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    Locations of parasite’s collection (1) Tak, (2) Kanchanaburi, (3) Ranong, (4) Srisaket, (5) Chanthaburi, and (6) Trat.

  • 1.

    World Health Organization, 2012. World Malaria Report 2012. Geneva, Switzerland: World Health Organization.

  • 2.

    Takala-Harrison S 2013. Genetic loci associated with delayed clearance of Plasmodium falciparum following artemisinin treatment in southeast Asia. Proc Natl Acad Sci USA 110: 240245.

    • Search Google Scholar
    • Export Citation
  • 3.

    Dondorp AM 2009. Artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med 361: 455467.

  • 4.

    Ashley EA.; Tracking Resistance to Artemisinin Collaboration, 2014. Spread of artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med 371: 411423.

    • Search Google Scholar
    • Export Citation
  • 5.

    Price RN 2004. Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number. Lancet 364: 438447.

  • 6.

    Chavchich M, Gerena L, Peters J, Chen N, Cheng Q, Kyle DE, 2010. Role of pfmdr1 amplification and expression in induction of resistance to artemisinin derivatives in Plasmodium falciparum. Antimicrob Agents Chemother 54: 24552464.

    • Search Google Scholar
    • Export Citation
  • 7.

    Slater HC, Griffin JT, Ghani AC, Okell LC, 2016. Assessing the potential impact of artemisinin and partner drug resistance in sub-Saharan Africa. Malar J 15: 10.

    • Search Google Scholar
    • Export Citation
  • 8.

    Croft SL, Duparc S, Arbe-Barnes SJ, Craft JC, Shin CS, Fleckenstein L, Borghini-Fuhrer I, Rim HJ, 2012. Review of pyronaridine anti-malarial properties and product characteristics. Malar J 11: 270.

    • Search Google Scholar
    • Export Citation
  • 9.

    Rueangweerayut R Pyronaridine-Artesunate Study Team, 2012. Pyronaridine-artesunate versus mefloquine plus artesunate for malaria. N Engl J Med 366: 12981309.

    • Search Google Scholar
    • Export Citation
  • 10.

    Kayentao K 2012. Pyronaridine-artesunate granules versus artemether-lumefantrine crushed tablets in children with Plasmodium falciparum malaria: a randomized controlled trial. Malar J 11: 364.

    • Search Google Scholar
    • Export Citation
  • 11.

    Poravuth Y 2011. Pyronaridine-artesunate versus chloroquine in patients with acute Plasmodium vivax malaria: a randomized, double-blind, non-inferiority trial. PLoS One 6: e14501.

    • Search Google Scholar
    • Export Citation
  • 12.

    Tshefu AK.; Pyronaridine-artesunate Study Team, 2010. Efficacy and safety of a fixed-dose oral combination of pyronaridine-artesunate compared with artemether-lumefantrine in children and adults with uncomplicated Plasmodium falciparum malaria: a randomised non-inferiority trial. Lancet 375: 14571467.

    • Search Google Scholar
    • Export Citation
  • 13.

    Duparc S, Borghini-Fuhrer I, Craft CJ, Arbe-Barnes S, Miller RM, Shin CS, Fleckenstein L, 2013. Safety and efficacy of pyronaridine-artesunate in uncomplicated acute malaria: an integrated analysis of individual patient data from six randomized clinical trials. Malar J 12: 70.

    • Search Google Scholar
    • Export Citation
  • 14.

    Ramharter M 2008. Fixed-dose pyronaridine-artesunate combination for treatment of uncomplicated falciparum malaria in pediatric patients in Gabon. J Infect Dis 198: 911919.

    • Search Google Scholar
    • Export Citation
  • 15.

    Pradines B, Tall A, Fusai T, Spiegel A, Hienne R, Rogier C, Trape JF, Le Bras J, Parzy D, 1999. In vitro activities of benflumetol against 158 Senegalese isolates of Plasmodium falciparum in comparison with those of standard antimalarial drugs. Antimicrob Agents Chemother 43: 418420.

    • Search Google Scholar
    • Export Citation
  • 16.

    Pradines B, Tall A, Parzy D, Spiegel A, Fusai T, Hienne R, Trape JF, Doury JC, 1998. In-vitro activity of pyronaridine and amodiaquine against African isolates (Senegal) of Plasmodium falciparum in comparison with standard antimalarial agents. J Antimicrob Chemother 42: 333339.

    • Search Google Scholar
    • Export Citation
  • 17.

    Schildbach S, Wernsdorfer WH, Suebsaeng L, Rooney W, 1990. In vitro sensitivity of multiresistant Plasmodium falciparum to new candidate antimalarial drugs in western Thailand. Southeast Asian J Trop Med Public Health 21: 2938.

    • Search Google Scholar
    • Export Citation
  • 18.

    Warsame M, Wernsdorfer WH, Payne D, Bjorkman A, 1991. Positive relationship between the response of Plasmodium falciparum to chloroquine and pyronaridine. Trans R Soc Trop Med Hyg 85: 570571.

    • Search Google Scholar
    • Export Citation
  • 19.

    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
  • 20.

    Djimde A 2001. A molecular marker for chloroquine-resistant falciparum malaria. N Engl J Med 344: 257263.

  • 21.

    Duraisingh MT, Jones P, Sambou I, von Seidlein L, Pinder M, Warhurst DC, 2000. The tyrosine-86 allele of the pfmdr1 gene of Plasmodium falciparum is associated with increased sensitivity to the anti-malarials mefloquine and artemisinin. Mol Biochem Parasitol 108: 1323.

    • Search Google Scholar
    • Export Citation
  • 22.

    Reed MB, Saliba KJ, Caruana SR, Kirk K, Cowman AF, 2000. Pgh1 modulates sensitivity and resistance to multiple antimalarials in Plasmodium falciparum. Nature 403: 906909.

    • Search Google Scholar
    • Export Citation
  • 23.

    Setthaudom C, Tan-ariya P, Sitthichot N, Khositnithikul R, Suwandittakul N, Leelayoova S, Mungthin M, 2011. Role of Plasmodium falciparum chloroquine resistance transporter and multidrug resistance 1 genes on in vitro chloroquine resistance in isolates of Plasmodium falciparum from Thailand. Am J Trop Med Hyg 85: 606611.

    • Search Google Scholar
    • Export Citation
  • 24.

    Cowman AF, Galatis D, Thompson JK, 1994. Selection for mefloquine resistance in Plasmodium falciparum is linked to amplification of the pfmdr1 gene and cross-resistance to halofantrine and quinine. Proc Natl Acad Sci USA 91: 11431147.

    • Search Google Scholar
    • Export Citation
  • 25.

    Wilson CM, Volkman SK, Thaithong S, Martin RK, Kyle DE, Milhous WK, Wirth DF, 1993. Amplification of pfmdr1 associated with mefloquine and halofantrine resistance in Plasmodium falciparum from Thailand. Mol Biochem Parasitol 57: 151160.

    • Search Google Scholar
    • Export Citation
  • 26.

    Pickard AL, Wongsrichanalai C, Purfield A, Kamwendo D, Emery K, Zalewski C, Kawamoto F, Miller RS, Meshnick SR, 2003. Resistance to antimalarials in southeast Asia and genetic polymorphisms in pfmdr1. Antimicrob Agents Chemother 47: 24182423.

    • Search Google Scholar
    • Export Citation
  • 27.

    Sidhu AB, Valderramos SG, Fidock DA, 2005. pfmdr1 mutations contribute to quinine resistance and enhance mefloquine and artemisinin sensitivity in Plasmodium falciparum. Mol Microbiol 57: 913926.

    • Search Google Scholar
    • Export Citation
  • 28.

    Uhlemann AC, Ramharter M, Lell B, Kremsner PG, Krishna S, 2005. Amplification of Plasmodium falciparum multidrug resistance gene 1 in isolates from Gabon. J Infect Dis 192: 18301835.

    • Search Google Scholar
    • Export Citation
  • 29.

    Pradines B, Briolant S, Henry M, Oeuvray C, Baret E, Amalvict R, Didillon E, Rogier C, 2010. Absence of association between pyronaridine in vitro responses and polymorphisms in genes involved in quinoline resistance in Plasmodium falciparum. Malar J 9: 339.

    • Search Google Scholar
    • Export Citation
  • 30.

    Trager W, Jensen JB, 1976. Human malaria parasites in continuous culture. Science 193: 673675.

  • 31.

    Desjardins RE, Canfield CJ, Haynes JD, Chulay JD, 1979. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob Agents Chemother 16: 710718.

    • Search Google Scholar
    • Export Citation
  • 32.

    Wooden J, Kyes S, Sibley CH, 1993. PCR and strain identification in Plasmodium falciparum. Parasitol Today 9: 303305.

  • 33.

    Ariey F 2014. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature 505: 5055.

  • 34.

    Vijaykadga S, Rojanawatsirivej C, Cholpol S, Phoungmanee D, Nakavej A, Wongsrichanalai C, 2006. In vivo sensitivity monitoring of mefloquine monotherapy and artesunate-mefloquine combinations for the treatment of uncomplicated falciparum malaria in Thailand in 2003. Trop Med Int Health 11: 211219.

    • Search Google Scholar
    • Export Citation
  • 35.

    Looareesuwan S, Kyle DE, Viravan C, Vanijanonta S, Wilairatana P, Wernsdorfer WH, 1996. Clinical study of pyronaridine for the treatment of acute uncomplicated falciparum malaria in Thailand. Am J Trop Med Hyg 54: 205209.

    • Search Google Scholar
    • Export Citation
  • 36.

    Childs GE, Hausler B, Milhous W, Chen C, Wimonwattrawatee T, Pooyindee N, Boudreau EF, 1988. In vitro activity of pyronaridine against field isolates and reference clones of Plasmodium falciparum. Am J Trop Med Hyg 38: 2429.

    • Search Google Scholar
    • Export Citation
  • 37.

    Witkowski B 2013. Novel phenotypic assays for the detection of artemisinin-resistant Plasmodium falciparum malaria in Cambodia: in-vitro and ex-vivo drug-response studies. Lancet Infect Dis 13: 10431049.

    • Search Google Scholar
    • Export Citation
  • 38.

    Elueze EI, Croft SL, Warhurst DC, 1996. Activity of pyronaridine and mepacrine against twelve strains of Plasmodium falciparum in vitro. J Antimicrob Chemother 37: 511518.

    • Search Google Scholar
    • Export Citation
  • 39.

    Pradines B, Mabika Mamfoumbi M, Parzy D, Owono Medang M, Lebeau C, Mourou Mbina JR, Doury JC, Kombila M, 1999. In vitro susceptibility of African isolates of Plasmodium falciparum from Gabon to pyronaridine. Am J Trop Med Hyg 60: 105108.

    • Search Google Scholar
    • Export Citation
  • 40.

    Price RN, Marfurt J, Chalfein F, Kenangalem E, Piera KA, Tjitra E, Anstey NM, Russell B, 2010. In vitro activity of pyronaridine against multidrug-resistant Plasmodium falciparum and Plasmodium vivax. Antimicrob Agents Chemother 54: 51465150.

    • Search Google Scholar
    • Export Citation
  • 41.

    Okombo J, Kiara SM, Mwai L, Pole L, Ohuma E, Ochola LI, Nzila A, 2012. Baseline in vitro activities of the antimalarials pyronaridine and methylene blue against Plasmodium falciparum isolates from Kenya. Antimicrob Agents Chemother 56: 11051107.

    • Search Google Scholar
    • Export Citation
  • 42.

    Kurth F, Pongratz P, Belard S, Mordmuller B, Kremsner PG, Ramharter M, 2009. In vitro activity of pyronaridine against Plasmodium falciparum and comparative evaluation of anti-malarial drug susceptibility assays. Malar J 8: 79.

    • Search Google Scholar
    • Export Citation
  • 43.

    Pascual A, Madamet M, Briolant S, Gaillard T, Amalvict R, Benoit N, Travers D, Pradines B; French National Reference Centre for Imported Malaria Study Group, 2015. Multinormal in vitro distribution of Plasmodium falciparum susceptibility to piperaquine and pyronaridine. Malar J 14: 49.

    • Search Google Scholar
    • Export Citation
  • 44.

    White NJ, 2004. Antimalarial drug resistance. J Clin Invest 113: 10841092.

  • 45.

    Madamet M, Briolant S, Amalvict R, Benoit N, Bouchiba H, Cren J, Pradines B; French National Centre for Imported Malaria Study Group, 2016. The Plasmodium falciparum chloroquine resistance transporter is associated with the ex vivo P. falciparum African parasite response to pyronaridine. Parasit Vectors 9: 77.

    • Search Google Scholar
    • Export Citation
  • 46.

    Leang R 2015. Evidence of Plasmodium falciparum malaria multidrug resistance to artemisinin and piperaquine in western Cambodia: dihydroartemisinin-piperaquine open-label multicenter clinical assessment. Antimicrob Agents Chemother 59: 47194726.

    • Search Google Scholar
    • Export Citation
  • 47.

    Amaratunga C 2016. Dihydroartemisinin-piperaquine resistance in Plasmodium falciparum malaria in Cambodia: a multisite prospective cohort study. Lancet Infect Dis 16: 357365.

    • Search Google Scholar
    • Export Citation
  • 48.

    Mungthin M, Suwandittakul N, Chaijaroenkul W, Rungsrihirunrat K, Harnyuttanakorn P, Seugorn A, Na Bangchang K, 2010. The patterns of mutation and amplification of Plasmodium falciparum pfcrt and pfmdr1 genes in Thailand during the year 1988 to 2003. Parasitol Res 107: 539545.

    • Search Google Scholar
    • Export Citation
  • 49.

    Mungthin M, Intanakom S, Suwandittakul N, Suida P, Amsakul S, Sitthichot N, Thammapalo S, Leelayoova S, 2014. Distribution of pfmdr1 polymorphisms in Plasmodium falciparum isolated from southern Thailand. Malar J 13: 117.

    • Search Google Scholar
    • Export Citation

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

In Vitro Sensitivity of Pyronaridine in Thai Isolates of Plasmodium falciparum

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  • 1 Department of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand;
  • 2 Department of Parasitology, Phramongkutklao College of Medicine, Bangkok 10400, Thailand

Pyronaridine, a Mannich base antimalarial agent with a high activity against chloroquine-resistant Plasmodium falciparum, has been combined with artesunate as a new artemisinin based combination therapy (ACT). Pyronaridine–artesunate combination could be one of the choices for the treatment of uncomplicated falciparum malaria in multidrug-resistant areas including Thailand. The aim of this study was to determine in vitro sensitivity and cross-resistance pattern of pyronaridine in Thai isolates of P. falciparum. In addition, the influence of resistant genes concerning in vitro pyronaridine sensitivity was determined. The mean pyronaridine 50% inhibitory concentration (IC50) of 118 parasite isolates was 5.6 ± 3.1 nM (range = 0.2–15.4 nM) with a significant positive correlation with artesunate IC50 (r = 0.246, P = 0.008) and amodiaquine IC50 (r = 0.220, P = 0.042) and a significant negative correlation with quinine IC50 (r = 0.185, P = 0.047). Parasites containing the pfmdr1 86Y allele exhibited significantly reduced pyronaridine sensitivity compared with those with the pfmdr1 N86 allele (7.6 ± 3.3 nM and 5.4 ± 3.0 nM, respectively, P = 0.032, independent t test); however, the difference may not be clinically relevant. Pyronaridine–artesunate could be the candidate ACT to treat multidrug-resistant falciparum malaria in Thailand with careful monitoring.

INTRODUCTION

Artemisinin-based combination therapy (ACT) has been recommended as the first-line treatment of uncomplicated falciparum malaria.1 Recently, partial artemisinin resistance indicated by delayed parasite clearance has been reported in the Greater Mekong Subregion.2,3 This artemisinin resistance phenotype is associated with point mutations in the Kelch 13 (K13) propeller gene. A few point mutations after amino acid position 440 are associated with delayed parasite clearance time.4 However, the treatment failure of ACT has been associated with resistance to the partner drugs rather than artemisinin-delayed parasite clearance.57 Thus, an ACT with an effective partner drug should be chosen rationally.

Pyronaridine, a Mannich base antimalarial drug, was first synthesized in 1970 at the Institute of Chinese Parasitic Disease and had been used in China as a monotherapy for more than 30th years.8 Recently, pyronaridine has been developed as a fixed-dose ACT, in a 3:1 ratio, with artesunate to treat acute uncomplicated falciparum and vivax malaria.8 A few clinical studies have indicated that the efficacy of pyronaridine–artesunate was high for treating uncomplicated falciparum and vivax malaria.914 Pyronaridine–artesunate is well tolerated. Although transient increases in transaminases were reported in a small proportion of patients after pyronaridine–artesunate treatment, no indication of serious hepatic side effects was observed.914

In vitro studies showed an excellent activity by pyronaridine against Plasmodium falciparum compared with a structurally related quinoline, chloroquine, even with possible cross-resistance with chloroquine.1518 Chloroquine resistance in P. falciparum has been linked to at least two genes, the P. falciparum chloroquine resistance transporter (pfcrt) gene and P. falciparum multidrug resistance 1 (pfmdr1) gene.1921 A mutation at position 76 (K76T) has been used as a molecular marker for chloroquine-resistant genotypes.20 The mutations and amplification of the pfmdr1 have been linked to different levels of chloroquine resistance.19,22,23 Moreover, the pfmdr1 gene has also been implicated in several drug-resistant phenotypes. In vitro and in vivo mefloquine resistance is associated with pfmdr1 polymorphisms.5,21 A few studies have shown the influence of pfmdr1 polymorphisms on altered in vitro sensitivity to quinolines including quinine, halofantrine, lumefantrine, piperaquine and also artemisinin derivatives.5,6,2428 Evaluation of the association between in vitro pyronaridine sensitivity and the pfmdr1 polymorphisms remains limited.29 In addition, recently adapted Thai isolates of P. falciparum have not been evaluated for in vitro pyronaridine sensitivity. In this study, we aimed to determine in vitro sensitivity and cross-resistance pattern of pyronaridine in adapted Thai isolates of P. falciparum. Influence of the known resistant genes on in vitro pyronaridine sensitivity was also determined. This study could provide baseline information for in vitro pyronaridine sensitivity and also help to determine the appropriate threshold values for parasite resistance to pyronaridine.

MATERIALS AND METHODS

Plasmodium falciparum cultivation.

One hundred and eighteen parasite isolates were collected from endemic areas of Thailand along the Thai–Cambodian border including Srisaket, Chanthaburi, and Trat Provinces and Thai–Myanmar border including Tak, Kanchanaburi, and Ranong Provinces (Figure 1) from 1989 to 2014. Recently adapted isolates were maintained in a continuous culture for 2–5 cycles using a method of Trager and Jensen30 before in vitro sensitivity assays. Plasmodium falciparum cultures were maintained in human O+ erythrocytes with 10% human AB serum in RPMI 1640 medium under an atmosphere of 90% N2, 5% O2, and 5% CO2. Laboratory strains used in this study comprised 3D7 and K1. The study was approved by the Ethics Committee of the Royal Thai Army Medical Department and Mahidol University-Center of Ethical Reinforcement for Human Research.

Figure 1.
Figure 1.

Locations of parasite’s collection (1) Tak, (2) Kanchanaburi, (3) Ranong, (4) Srisaket, (5) Chanthaburi, and (6) Trat.

Citation: The American Journal of Tropical Medicine and Hygiene 98, 1; 10.4269/ajtmh.17-0286

In vitro sensitivity assays.

In vitro sensitivity assay of P. falciparum to pyronaridine and other antimalarial drugs, that is, choloroquine, amodiaquine, piperaquine, mefloquine, quinine, artesunate, and dihydroartemisinin, were determined by measuring [3H] hypoxanthine uptake as previously described.31 In vitro sensitivity assays of these drugs were performed at the same time. Pyronaridine, chloroquine, amodiaquine, mefloquine, quinine, and artesunate were purchased from Sigma-Aldrich® (St. Louis, MO). Piperaquine and dihydroartemisinin were gifts from Professor Stephen A. Ward, Liverpool School of Tropical Medicine, Liverpool, United Kingdom. Pyronaridine, chloroquine, amodiaquine, mefloquine, and quinine were also dissolved in 50% ethanol. Each drug was diluted in complete media to obtain final concentrations ranging from 0.1 to 25 nM for pyronaridine, 1 to 250 nM for chloroquine, 0.1 to 50 nM for amodiaquine, 1 to 200 nM for mefloquine, and 5 to 1,000 nM for quinine. Artesunate and dihydroartemisinin were dissolved in dimethyl sulfoxide and diluted in complete medium to obtain final concentrations ranging from 0.1 to 50 nM. Piperaquine was dissolved in methanol and diluted to 1 to 100 nM. For each assay, ring-stage parasite preparation containing 1% parasitemia and 2% hematocrit was incubated with different concentrations of tested drugs at 37°C for 24 hours before [3H] hypoxanthine preparation was added. The plate was then incubated at 37°C for another 24 hours before harvested. Each assay was performed in triplicate. The 50% inhibitory concentration (IC50) values were calculated using GraFit data analysis software (Erithacus Software, Kent, United Kingdom). The IC50 values of these drugs were used to determine cross-resistance patterns between pyronaridine and other antimalarial drugs.

Genotypic characterization for the pfcrt, pfmdr1, and K13 propeller genes.

Parasite DNA was extracted using the Chelex resin method32 at the same time when the in vitro sensitivity assay was performed. Polymerase chain reaction (PCR) and restriction fragment length polymorphism were used to detect pfcrt and pfmdr1 mutations. The pfcrt K76T mutation method was developed by Djimde et al.20 Genomic DNA extraction from 3D7 and K1 were used as the positive control. The pfmdr1 mutations at codons 86, 184, 1034, 1042, and 1246 were detected using the method developed by Duraisingh et al.21 Genomic DNA extraction from K1 (YYSND) and 7G8 (NFCDY) were used as the positive control. Results with a combined band pattern of undigested and digested fragments were considered mixed alleles. The pfmdr1 copy number was assessed by TaqMan real-time PCR (CFX96 Touch™; Bio-Rad, Hercules, CA) as developed by Price et al.5 The pfmdr1 and β-tubulin amplification was performed as multiplex PCR. DNA samples from 3D7 and DD2 containing 1 and 4 pfmdr1 copy numbers, respectively, were used as the reference DNA sample. All reactions were performed in triplicate. The relative measure of the concentration was analyzed by the comparative threshold cycle (Ct). The copy number was calculated using the formula 2∆∆Ct as previously described.5 PCR sequencing for genotyping single nucleotide polymorphisms of the K13 propeller domain was performed using the method of Ariey et al.33

Statistical analysis.

Data were analyzed using SPSS for Windows (SPSS Inc., Chicago, IL). Each IC50 value represented the mean of at least three independent experiments. Normal distribution of data was assessed using the Kolmogorov–Smirnov test. Correlation of in vitro sensitivities was assessed by Pearson’s correlation. Differences of the mean IC50 and copy number of the pfmdr1 among parasites from different areas were analyzed using the independent t test. Associations between pfmdr1 polymorphisms and mean IC50 were determined by the independent t test and one-way analysis of variance (ANOVA). Statistical significance level was set at P value < 0.05 for all tests.

RESULTS

In vitro pyronaridine sensitivity.

The mean IC50 values of tested antimalarial drugs are shown in Table 1.The mean ±SD IC50 value of 118 P. falciparum isolates for pyronaridine was 5.6 ± 3.1 nM (range = 0.2–15.4 nM). Laboratory strains, 3D7 and K1 showed pyronaridine IC50 values of 9.7 and 5.1 nM, respectively. Table 2 shows the mean pyronaridine IC50 value of 118 parasites isolated from Thai–Myanmar and Thai–Cambodian areas. No significant difference was found in pyronaridine IC50 values among the parasites isolated from different areas (independent t test, P = 0.601). Table 3 shows the correlation between in vitro sensitivities to pyronaridine and other antimalarial drugs, that is, chloroquine, amodiaquine, piperaquine, mefloquine, quinine, artesunate, and dihydroartemisinin. Significantly positive correlations were found between IC50 values of pyronaridine and amodiaquine (r = 0.220, P = 0.042) and pyronaridine and artesunate (r = 0.246, P = 0.008). A negative correlation was found between IC50 values of pyronaridine and quinine (r = −0.185, P = 0.047).

Table 1

In vitro sensitivity of Thai and standard laboratory isolates of Plasmodium falciparum against antimalarial drugs

Antimalarial drugsThai isolatesLaboratory isolates
No.Mean IC50 ± SD (nM)Min–Max IC50 (nM)3D7 IC50 (nM)K1 IC50 (nM)
Pyronaridine1185.6 ± 3.10.2–15.49.75.1
Chloroquine11790.7 ± 43.418.2–230.318.2130.9
Amodiaquine869.2 ± 4.21.4–26.89.612.0
Piperaquine11418.4 ± 8.46.4–55.68.137.8
Quinine115205.7 ± 127.029.7–737.980133.2
Mefloquine11727.8 ± 21.62.0–130.948.813.9
Artesunate1163.4 ± 1.90.6–9.74.84.8
Dihyroartemisinin1132.1 ± 1.20.2–5.61.11.7
Table 2

In vitro pyronaridine sensitivity and distribution of the pfmdr1 mutations of parasites from Thai–Myanmar and Thai–Cambodian areas

AreaNPyronaridine IC50 (nM)pfmdr1 copy no.pfmdr1 mutation no. (%)
86Y184F1034C1042D1246Y
Thai–Myanmar615.8 ± 3.22.9 ± 1.44 (6.6)19 (31.1)3 (4.9)3 (4.9)
Thai–Cambodian575.5 ± 2.91.3 ± 0.96 (10.5)50 (87.7)3 (5.3)5 (8.8)
Total1185.6 ± 3.12.1 ± 1.410 (8.5)69 (58.5)6 (5.1)8 (6.8)
Table 3

Correlation of in vitro sensitivity of Plasmodium falciparum to pyronaridine and other antimalarial drugs

Drug pairrr2P value
PyronaridineChloroquine−0.1130.0130.224
Amodiaquine0.2200.0480.042*
Piperaquine0.004< 0.0010.966
Mefloquine0.0380.0010.686
Quinine−0.1850.0340.047*
Artesunate0.2460.0610.008*
Dihydroartemisinin−0.0860.0070.364

Significant difference determined by Pearson’s correlation.

Characterization of the pfcrt, pfmdr1, and K13 propeller genes.

All parasite isolates contained the pfcrt 76T allele. Distribution of the pfmdr1 polymorphisms in the parasite isolates from two different areas is shown in Table 2. All parasite isolates contained 100% D1246 allele. In all, 10 (8.5%), 69 (58.5%), 6 (5.1%) and 8 (6.8%) parasite isolates contained the pfmdr1 86Y, 184F, 1034C, and 1042D mutation, respectively. Approximately 60% of the parasite isolates contained the pfmdr1 184F allele and was more commonly found in the parasite isolates from the Thai–Cambodian area than in the isolates from Thai–Myanmar. No mixed alleles were detected. The mean pfmdr1 copy number value was 2.1 ± 1.4 (range = 0.8–5.5). The parasite isolates from Thai–Myanmar had significantly higher copy number values (independent t test, P < 0.001). A total of 85 parasite isolates were characterized for the K13 propeller gene. Fifty-eight (68.2%), 22 (25.9%), and 5 (5.9%) isolates contained wild-type, 580Y, and 539T mutations of the K13 propeller gene, respectively.

The association between in vitro pyronaridine sensitivity and the pfmdr1 and K13 propeller genes.

Table 4 shows the mean pyronaridine IC50 value of 118 parasites with different pfmdr1 genotypes. Decreased in vitro pyronaridine sensitivity was observed in the parasites containing the pfmdr1 86Y allele. No significant differences were observed in the pyronaridine IC50 values among the parasite isolates with different copy number (one-way ANOVA, P = 0.135). Table 5 shows seven groups of different haplotypes, obtained from the analysis of all parasite isolates. They comprised the wild-type, single mutation, double mutation and triple mutation, that is, (I) wild-type, (II) 86Y allele, (III) 184F allele, (IV) 86Y + 184F, (V) 184F + 1042D, (VI) 1034C + 1042D, and (VII) 184F + 1034C + 1042D. No significant difference was found in pyronaridine IC50 values among these different haplotypes (one-way ANOVA, P = 0.378). No significant difference was found in pyronaridine IC50 values among parasites with wild-type, 580Y and 539T mutations of the K13 propeller gene (6.2 ± 3.3, 4.6 ± 2.1, and 5.1 ± 2.2 nM, respectively, one-way ANOVA, P = 0.104) as well.

Table 4

Comparison of in vitro pyronaridine sensitivity among Plasmodium falciparum with different pfmdr1 genotypes

pfmdr1 genotypesN (%)Pyronaridine IC50 (nM)P value
Mutations
86N86108 (91.5)5.4 ± 3.00.032*
86Y10 (8.5)7.6 ± 3.3
184Y18449 (41.5)6.2 ± 3.40.074
184F69 (58.5)5.2 ± 2.8
1034S1034112 (94.9)5.6 ± 3.10.805
1034C6 (5.1)5.3 ± 1.8
1042N1042110 (93.2)5.6 ± 3.20.903
1042D8 (6.8)5.4 ± 1.6
Copy no.≤ 147 (39.8)5.0 ± 2.60.135
> 1–223 (19.5)6.4 ± 3.4
> 2–319 (16.1)6.6 ± 4.1
> 3–413 (11.0)4.6 ± 1.6
> 416 (13.6)5.8 ± 3.0

Significant difference determined by independent t test.

Table 5

In vitro pyronaridine sensitivity of different genotyped subgroups

Grouppfmdr1 haplotypeNPyronaridine IC50 (nM)
861841,0341,042Copy no.
INYSN3.0 ± 1.5395.9 ± 3.4
IINYCD0.8915.31
IIINFSN1.6 ± 1.0615.1 ± 2.9
IVNFSD2.8 ± 2.626.0 ± 0.5
VNFCD2.0 ± 1.255.3 ± 2.0
VIYYSN1.9 ± 1.297.8 ± 3.4
VIIYFSN1.4015.56

Significant difference among groups (P = 0.378, one-way analysis of variance).

DISCUSSION

Multidrug-resistant falciparum malaria has emerged and spread in Thailand. Artesunate–mefloquine is currently used as the first-line combination to treat uncomplicated falciparum malaria. The efficacy of artesunate–mefloquine has decreased in some regions, particularly Southeast Asia.34 New ACTs including pyronaridine–artesunate could be a candidate to replace artesunate–mefloquine. An efficacy and safety study of pyronaridine among patients with falciparum malaria was conducted in Thailand 20 years ago.35 In vitro sensitivity of pyronaridine against field isolates in Thailand was also evaluated 30 years ago.36 We determined in vitro sensitivity of recently adapted Thai isolates to pyronaridine and in vitro cross-resistance with other antimalarial as baseline information. Although another method, that is a ring-stage survival assay, has been used to characterize artemisinin resistance,37 determining the usefulness of this method for pyronaridine resistance has not been performed. In addition, we would like to compare the pyronaridine IC50 in our study with others. Our study shows an excellent activity of pyronaridine against all P. falciparum isolates from both Thai–Myanmar and Thai–Cambodian areas. This result is similar to previous studies conducted in Thailand 30 years ago and elsewhere at the present time.16,29,35,36,3843 The clear standard cut-off point of pyronaridine is still unavailable. A related study suggested estimating using the geometric mean plus 2 SDs.16,38,43 When we used this criteria for the reduced pyronaridine sensitivity (IC50 plus 2 SDs = 11.8 nM), seven (5.9%) Thai isolates exhibited reduced sensitivity to in vitro pyronaridine (range = 12.7–15.4 nM). However, when we used a 3-fold decrease in sensitivity to pyronaridine of 3D7 (29.1 nM), no parasite isolate exhibited reduced pyronaridine sensitivity. Nevertheless, when we used a 3-fold decrease in sensitivity to pyronaridine of K1 (15.3 nM), only one isolate exhibited reduced sensitivity to pyronaridine. However, this cut-off point might not be comparable because different in vitro methods were used. In addition, the data should be considered with clinical observation of recrudescence after treating with pyronaridine. As a result from this study, pyronaridine still showed high activity against Thai isolates of P. falciparum and seemed to be equally active against isolates from both areas where the multidrug resistance has spread.

One of the factors causing the emergence of drug resistance is the cross-resistance in structurally related antimalarial drugs.44 A correlation between in vitro pyronaridine and chloroquine sensitivity has been reported in some but not all studies.1518 In addition, a recent study showed the influence of the pfcrt mutation on ex vivo pyronaridine sensitivity.45 In the present study, no significant correlation was observed between in vitro pyronaridine and chloroquine sensitivity. Because all parasites contained the pfcrt 76T allele, the association between this chloroquine-resistant genotype and in vitro pyronaridine sensitivity cannot be tested. Compelling evidence shows that the pfmdr1 gene also contributes to chloroquine resistance.22,23 Our recent study showed that both Y184F mutation and copy number of the pfmdr1 gene were associated with the level of chloroquine resistance.23 However, these two pfmdr1 genotypes did not influence in vitro pyronaridine sensitivity. Correlations between pyronaridine and other quinolines including amodiaquine and quinine were identified. Pyronaridine IC50 was also positively correlated with artesunate IC50 which is similar to two related studies.29,41 However, a statistically significant result (P < 0.05) can often be seen when comparing the drug activities against a large number of isolates. The r value > 0.7–0.8 or r2 value > 0.5 is more suggestive of cross-resistance. Thus, the correlation with artesunate, amodiaquine, and quinine in this study was weak.

Dihydroartemisinin–piperaquine, a fixed-dose ACT, has been used in Southeast Asia including Thailand, Myanmar, and Cambodia. Unfortunately, the efficacy of dihydroartemisinin–piperaquine has rapidly declined in Cambodia, partly, because of the emergence of piperaquine resistance.46,47 Thus, pyronaridine–artesunate might be one of the candidates to treat uncomplicated falciparum malaria in these areas. Because pyronaridine and piperaquine are structurally related, cross-resistance between these two drugs should be carefully investigated. From our information, no significant correlation was found between pyronaridine and piperaquine IC50 (Table 2). To date, no available cut-off point has been established for in vitro piperaquine resistance. When the cut-off point for reduced in vitro piperaquine sensitivity was estimated using the mean IC50 plus 2 SDs, that is 35.2 nM, six of 114 parasite isolates in the present study exhibited reduced piperaquine sensitivity. However, the parasites with reduced piperaquine sensitivity showed no significant difference in the pyronaridine IC50 compared with those exhibiting no reduced piperaquine sensitivity (4.6 and 5.7 nM, P = 0.428).

Although we did not find mixed alleles of the pfcrt and pfmdr1 gene in these parasites, multiple clones in these cultures cannot be ruled out. Thus, the phenotypes and also genotypes may represent the dominant clones. In Thailand, P. falciparum isolates from different areas contained different patterns of pfmdr1 haplotypes.48 The parasites from the Thai–Cambodian border contained the pfmdr1 184F allele with one copy number, whereas parasites collected from the Thai–Myanmar border usually contained either the pfmdr1 184Y or 184F allele with more than one copy number.48 In contrast, the parasites from the Thai–Malaysian border predominantly contained the pfmdr1 86Y allele.49 In Thailand, different antimalarial regimens were used to treat falciparum malaria in different areas according to the level of mefloquine resistance.34 Consequently, different medications might promote different patterns of the pfmdr1 haplotypes. In addition, the pfmdr1 polymorphisms in P. falciparum might be due to human migration across borders. These pfmdr1 haplotypes showed different responses to antimalarial drugs.5,6,2428 For the partner drugs in ACT, related studies also confirmed that the mutations and amplification of the pfmdr1 gene influenced the parasite’s sensitivity to mefloquine, lumefantrine, and piperaquine.5,2428 A significant difference of in vitro pyronaridine sensitivity was observed between the parasites containing the pfmdr1 N86 and 86Y alleles. However, the difference seemed not to be clinically significance because it was in the nanomolar range. In the Greater Mekong Subregion, treatment failure of the ACT has been observed when the parasites are resistant to the partner drug. Thus, an ACT with the effective partner drug should be chosen rationally. In the present study, no alteration of pyronaridine IC50 was observed in the parasites exhibiting the artemisinin-resistant genotype, 580Y and 539T mutations of the K13 propeller gene compared with those containing the wild type.

In conclusion, the present study determined the baseline in vitro pyronaridine sensitivity in Thai isolates of P. falciparum. The results indicated that pyronaridine is still active against P. falciparum isolates from both the Thai–Myanmar and Thai–Cambodian areas. Weak correlations with other quinolines, quinine, and amodiaquine and artesunate were found. Parasites with the pfmdr1 86Y allele showed significantly reduced pyronaridine sensitivity; however, the reduced IC50 might not be clinically relevant. The cut-off value for pyronaridine resistance or reduced pyronaridine sensitivity should be further investigated. To date, efficacy and safety of pyronaridine–artesunate was studied only in the Thai–Myanmar area. This combination should be further investigated in different areas of Thailand where parasites with different resistant genotypes exist.

REFERENCES

  • 1.

    World Health Organization, 2012. World Malaria Report 2012. Geneva, Switzerland: World Health Organization.

  • 2.

    Takala-Harrison S 2013. Genetic loci associated with delayed clearance of Plasmodium falciparum following artemisinin treatment in southeast Asia. Proc Natl Acad Sci USA 110: 240245.

    • Search Google Scholar
    • Export Citation
  • 3.

    Dondorp AM 2009. Artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med 361: 455467.

  • 4.

    Ashley EA.; Tracking Resistance to Artemisinin Collaboration, 2014. Spread of artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med 371: 411423.

    • Search Google Scholar
    • Export Citation
  • 5.

    Price RN 2004. Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number. Lancet 364: 438447.

  • 6.

    Chavchich M, Gerena L, Peters J, Chen N, Cheng Q, Kyle DE, 2010. Role of pfmdr1 amplification and expression in induction of resistance to artemisinin derivatives in Plasmodium falciparum. Antimicrob Agents Chemother 54: 24552464.

    • Search Google Scholar
    • Export Citation
  • 7.

    Slater HC, Griffin JT, Ghani AC, Okell LC, 2016. Assessing the potential impact of artemisinin and partner drug resistance in sub-Saharan Africa. Malar J 15: 10.

    • Search Google Scholar
    • Export Citation
  • 8.

    Croft SL, Duparc S, Arbe-Barnes SJ, Craft JC, Shin CS, Fleckenstein L, Borghini-Fuhrer I, Rim HJ, 2012. Review of pyronaridine anti-malarial properties and product characteristics. Malar J 11: 270.

    • Search Google Scholar
    • Export Citation
  • 9.

    Rueangweerayut R Pyronaridine-Artesunate Study Team, 2012. Pyronaridine-artesunate versus mefloquine plus artesunate for malaria. N Engl J Med 366: 12981309.

    • Search Google Scholar
    • Export Citation
  • 10.

    Kayentao K 2012. Pyronaridine-artesunate granules versus artemether-lumefantrine crushed tablets in children with Plasmodium falciparum malaria: a randomized controlled trial. Malar J 11: 364.

    • Search Google Scholar
    • Export Citation
  • 11.

    Poravuth Y 2011. Pyronaridine-artesunate versus chloroquine in patients with acute Plasmodium vivax malaria: a randomized, double-blind, non-inferiority trial. PLoS One 6: e14501.

    • Search Google Scholar
    • Export Citation
  • 12.

    Tshefu AK.; Pyronaridine-artesunate Study Team, 2010. Efficacy and safety of a fixed-dose oral combination of pyronaridine-artesunate compared with artemether-lumefantrine in children and adults with uncomplicated Plasmodium falciparum malaria: a randomised non-inferiority trial. Lancet 375: 14571467.

    • Search Google Scholar
    • Export Citation
  • 13.

    Duparc S, Borghini-Fuhrer I, Craft CJ, Arbe-Barnes S, Miller RM, Shin CS, Fleckenstein L, 2013. Safety and efficacy of pyronaridine-artesunate in uncomplicated acute malaria: an integrated analysis of individual patient data from six randomized clinical trials. Malar J 12: 70.

    • Search Google Scholar
    • Export Citation
  • 14.

    Ramharter M 2008. Fixed-dose pyronaridine-artesunate combination for treatment of uncomplicated falciparum malaria in pediatric patients in Gabon. J Infect Dis 198: 911919.

    • Search Google Scholar
    • Export Citation
  • 15.

    Pradines B, Tall A, Fusai T, Spiegel A, Hienne R, Rogier C, Trape JF, Le Bras J, Parzy D, 1999. In vitro activities of benflumetol against 158 Senegalese isolates of Plasmodium falciparum in comparison with those of standard antimalarial drugs. Antimicrob Agents Chemother 43: 418420.

    • Search Google Scholar
    • Export Citation
  • 16.

    Pradines B, Tall A, Parzy D, Spiegel A, Fusai T, Hienne R, Trape JF, Doury JC, 1998. In-vitro activity of pyronaridine and amodiaquine against African isolates (Senegal) of Plasmodium falciparum in comparison with standard antimalarial agents. J Antimicrob Chemother 42: 333339.

    • Search Google Scholar
    • Export Citation
  • 17.

    Schildbach S, Wernsdorfer WH, Suebsaeng L, Rooney W, 1990. In vitro sensitivity of multiresistant Plasmodium falciparum to new candidate antimalarial drugs in western Thailand. Southeast Asian J Trop Med Public Health 21: 2938.

    • Search Google Scholar
    • Export Citation
  • 18.

    Warsame M, Wernsdorfer WH, Payne D, Bjorkman A, 1991. Positive relationship between the response of Plasmodium falciparum to chloroquine and pyronaridine. Trans R Soc Trop Med Hyg 85: 570571.

    • Search Google Scholar
    • Export Citation
  • 19.

    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
  • 20.

    Djimde A 2001. A molecular marker for chloroquine-resistant falciparum malaria. N Engl J Med 344: 257263.

  • 21.

    Duraisingh MT, Jones P, Sambou I, von Seidlein L, Pinder M, Warhurst DC, 2000. The tyrosine-86 allele of the pfmdr1 gene of Plasmodium falciparum is associated with increased sensitivity to the anti-malarials mefloquine and artemisinin. Mol Biochem Parasitol 108: 1323.

    • Search Google Scholar
    • Export Citation
  • 22.

    Reed MB, Saliba KJ, Caruana SR, Kirk K, Cowman AF, 2000. Pgh1 modulates sensitivity and resistance to multiple antimalarials in Plasmodium falciparum. Nature 403: 906909.

    • Search Google Scholar
    • Export Citation
  • 23.

    Setthaudom C, Tan-ariya P, Sitthichot N, Khositnithikul R, Suwandittakul N, Leelayoova S, Mungthin M, 2011. Role of Plasmodium falciparum chloroquine resistance transporter and multidrug resistance 1 genes on in vitro chloroquine resistance in isolates of Plasmodium falciparum from Thailand. Am J Trop Med Hyg 85: 606611.

    • Search Google Scholar
    • Export Citation
  • 24.

    Cowman AF, Galatis D, Thompson JK, 1994. Selection for mefloquine resistance in Plasmodium falciparum is linked to amplification of the pfmdr1 gene and cross-resistance to halofantrine and quinine. Proc Natl Acad Sci USA 91: 11431147.

    • Search Google Scholar
    • Export Citation
  • 25.

    Wilson CM, Volkman SK, Thaithong S, Martin RK, Kyle DE, Milhous WK, Wirth DF, 1993. Amplification of pfmdr1 associated with mefloquine and halofantrine resistance in Plasmodium falciparum from Thailand. Mol Biochem Parasitol 57: 151160.

    • Search Google Scholar
    • Export Citation
  • 26.

    Pickard AL, Wongsrichanalai C, Purfield A, Kamwendo D, Emery K, Zalewski C, Kawamoto F, Miller RS, Meshnick SR, 2003. Resistance to antimalarials in southeast Asia and genetic polymorphisms in pfmdr1. Antimicrob Agents Chemother 47: 24182423.

    • Search Google Scholar
    • Export Citation
  • 27.

    Sidhu AB, Valderramos SG, Fidock DA, 2005. pfmdr1 mutations contribute to quinine resistance and enhance mefloquine and artemisinin sensitivity in Plasmodium falciparum. Mol Microbiol 57: 913926.

    • Search Google Scholar
    • Export Citation
  • 28.

    Uhlemann AC, Ramharter M, Lell B, Kremsner PG, Krishna S, 2005. Amplification of Plasmodium falciparum multidrug resistance gene 1 in isolates from Gabon. J Infect Dis 192: 18301835.

    • Search Google Scholar
    • Export Citation
  • 29.

    Pradines B, Briolant S, Henry M, Oeuvray C, Baret E, Amalvict R, Didillon E, Rogier C, 2010. Absence of association between pyronaridine in vitro responses and polymorphisms in genes involved in quinoline resistance in Plasmodium falciparum. Malar J 9: 339.

    • Search Google Scholar
    • Export Citation
  • 30.

    Trager W, Jensen JB, 1976. Human malaria parasites in continuous culture. Science 193: 673675.

  • 31.

    Desjardins RE, Canfield CJ, Haynes JD, Chulay JD, 1979. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob Agents Chemother 16: 710718.

    • Search Google Scholar
    • Export Citation
  • 32.

    Wooden J, Kyes S, Sibley CH, 1993. PCR and strain identification in Plasmodium falciparum. Parasitol Today 9: 303305.

  • 33.

    Ariey F 2014. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature 505: 5055.

  • 34.

    Vijaykadga S, Rojanawatsirivej C, Cholpol S, Phoungmanee D, Nakavej A, Wongsrichanalai C, 2006. In vivo sensitivity monitoring of mefloquine monotherapy and artesunate-mefloquine combinations for the treatment of uncomplicated falciparum malaria in Thailand in 2003. Trop Med Int Health 11: 211219.

    • Search Google Scholar
    • Export Citation
  • 35.

    Looareesuwan S, Kyle DE, Viravan C, Vanijanonta S, Wilairatana P, Wernsdorfer WH, 1996. Clinical study of pyronaridine for the treatment of acute uncomplicated falciparum malaria in Thailand. Am J Trop Med Hyg 54: 205209.

    • Search Google Scholar
    • Export Citation
  • 36.

    Childs GE, Hausler B, Milhous W, Chen C, Wimonwattrawatee T, Pooyindee N, Boudreau EF, 1988. In vitro activity of pyronaridine against field isolates and reference clones of Plasmodium falciparum. Am J Trop Med Hyg 38: 2429.

    • Search Google Scholar
    • Export Citation
  • 37.

    Witkowski B 2013. Novel phenotypic assays for the detection of artemisinin-resistant Plasmodium falciparum malaria in Cambodia: in-vitro and ex-vivo drug-response studies. Lancet Infect Dis 13: 10431049.

    • Search Google Scholar
    • Export Citation
  • 38.

    Elueze EI, Croft SL, Warhurst DC, 1996. Activity of pyronaridine and mepacrine against twelve strains of Plasmodium falciparum in vitro. J Antimicrob Chemother 37: 511518.

    • Search Google Scholar
    • Export Citation
  • 39.

    Pradines B, Mabika Mamfoumbi M, Parzy D, Owono Medang M, Lebeau C, Mourou Mbina JR, Doury JC, Kombila M, 1999. In vitro susceptibility of African isolates of Plasmodium falciparum from Gabon to pyronaridine. Am J Trop Med Hyg 60: 105108.

    • Search Google Scholar
    • Export Citation
  • 40.

    Price RN, Marfurt J, Chalfein F, Kenangalem E, Piera KA, Tjitra E, Anstey NM, Russell B, 2010. In vitro activity of pyronaridine against multidrug-resistant Plasmodium falciparum and Plasmodium vivax. Antimicrob Agents Chemother 54: 51465150.

    • Search Google Scholar
    • Export Citation
  • 41.

    Okombo J, Kiara SM, Mwai L, Pole L, Ohuma E, Ochola LI, Nzila A, 2012. Baseline in vitro activities of the antimalarials pyronaridine and methylene blue against Plasmodium falciparum isolates from Kenya. Antimicrob Agents Chemother 56: 11051107.

    • Search Google Scholar
    • Export Citation
  • 42.

    Kurth F, Pongratz P, Belard S, Mordmuller B, Kremsner PG, Ramharter M, 2009. In vitro activity of pyronaridine against Plasmodium falciparum and comparative evaluation of anti-malarial drug susceptibility assays. Malar J 8: 79.

    • Search Google Scholar
    • Export Citation
  • 43.

    Pascual A, Madamet M, Briolant S, Gaillard T, Amalvict R, Benoit N, Travers D, Pradines B; French National Reference Centre for Imported Malaria Study Group, 2015. Multinormal in vitro distribution of Plasmodium falciparum susceptibility to piperaquine and pyronaridine. Malar J 14: 49.

    • Search Google Scholar
    • Export Citation
  • 44.

    White NJ, 2004. Antimalarial drug resistance. J Clin Invest 113: 10841092.

  • 45.

    Madamet M, Briolant S, Amalvict R, Benoit N, Bouchiba H, Cren J, Pradines B; French National Centre for Imported Malaria Study Group, 2016. The Plasmodium falciparum chloroquine resistance transporter is associated with the ex vivo P. falciparum African parasite response to pyronaridine. Parasit Vectors 9: 77.

    • Search Google Scholar
    • Export Citation
  • 46.

    Leang R 2015. Evidence of Plasmodium falciparum malaria multidrug resistance to artemisinin and piperaquine in western Cambodia: dihydroartemisinin-piperaquine open-label multicenter clinical assessment. Antimicrob Agents Chemother 59: 47194726.

    • Search Google Scholar
    • Export Citation
  • 47.

    Amaratunga C 2016. Dihydroartemisinin-piperaquine resistance in Plasmodium falciparum malaria in Cambodia: a multisite prospective cohort study. Lancet Infect Dis 16: 357365.

    • Search Google Scholar
    • Export Citation
  • 48.

    Mungthin M, Suwandittakul N, Chaijaroenkul W, Rungsrihirunrat K, Harnyuttanakorn P, Seugorn A, Na Bangchang K, 2010. The patterns of mutation and amplification of Plasmodium falciparum pfcrt and pfmdr1 genes in Thailand during the year 1988 to 2003. Parasitol Res 107: 539545.

    • Search Google Scholar
    • Export Citation
  • 49.

    Mungthin M, Intanakom S, Suwandittakul N, Suida P, Amsakul S, Sitthichot N, Thammapalo S, Leelayoova S, 2014. Distribution of pfmdr1 polymorphisms in Plasmodium falciparum isolated from southern Thailand. Malar J 13: 117.

    • Search Google Scholar
    • Export Citation

Author Notes

Address correspondence to Mathirut Mungthin, Department of Parasitology, Phramongkutklao College of Medicine, Ratchawithi Rd., Bangkok 10400, Thailand. E-mail: mathirut@hotmail.com

Financial support: This study was financially supported by the Health System Research Institute/National Science and Technology Development Agency (P-13-50112), the Phramongkutklao Research Fund, and the Science Achievement Scholarship of Thailand.

Authors’ addresses: Kittiya Mahotorn, Peerapan Tan-ariya, and Thunyapit Thita, Department of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand, E-mails: k.mahotorn@gmail.com, peerapan.tan@mahidol.ac.th, and aomay_ao@hotmail.com. Toon Ruang-areerate, Naruemon Sitthichot, Nantana Suwandittakul, and Mathirut Mungthin, Department of Parasitology, Phramongkutklao College of Medicine, Bangkok 10400, Thailand, E-mails: youangtr@yahoo.com, mude_143@hotmail.com, suwanna_b@hotmail.com, and mathirut@hotmail.com.

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