 3-day ACT group in comparison with the 2-day ACT group (P < 0.05, randomization test). By contrast, there was no significant difference in the number of haplotypes of Pfdhfr, Pfmsp-1, and Pfmsp-2 between these groups (Supplemental Table 3). The distribution of Pfcrt haplotypes was more skewed toward few haplotypes in the pre-3-day ACT than in the 3-day ACT groups as viewed from haplotype diversity values of total samples. The haplotype diversity in Pfdhfr, Pfmsp-1, and Pfmsp-2 of the corresponding populations was almost comparable (Supplemental Table 3). The number of mutations and nucleotide diversity of Pfcrt in the 3-day ACT group exceeded those in the pre-3-day ACT group. Nonsynonymous nucleotide diversity consistently outnumbered synonymous nucleotide diversity for all comparisons shown in Table 3.

Table 3

Nucleotide diversity in Plasmodium falciparum chloroquine resistance transporter of Plasmodium falciparum populations in relation to sample collection period

Population borderingYearnMhπ ± SE (×10−3)πS ± SE (×10−3)πN ± SE (×10−3)
Myanmar1991–2008411020.038 ± 0.0370.000 ± 0.0000.049 ± 0.045
2009–2016561570.197 ± 0.0740.131 ± 0.1270.215 ± 0.084
Cambodia1991–2008411540.340 ± 0.1400.179 ± 0.1660.384 ± 0.180
2009–2016282091.488 ± 0.392##0.000 ± 0.0001.896 ± 0.571*
Malaysia1991–2008101020.157 ± 0.1540.000 ± 0.0000.200 ± 0.190
2009–2016131951.701 ± 0.409###0.565 ± 0.5372.013 ± 0.572***
Total1991–2008921760.187 ± 0.0710.080 ± 0.0780.217 ± 0.087
2009–2016972717¶0.780 ± 0.228#0.151 ± 0.1060.951 ± 0.255**

h = number of haplotypes; M = number of mutations relative to the 3D7 coding regions; π = nucleotide diversity; πS = nucleotide diversity at synonymous sites; πN = nucleotide diversity at nonsynonymous sites. Test of significant difference between π for population during 1991 and 2008 and those during 2009 and 2016: #P < 0.05; ##P < 0.01; ###P < 0.005. Test of significant difference between πN for population during 1991 and 2008 and those during 2009 and 2016: *P < 0.05; **P < 0.01; ***P < 0.005. Randomization test of the hypothesis that the number of haplotypes for the population during 1991 and 2008 equals those during 2009 and 2016: ¶P < 0.05.

Domain-specific selection in Pfcrt.

A signature of positive selection in Pfcrt was evidenced by a significantly greater dN (0.0036 ± 0.0009) than dS (0.0010 ± 0.0006) (P < 0.05). The transmembrane domains of PfCRT reportedly dictate the transport of chloroquine and other charged molecules across parasite’s acid vesicles, thereby affecting drug accumulation and heme detoxification process.3 Hence, we analyzed nucleotide diversity for the putative transmembrane domain-encoding regions containing 199 codons and compared with that for the remainders of the coding regions spanning 225 codons. The mean nucleotide diversity for the transmembrane domains was 7-fold greater than that for the remaining regions and the difference was statistically significant (P < 0.05) (Table 4). The nucleotide diversity at nonsynonymous sites (πN) of 189 isolates significantly outnumbered that at synonymous sites (πS) for transmembrane domains (P < 0.05), but not for the remaining regions, and occurred only in the 3-day ACT group. There was no significant difference in πS between these groups. Meanwhile, Tajima’s D statistics yielded significantly negative values (Table 5). Importantly, significant negative Tajima’s D values were observed at nonsynonymous sites but not at synonymous sites. Meanwhile, no evidence of recombination was discernible in Pfcrt by all tests analyzed.

Table 4

Nucleotide diversity (π) at synonymous sites (πS) and nonsynonymous sites (πN) of the Pfcrt coding sequences of Thai isolates collected during 1991 and 2016

Collection period/Pfcrt domainπ (×10−3)πS (×10−3)πN (×10−3)
1991–2008 (N = 92)
 All coding region0.187 ± 0.0710.080 ± 0.0780.217 ± 0.084
  Transmembrane domains0.363 ± 0.154♦0.162 ± 0.1630.422 ± 0.187
  Remaining regions0.032 ± 0.0320.000 ± 0.0000.041 ± 0.039
2009–2016 (N = 97)
 All coding region0.780 ± 0.228•0.151 ± 0.1020.951 ± 0.245##**
  Transmembrane domains1.427 ± 0.431♦0.307 ± 0.2101.753 ± 0.523#*
  Remaining regions0.211 ± 0.1260.000 ± 0.0000.265 ± 0.159
All
 All coding region0.494 ± 0.1280.117 ± 0.0670.597 ± 0.160#
  Transmembrane domains0.915 ± 0.253♦0.236 ± 0.1341.113 ± 0.330#
  Remaining regions0.124 ± 0.0760.000 ± 0.0000.156 ± 0.094

Pfcrt = Plasmodium falciparum chloroquine resistance transporter. Test of the hypothesis that mean π for transmembrane domains equals that for the remaining regions: ♦P < 0.05. Test of the hypothesis that mean π for all coding regions of population during 1991 and 2008 equals the corresponding value of population during 2009 and 2016: •P < 0.05. Test of the hypothesis that mean πS equals the corresponding mean πN: #P < 0.05; ##P < 0.01. Test of the hypothesis that mean πN for population during 1991 and 2008 equals mean πN for population during 2009 and 2016: *P < 0.05; **P < 0.01.

Table 5

Genetic diversity in Plasmodium falciparum chloroquine resistance transporter of Plasmodium falciparum populations in Thailand

PopulationSampling periodEndemic areas borderingTotal
1991–20082009–2016MyanmarCambodiaMalaysia
N9297976923189
No. haplotypes617811621
No. polymorphic sites9228161525
No. segregating sites9208141523
Tajima’s D−2.187###−2.264###−2.178###−2.026#−2.423###−2.376###
D (synonymous sites)−1.037−1.382−1.031−1.069−1.161−1.516
D (nonsynonymous sites)−2.119#−2.198###−2.105#−1.972#−2.403###−2.302###

#, ##, and ### denote significant values at P < 0.05, P < 0.02, and P < 0.01, respectively.

Discussion

The complete Pfcrt coding sequences of 198 isolates collected over two decades from seven endemic provinces contained 21 distinct genotypes, 13 of which were novel. Together with previous reports, at least 67 naturally occurring Pfcrt haplotypes have been documented.7,8 Haplotypes I–III in this study were previously detected among Thai isolates collected over three decades ago, suggesting temporal stability of these haplotypes in this country. Likewise, identical sequences of isolate Cam734 from Cambodia collected a decade ago and haplotype IV, and isolate GB4 from Ghana and haplotype V could imply local and intercontinental spread of chloroquine-resistant P. falciparum. Spatiotemporal persistence of particular Pfcrt haplotypes has suggested adaptive evolutionary success of parasites carrying these variants in response to antimalarial drug pressure across endemic areas.

Twelve novel amino acid substitutions identified in this study have contributed to a total of 34 naturally occurring amino acid substitution sites in PfCRT so far identified. Most novel amino acid substitutions were found in single isolates and were not confined to particular endemic areas. Therefore, it is likely that mutations in Pfcrt could have arisen as independent events, probably as a result of the stochastic nature of a de novo point mutation. The significance of these novel mutations in PfCRT remains to be elucidated. However, a mutation at K76I in Pfcrt of two laboratory lines (J3D4 and C5K76I) induced by in vitro chloroquine selective pressure has been observed in isolate YL2823 from Yala collected in 2015.20 Although mutations in Pfcrt could have arisen initially from a stochastic mutational process, the shared mutations between a clinical isolate and drug-induced laboratory clones could suggest a common evolutionary pathway exerted by antimalarial selective pressure.

The interplay between antimalarial drug treatment regimens and genetics of malaria parasites has been documented. A timely withdrawal of chloroquine as a standard treatment of falciparum malaria has resulted in reemergence and expansion of chloroquine-sensitive parasites that still remain in the containment areas.37 By contrast, an increase in drug pressure could enhance the emergence of drug resistance genotypes as nationwide modification of ACT from 2-day to 3-day regimens in Thailand gave rise to a remarkable increase in the prevalence of artemisinin resistance genotypes. Several lines of evidence have suggested that mutations in Pfcrt can modulate susceptibility of artemisinin and some partner drugs used in ACT such as piperaquine, mefloquine, lumefantrine, and amodiaquine.6,21 Importantly, some of these compounds share with chloroquine a quinolone scaffold and affect heme detoxification process of the parasites.3 This in turn suggests that some antimalarials other than chloroquine could mediate selective pressure on Pfcrt as evidenced by the emergence of Pfcrt mutations induced by stepwise selection with amantadine or halofantrine.20 Therefore, cessation of chloroquine usage against falciparum malaria may not prevent ongoing selection in the Pfcrt locus because ACT may influence the evolution of this gene. A significant increase in the number of haplotypes and nonsynonymous mutations in Pfcrt across malaria-endemic areas in Thailand after nationwide change in ACT to 3-day regimen could be exerted by an increase in intensity of drug pressure from either increased duration of artesunate administration or long-term use and cumulative effect of mefloquine. This phenomenon occurred neither by chance nor by population process because no significant change in the number of haplotypes was observed in other unrelated genetic loci (Pfdhfr, Pfmsp-1, and Pfmsp-2), whereas gene flow was limited across endemic areas. Furthermore, intragenic recombination seems not to play an important role in generating sequence variation in Pfcrt among samples analyzed.

Natural selection exerted by antimalarial drugs could have influenced sequence variation in genes conferring drug resistance.12,38 Our study has shown that positive Darwinian selection favoring amino acid variants in Pfcrt was evidenced in the 3-day ACT group but not in isolates collected previously, suggesting that selective pressure has been triggered by an increase in duration of treatment regimen. Evidence of positive selection occurred exclusively in the transmembrane domains of PfCRT for the 3-day ACT group (Table 3). Tajima’s D test for nonsynonymous sites in Pfcrt yielded significant deviation from zero in negative direction, whereas a nonsignificant D value was found at synonymous sites (Table 4). The absence of recombination event along with the low level of gene (haplotype) diversity in the Pfcrt locus and the excess of low-frequency variants at nonsynonymous sites (strong negative Tajima’s D value) are consistent with the presence of slightly deleterious mutations in coding regions of this gene. In fact, variation in the growth rate of Pfcrt mutants has been documented among different haplotypes.5 Therefore, slightly deleterious mutations in Pfcrt will be subject to ongoing purifying selection which will either eliminate the change from the population or drive them to low frequencies.38 On the other hand, some compensatory mutations seem to be required to restore the fitness of some mutants. For example, the Cam734 haplotype (haplotype IV in this study) reportedly acquired the mutations A144F, I148L, and S333T with dual chloroquine resistance phenotype and compensatory restoration of fitness.6 A prolonged bottleneck effect or selective sweeps on the Pfcrt locus could occur during several decades of chloroquine selective pressure in Thailand as evidenced by analysis of flanking microsatellite loci.39 Several lines of evidence have supported the prediction that slightly deleterious variants will accumulate in a species that has undergone a severe bottleneck or in cases where recombination is reduced or absent.40,41 A number of singleton substitutions and the presence of synonymous polymorphism in Pfcrt of Thai isolates could have arisen from genetic drift, whereas the emergence of slightly deleterious mutations is a transient feature of evolution and these nonsynonymous mutations are subject to be eliminated by ongoing purifying selection. Importantly, integrated malaria control strategies in Thailand other than antimalarial treatment policy during the sample collection period were unchanged. Taken together, it is likely that an increase in duration of artesunate as a 3-day ACT regimen could provoke further selective pressure on the Pfcrt locus among P. falciparum isolates in Thailand. The impact of these novel haplotypes on parasite growth rate and drug susceptibility status would require further elucidation such as by transfection-based approaches.

Supplementary Material

Acknowledgments:

We are grateful to all patients who donated their blood samples for this study. We thank the staff of the Bureau of Vector Borne Disease, Department of Disease Control, Ministry of Public Health, Thailand, for assistance in field work.

REFERENCES

  • 1.

    Wellems TE, Plowe CV, 2001. Chloroquine-resistant malaria. J Infect Dis 184: 770776.

  • 2.

    Wongsrichanalai C, Pickard AL, Wernsdorfer WH, Meshnick SR, 2002. Epidemiology of drug-resistant malaria. Lancet Infect Dis 2: 209218.

  • 3.

    Fidock DA 2000. Mutations in the P. falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Mol Cell 6: 861871.

    • Search Google Scholar
    • Export Citation
  • 4.

    Ecker A, Lehane AM, Clain J, Fidock DA, 2012. PfCRT and its role in antimalarial drug resistance. Trends Parasitol 28: 504514.

  • 5.

    Petersen I 2015. Balancing drug resistance and growth rates via compensatory mutations in the Plasmodium falciparum chloroquine resistance transporter. Mol Microbiol 97: 381395.

    • Search Google Scholar
    • Export Citation
  • 6.

    Gabryszewski SJ 2016. Evolution of fitness cost-neutral mutant PfCRT conferring P. falciparum 4-aminoquinoline drug resistance is accompanied by altered parasite metabolism and digestive vacuole physiology. PLoS Pathog 12: e1005976.

    • Search Google Scholar
    • Export Citation
  • 7.

    Callaghan PS, Hassett MR, Roepe PD, 2015. Functional comparison of 45 naturally occurring isoforms of the Plasmodium falciparum chloroquine resistance transporter (PfCRT). Biochemistry 54: 50835094.

    • Search Google Scholar
    • Export Citation
  • 8.

    Agrawal S 2017. Association of a novel mutation in the Plasmodium falciparum chloroquine resistance transporter with decreased piperaquine sensitivity. J Infect Dis 216: 468476.

    • Search Google Scholar
    • Export Citation
  • 9.

    Parker D, Lerdprom R, Srisatjarak W, Yan G, Sattabongkot J, Wood J, Sirichaisinthop J, Cui L, 1994. Longitudinal in vitro surveillance of Plasmodium falciparum sensitivity to common anti-malarials in Thailand between 1994 and 2010. Malar J 11: 290.

    • Search Google Scholar
    • Export Citation
  • 10.

    Chaijaroenkul W, Ward SA, Mungthin M, Johnson D, Owen A, Bray PG, Na-Bangchang K, 2011. Sequence and gene expression of chloroquine resistance transporter (Pfcrt) in the association of in vitro drugs resistance of Plasmodium falciparum. Malar J 10: 42.

    • Search Google Scholar
    • Export Citation
  • 11.

    Takahashi N 2012. Large-scale survey for novel genotypes of Plasmodium falciparum chloroquine-resistance gene Pfcrt. Malar J 11: 92.

  • 12.

    Harinasuta T, Suntharasamai P, Viravan C, 1965. Chloroquine-resistant falciparum malaria in Thailand. Lancet 2: 657660.

  • 13.

    Malikul S, 2000. Malariology 1999 in Commemoration of 50 Years of Malaria Control in Thailand. Bangkok, Thailand: The Agricultural Co-operative Federation of Thailand Press (in Thai).

  • 14.

    Thimasarn K, Sirichaisinthop J, Vijaykadga S, Tansophalaks S, Yamokgul P, Laomiphol A, Palananth C, Thamewat U, Tháithong S, Rooney W, 1995. In vivo study of the response of Plasmodium falciparum to standard mefloquine/sulfadoxine/pyrimethamine (MSP) treatment among gem miners returning from Cambodia. Southeast Asian J Trop Med Public Health 26: 204212.

    • Search Google Scholar
    • Export Citation
  • 15.

    Wongsrichanalai C, Prajakwong S, Meshnick SR, Shanks GD, Thimasarn K, 2004. Mefloquine-its 20 years in the Thai malaria control program. Southeast Asian J Trop Med Public Health 35: 300308.

    • Search Google Scholar
    • Export Citation
  • 16.

    Zhou G, Sirichaisinthop J, Sattabongkot J, Jones J, Bjørnstad ON, Yan G, Cui L, 2005. Spatio-temporal distribution of Plasmodium falciparum and P. vivax malaria in Thailand. Am J Trop Med Hyg 72: 256262.

    • Search Google Scholar
    • Export Citation
  • 17.

    World Health Organisation, 2014. Status Report on Artemisinin Resistance: September 2014. Geneva, Switzerland: WHO.

  • 18.

    Putaporntip C, Kuamsab N, Kosuwin R, Tantiwattanasub W, Vejakama P, Sueblinvong T, Seethamchai S, Jongwutiwes S, Hughes AL, 2016. Natural selection of K13 mutants of Plasmodium falciparum in response to artemisinin combination therapies in Thailand. Clin Microbiol Infect 285: e1e8.

    • Search Google Scholar
    • Export Citation
  • 19.

    Jongwutiwes S, Buppan P, Kosuvin R, Seethamchai S, Pattanawong U, Sirichaisinthop J, Putaporntip C, 2011. Plasmodium knowlesi malaria in humans and macaques, Thailand. Emerg Infect Dis 17: 17991806.

    • Search Google Scholar
    • Export Citation
  • 20.

    Sidhu AB, Verdier-Pinard D, Fidock DA, 2002. Chloroquine resistance in Plasmodium falciparum malaria parasites conferred by Pfcrt mutations. Science 298: 210213.

    • Search Google Scholar
    • Export Citation
  • 21.

    Johnson DJ, Fidock DA, Mungthin M, Lakshmanan V, Sidhu AB, Bray PG, Ward SA, 2004. Evidence for a central role for PfCRT in conferring Plasmodium falciparum resistance to diverse antimalarial agents. Mol Cell 15: 867877.

    • Search Google Scholar
    • Export Citation
  • 22.

    Dhingra SK 2017. A variant PfCRT isoform can contribute to Plasmodium falciparum resistance to the first-line partner drug piperaquine. MBio 8: e00303e00317.

    • Search Google Scholar
    • Export Citation
  • 23.

    Conrad MD 2014. Comparative impacts over 5 years of artemisinin-based combination therapies on Plasmodium falciparum polymorphisms that modulate drug sensitivity in Ugandan children. J Infect Dis 210: 344353.

    • Search Google Scholar
    • Export Citation
  • 24.

    Hemming-Schroeder E 2018. Impacts of antimalarial drugs on Plasmodium falciparum drug resistance markers, western Kenya, 2003–2015. Am J Trop Med Hyg 98: 692699.

    • Search Google Scholar
    • Export Citation
  • 25.

    Tumwebaze P 2017. Changing antimalarial drug resistance patterns identified by surveillance at three sites in Uganda. J Infect Dis 215: 631635.

    • Search Google Scholar
    • Export Citation
  • 26.

    Putaporntip C, Buppan P, Jongwutiwes S, 2011. Improved performance with saliva and urine as alternative DNA sources for malaria diagnosis by mitochondrial DNA-based PCR assays. Clin Microbiol Infect 17: 14841491.

    • Search Google Scholar
    • Export Citation
  • 27.

    Librado P, Rozas J, 2009. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25: 14511452.

  • 28.

    Tamura K, Stecher G, Peterson D, Filipski A, Kumar S, 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30: 27252729.

    • Search Google Scholar
    • Export Citation
  • 29.

    Martin DP, Murrell B, Khoosal A, Muhire B, 2017. Detecting and analyzing genetic recombination using RDP4. Methods Mol Biol 1525: 433460.

  • 30.

    Tajima F, 1983. Evolutionary relationship of DNA sequences in finite populations. Genetics 105: 437460.

  • 31.

    Excoffier L, Lischer HE, 2010. Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows. Mol Ecol Resour 10: 564567.

    • Search Google Scholar
    • Export Citation
  • 32.

    Looareesuwan S, Viravan C, Webster HK, Kyle DE, Hutchinson DB, Canfield CJ, 1996. Clinical studies of atovaquone, alone or in combination with other antimalarial drugs, for treatment of acute uncomplicated malaria in Thailand. Am J Trop Med Hyg 54: 6266.

    • Search Google Scholar
    • Export Citation
  • 33.

    Thaithong S, Beale GH, 1981. Resistance of ten Thai isolates of Plasmodium falciparum to chloroquine and pyrimethamine by in vitro tests. Trans R Soc Trop Med Hyg 75: 271273.

    • Search Google Scholar
    • Export Citation
  • 34.

    Foote SJ, Kyle DE, Martin RK, Oduola AM, Forsyth K, Kemp DJ, Cowman AF, 1990. Several alleles of the multidrug-resistance gene are closely linked to chloroquine resistance in Plasmodium falciparum. Nature 345: 255258.

    • Search Google Scholar
    • Export Citation
  • 35.

    Durrand V, Berry A, Sem R, Glaziou P, Beaudou J, Fandeur T, 2004. Variations in the sequence and expression of the Plasmodium falciparum chloroquine resistance transporter (Pfcrt) and their relationship to chloroquine resistance in vitro. Mol Biochem Parasitol 136: 273285.

    • Search Google Scholar
    • Export Citation
  • 36.

    Jongwutiwes S, Putaporntip C, Hughes AL, 2010. Bottleneck effects on vaccine-candidate antigen diversity of malaria parasites in Thailand. Vaccine 28: 31123117.

    • Search Google Scholar
    • Export Citation
  • 37.

    Kublin JG, Cortese JF, Njunju EM, Mukadam RA, Wirima JJ, Kazembe PN, Djimdé AA, Kouriba B, Taylor TE, Plowe CV, 2003. Reemergence of chloroquine-sensitive Plasmodium falciparum malaria after cessation of chloroquine use in Malawi. J Infect Dis 187: 18701875.

    • Search Google Scholar
    • Export Citation
  • 38.

    Hughes AL, 2008. Near neutrality: leading edge of the neutral theory of molecular evolution. Ann N Y Acad Sci 1133: 162179.

  • 39.

    Nash D, Nair S, Mayxay M, Newton PN, Guthmann JP, Nosten F, Anderson TJ, 2005. Selection strength and hitchhiking around two anti-malarial resistance genes. Proc Biol Sci 272: 11531161.

    • Search Google Scholar
    • Export Citation
  • 40.

    Hughes AL, Packer B, Welch R, Bergen AW, Chanock SJ, Yeager M, 2003. Widespread purifying selection at polymorphic sites in human protein-coding loci. Proc Natl Acad Sci USA 100: 1575415757.

    • Search Google Scholar
    • Export Citation
  • 41.

    Price MN, Arkin AP, 2015. Weakly deleterious mutations and low rates of recombination limit the impact of natural selection on bacterial genomes. MBio 6: e01302e01315.

    • Search Google Scholar
    • Export Citation

Author Notes

Address correspondence to Chaturong Putaporntip or Somchai Jongwutiwes, Molecular Biology of Malaria and Opportunistic Parasites Research Unit, Department of Parasitology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand. E-mails: p.chaturong@gmail.com or jongwutiwes@gmail.com

Financial support: The Thai Government Research Budgets (GRB-APS-12593011 and GBA-600093004) and Chulalongkorn University Research Grants (Fiscal Years 2006–2008) to S. J., C. P., and S. S.; The Thailand Research Fund (RSA5980054) to C. P.; and Ratchadapiseksompotch Fund, Faculty of Medicine, Chulalongkorn University (RA60/109) to C. P., S. J., and P. B.

Authors’ addresses: Pattakorn Buppan, Molecular Biology of Malaria and Opportunistic Parasites Research Unit, Department of Parasitology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand and Inter-Department Program of Biomedical Sciences, Faculty of Graduate School, Chulalongkorn University, Bangkok, Thailand, E-mail: pattakorn.b@gmail.com. Napaporn Kuamsab, Chaturong Putaporntip, and Somchai Jongwutiwes, Molecular Biology of Malaria and Opportunistic Parasites Research Unit, Department of Parasitology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand, E-mails: kuamsab@gmail.com, p.chaturong@gmail.com, and jongwutiwes@gmail.com. Sunee Seethamchai, Department of Biology, Faculty of Science, Naresuan University, Pitsanulok Province, Thailand, E-mail: sunoat@gmail.com. Pongchai Harnyuttanakorn, Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand, E-mail: hpongchai@gmail.com.

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