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Influence of the pfmdr1 Gene on In Vitro Sensitivities of Piperaquine in Thai Isolates of Plasmodium falciparum

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  • 1 Department of Parasitology, Phramongkutklao College of Medicine, Bangkok, Thailand.
  • 2 Mahidol University International College, Nakhon Pathom, Thailand.
  • 3 Division of Molecular and Biochemical Parasitology, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.

Piperaquine combined with dihydroartemisinin is one of the artemisinin derivative combination therapies, which can replace artesunate–mefloquine in treating uncomplicated falciparum malaria in Thailand. The aim of this study was to determine the in vitro sensitivity of Thai Plasmodium falciparum isolates against piperaquine and the influence of the pfmdr1 gene on in vitro response. One hundred and thirty-seven standard laboratory and adapted Thai isolates of P. falciparum were assessed for in vitro piperaquine sensitivity. Polymorphisms of the pfmdr1 gene were determined by polymerase chain reaction methods. The mean and standard deviation of the piperaquine IC50 in Thai isolates of P. falciparum were 16.7 ± 6.3 nM. The parasites exhibiting chloroquine IC50 of ≥ 100 nM were significantly less sensitive to piperaquine compared with the parasite with chloroquine IC50 of < 100 nM. No significant association between the pfmdr1 copy number and piperaquine IC50 values was found. In contrast, the parasites containing the pfmdr1 86Y allele exhibited significantly reduced piperaquine sensitivity. Before nationwide implementation of dihydroartemisinin–piperaquine as the first-line treatment in Thailand, in vitro and in vivo evaluations of this combination should be performed especially in areas where parasites containing the pfmdr1 86Y allele are predominant such as the Thai–Malaysian border.

Introduction

Emergence and spread of multidrug-resistant Plasmodium falciparum strains presents its most serious situation along the Thai–Myanmar and Thai–Cambodian borders.1,2 To combat this problem, Thailand was the first country to use an artemisinin combination therapy (ACT), artesunate–mefloquine to treat uncomplicated falciparum malaria.3 Unfortunately, evidence of artemisinin resistance, indicated by delayed parasite clearance has been reported in these areas.4,5 Although artemisinin resistance has been a matter of concern, ACTs are still the first line of choice to treat multidrug-resistant falciparum malaria due to its acceptable cure rate. Treatment failure of ACT has been associated with resistance to the partner drugs rather than delayed parasite clearance phenotype.68 Thus, an ACT with the effective partner drug should be chosen rationally.

Piperaquine, a bisquinoline antimalarial drug, was extensively used as a monotherapy for the treatment of chloroquine-resistant falciparum malaria in China.9 To date, dihydroartemisinin–piperaquine is one of the available ACTs to treat uncomplicated falciparum malaria. Short-course dihydroartemisinin–piperaquine has shown excellent efficacy in a few clinical trials and is considered as a promising fixed-dose formulation to treat multidrug-resistant falciparum malaria.1012 Currently, dihydroartemisinin–piperaquine has been used in southeast Asia including Myanmar and Cambodia. However, the development of piperaquine resistance in P. falciparum has been concerned as the resistance was reported in China after being used as a monotherapy. Rapid emergence of piperaquine resistance may be associated with its long half-life and cross-resistance to a structurally related 4-aminoquinoline, chloroquine.9 After implementing national policy to use dihydroartemisinin–piperaquine as the first-line treatment of uncomplicated falciparum malaria in Cambodia, rapid decline in the efficacy of dihydroartemisinin–piperaquine was reported.1317 The treatment failure observed with dihydroartemisinin–piperaquine has proved to be related to the presence of piperaquine resistance.1618

Recently, a few molecular markers have been identified for antimalarial resistance in P. falciparum. The P. falciparum chloroquine resistance transporter (pfcrt) has been identified as the main determinant of chloroquine resistance.19 A point mutation on the pfcrt gene resulting in replacement of lysine by threonine in the PfCRT at codon 76 has been linked to chloroquine resistance among parasite isolates collected worldwide.20 A few studies have shown a correlation between in vitro chloroquine and piperaquine sensitivity2124; however, the influence of the pfcrt 76T allele on the in vitro sensitivity of piperaquine is still controversial.2427 Plasmodium falciparum multidrug resistance 1 (pfmdr1), a gene on chromosome 5 encoding a P-glycoprotein homologue 1 (Pgh1) also contributes to chloroquine resistance.2831 At least five single-nucleotide polymorphisms (SNPs) on the pfmdr1 gene have been identified, that is, N86Y, Y184F, S1034C, N1042D, and D1246Y.28 Both SNPs and copy number variation (CNV) of the pfmdr1 gene influence in vitro and in vivo responses to mefloquine, an arylaminoalcohol.3236 Evidence suggests that the pfmdr1 gene plays a role in the in vitro response to other quinolines such as quinine, lumefantrine, and artemisinin derivatives.29,3740

Plasmodium falciparum isolates collected from different areas along the international border of Thailand have exhibited different resistant phenotypic and genotypic patterns.41 Different pfmdr1 polymorphism patterns exhibit varied antimalarial drug susceptibilities.41 Dihydroartemisinin–piperaquine is one of the ACTs of choice to replace artesunate–mefloquine to treat uncomplicated falciparum malaria in Thailand. Thus, we aimed to determine in vitro piperaquine sensitivity against both laboratory and recently adapted Thai isolates of P. falciparum and the influence of the pfmdr1 gene on in vitro piperaquine sensitivity.

Materials and Methods

Plasmodium falciparum strains and cultivation.

One hundred and thirty-seven isolates of P. falciparum including five standard laboratory isolates, that is, K1, T994, M12, 3D7, and G112 and adapted Thai isolates, obtained from malarial patients presenting for treatment from the Thai–Myanmar border, that is, Tak, Kanchanaburi, and Ranong and Thai–Cambodian border, that is, Chanthaburi, Trat, and Sisaket from 1989 to 2014. A total of 12, 9, 60, 27, and 24 isolates were collected in 1989, 1993, 1998, 2003, and 2009 to 2014, respectively. Parasites were maintained in continuous cultures for two to three cycles before in vitro sensitivity assays by modifying method of Trager and Jensen.42 Cultures were maintained under an atmosphere of 90% N2, 5% O2, and 5% CO2.

In vitro sensitivity assays.

Sensitivity of P. falciparum isolates to piperaquine and other antimalarial drugs including chloroquine, quinine, mefloquine, lumefantrine, artemether, artesunate, and dihydroartemisinin were determined by measuring [3H]hypoxanthine incorporation in parasite nucleic acids as previously described.43 For each experiment, parasite preparation containing 1% parasitemia and 2% hematocrit was incubated with different concentrations of the tested drug 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 harvesting. Drug IC50, that is, the concentration of a drug which inhibits parasite growth by 50%, was determined from the log dose/response relationship as fitted by GRAFIT (Erithacus Software, Kent, England).

Genotypic characterization for the pfcrt and pfmdr1 genes.

Parasite DNA was extracted simultaneously as the in vitro sensitivity assay was performed using the Chelex resin method.44 Polymerase chain reaction (PCR) and allele-specific restriction analysis were performed to detect the pfcrt mutations encoded amino acids at position 76.45 Nested PCR and restriction endonuclease digestion method, developed by Duraisingh and others (2000) was performed to detect the pfmdr1 mutations at codons 86, 184, 1034, 1042, and 1246. K1 and 7G8 strains were used as the positive control.37 Results with a combined band pattern of undigested and digested fragments were considered mixed alleles. The pfmdr1 gene copy number was determined by TaqMan real-time PCR (ABI sequence detector 7000; Applied Biosystems, Foster City, CA) as developed by Price and others (2004).36 Primers and fluorescence-labeled probes were used to amplify pfmdr1 and β-tubulin genes. PCR conditions and thermal cycling conditions were used as described. The K1 and DD2 clone containing one and four pfmdr1 copies, respectively, was used as the reference DNA sample. The pfmdr1 and β-tubulin amplification reactions were run in duplicate. The relative pfmdr1 copy number was assessed using the method described by Price and others (2004).36

Statistical analysis.

Data were analyzed using STATA/MP, Version 12 (StataCorp, College Station, TX). Each IC50 value represented the mean of at least three independent experiments. Normally distributed IC50 data were assessed by the Kolmogorov–Smirnov test. Correlations were assessed by Pearson's correlation. Differences of the mean IC50 and copy number of the pfmdr1 gene among groups were analyzed using independent t test and one-way analysis of variance (ANOVA). Post hoc test (Scheffe) for multiple comparisons was used to test differences between the two groups. Association between genotypes and P. falciparum from different areas was analyzed using χ2 test or Fisher's exact test. The level of significance was set at a P value of < 0.05.

Results

In vitro piperaquine sensitivity.

One hundred and thirty-seven isolates were tested for sensitivity to piperaquine. The piperaquine IC50s of the 132 adapted Thai isolates ranged from 6.4 to 33.7 nM. Laboratory strains including K1, T994, M12, 3D7, and G112 showed IC50s of 37.8, 24.7, 37.9, 18.9, and 14.2 nM, respectively. The mean and standard deviation (SD) of piperaquine IC50 in 132 Thai isolates were 16.7 ± 6.3 nM. Piperaquine IC50s of these isolates were normally distributed. Table 1 shows the mean piperaquine IC50s of 132 parasites isolated from Thai–Myanmar and Thai–Cambodian areas. No significant difference was found in piperaquine IC50s among the parasites isolated from the two different areas (independent t test, P = 0.223). The mean piperaquine IC50 of the parasites isolated from different years (14.9 ± 6.1 nM in 1989, 16.1 ± 9.0 nM in 1993, 16.4 ± 5.9 nM in 1998, 16.8 ± 5.4 nM in 2003, and 18.6 ± 7.0 nM in 2009–2014) showed no significant difference (one-way ANOVA, P = 0.438). The correlations between the IC50s of piperaquine and other antimalarial drugs, that is, chloroquine, quinine, mefloquine, lumefantrine, artemether, artesunate, and dihydroartemisinin were insignificant (data not shown). However, chloroquine-resistant parasites (IC50 ≥ 100 nM) exhibited less sensitivity to piperaquine (19.1 ± 7.8 nM, N = 50) compared with parasites exhibiting chloroquine IC50 of < 100 nM (15.9 ± 5.7, N = 87) (independent t test, P = 0.014).

Table 1

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

AreaNPiperaquine IC50 (nM)pfmdr1 copy no.pfmdr1 mutations n (%)
86Y184F1034C1042D1246Y
Thai–Myanmar7216.5 ± 5.82.8 ± 1.45 (6.9)25 (34.7)4 (5.6)4 (5.6)
Thai–Cambodian6016.6 ± 5.21.4 ± 0.97 (11.7)52 (86.7)6 (10.0)10 (16.7)
Total13216.7 ± 6.32.2 ± 1.412 (9.1)77 (58.3)10 (7.6)14 (10.6)

Characterization of the pfmdr1 gene.

One hundred and thirty-two adapted Thai isolates were analyzed for mutations in the pfcrt and pfmdr1 genes. All 132 isolates contained the pfcrt 76T allele. Mixed K76 and 76T alleles were unidentified. Distribution of the pfmdr1 polymorphisms in the parasite isolates from two different areas is shown in Table 1. Distribution of the pfmdr1 alleles had changed significantly over time among the parasites from the Thai–Cambodian border. The pfmdr1 184F allele increased from 66.7% in 1989 and 1993 to 93.3% in 2003 and 100% after 2009. The mean pfmdr1 copy number of these isolates was 2.2 ± 1.4 (range = 0.76–5.5). For laboratory isolates, only K1 and M12 contained the pfcrt 76T allele. K1, T994, and M12 contained the pfmdr1 86N allele. Isolate 3D7 contained the pfmdr1 184F allele, whereas G112 showed no mutation.

Association between in vitro piperaquine sensitivity and the pfmdr1 gene.

Table 2 shows in vitro piperaquine sensitivities of P. falciparum isolates with different pfmdr1 genotypes. Reduced piperaquine sensitivity was observed in those parasites containing 86Y and 1042N alleles. The pfmdr1 copy number had no impact on piperaquine susceptibility. The parasite isolates were categorized in seven groups according to their genotype of the pfmdr1 gene (Table 3), that is, (I) 86N allele, (II) 184F allele, (III) 1034C + 1042D alleles, (IV) 1042D allele, (V) 184F + 1034C + 1042D alleles, (VI) wild type with the copy number of ≤ 1, and (VII) wild type with the copy number of > 1. Significant differences were observed in the piperaquine IC50s among these seven haplotypes (P < 0.001, one-way ANOVA). Post hoc analysis showed that the piperaquine IC50s of the parasites in group I was significantly higher than those of groups II (P < 0.001), III (P = 0.003), IV (P = 0.003), and VII (P < 0.001). Because the parasites in groups I–IV contained varied pfmdr1 copy numbers, influence of the pfmdr1 copy number on the piperaquine IC50s in each group was analyzed. No significant difference was found in the piperaquine IC50s among the parasites with different copy numbers in these four groups.

Table 2

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

pfmdr1 genotypesN (%)Mean piperaquine IC50 (nM)P value
Mutations   
86N86122 (89.1)16.0 ± 5.7< 0.001*
86Y15 (10.9)25.8 ± 8.2
184Y18459 (43.1)17.3 ± 7.60.252
184F78 (56.9)16.5 ± 5.9
1034S1034127 (92.7)17.3 ± 6.80.177
1034C10 (7.3)14.3 ± 3.8
1042N1042123 (89.8)17.5 ± 6.80.003*
1042D14 (10.2)13.5 ± 3.9
Copy no.≤ 141 (30.0)17.3 ± 7.20.365
> 1–235 (25.5)17.8 ± 7.0
> 2–324 (17.5)16.8 ± 6.3
> 3–418 (13.1)18.4 ± 6.7
≥ 419 (13.9)14.3 ± 4.8

Significant difference determined by independent t test.

Table 3

In vitro piperaquine sensitivities of different genotyped subgroups

Grouppfmdr1 haplotypeNPiperaquine IC50 (nM)
8618410341042Copy no.
IYYSN0.87–5.01525.8 ± 8.2
IINFSN0.76–5.06517.1 ± 6.1*
IIINYCD0.87–3.4914.8 ± 3.7
IVNYSD1.0–4.7411.3 ± 3.8
VNFCD1.0110.1
VINYCN≤ 1416.5 ± 5.9
VIINYCN> 13915.1 ± 5.1*

Significant difference among groups (P < 0.001, one-way analysis of variance).

Significant difference compared with group I (P < 0.001, post hoc analysis).

Significant difference compared with group I (P = 0.003, post hoc analysis).

Using 28.3 nM (the mean IC50 plus two SDs) as the cutoff point for reduced piperaquine sensitivity, 11 of 137 (8.3%) isolates exhibited reduced piperaquine sensitivity. Univariate and multivariate analysis identified the pfmdr1 86Y allele as a significant factor associated with reduced piperaquine sensitivity (odds ratio = 15.7, 95% confidence interval = 4.0–60.8, P < 0.001), adjusted for the pfmdr1 N1042D mutation, pfmdr1 copy number, and chloroquine resistance (IC50 ≥ 100 nM).

Discussion

In vitro sensitivities of piperaquine of laboratory and adapted Thai isolates were determined. To date, the cutoff point for in vitro piperaquine resistance remains undetermined. Different criteria for reduced sensitivity to piperaquine in vitro were used in a few studies.23,46,47 When we used a 3-fold decrease in sensitivity to piperaquine IC50 (56.7 nM) of 3D7, the laboratory standard indicated in the study of China–Myanmar isolates,23 no parasite isolate exhibited reduced piperaquine sensitivity. When the cutoff point for reduced in vitro piperaquine sensitivity was estimated using the mean IC50 plus 2 SDs (28.3 nM), 11 Thai isolates (8.3%) exhibited reduced sensitivity to piperaquine in vitro (28.9–33.7 nM). The mean piperaquine IC50 in the present study was in the same range as reported in other studies from southeast Asia.22,23,4850 However, Thai isolates in this study showed up to five times more sensitivity to piperaquine compared with African isolates.21,22,51,52 In 2010, dihydroartemisinin–piperaquine was adopted as the nationwide drug used for multidrug-resistant falciparum malaria in Cambodia.13 Unfortunately, treatment failure of dihydroartemisinin–piperaquine was reported right after the implementation possibly due to the existing parasites with reduced piperaquine susceptibility because piperaquine monotherapy was used in Cambodia in the 1990s.9 Some but not all reports showed that treatment failure of dihydroartemisinin–piperaquine was associated with higher piperaquine IC50s.14,17,18

The rapid emergence of piperaquine resistance in China might be explained by the cross-resistance between piperaquine and chloroquine.9 A significant correlation between in vitro piperaquine and chloroquine sensitivity has been reported in some but not all studies.2126,51 From the analysis of 137 laboratory and adapted Thai isolates, no significant correlation was observed between in vitro piperaquine and chloroquine sensitivity (Pearson's correlation coefficient = 0.13, P = 0.13). The parasites exhibiting chloroquine IC50 ≥ 100 nM showed slightly but significantly higher piperaquine IC50s (19.1 nM) compared with those exhibited chloroquine IC50 of < 100 nM (15.9 nM). This could explain the result of multivariate analysis showing that a chloroquine-resistant phenotype was not the associated factor of reduced piperaquine sensitivity.

Polymorphisms of the pfcrt and pfmdr1 genes have been linked to chloroquine resistance.19,20,2831 The K76T mutation in the pfcrt gene is a key determinant for chloroquine resistance.19,20 However, a few studies have shown parasite isolates containing the pfcrt 76T allele exhibited chloroquine sensitivity.53,54 In the present study, we found three Thai chloroquine-sensitive isolates containing the pfcrt 76T allele. The discordance between the in vitro sensitivity and the K76T mutation may be due to the interaction of resistant genes. Indeed, it has been shown that polymorphisms of the pfmdr1 gene could modulate the level of chloroquine resistance.2831 In contrast to chloroquine, the role of these resistant genes on piperaquine sensitivity is controversial. Using genetically modified parasites, Muangnoicharoen and others showed that the K76T mutation in the pfcrt gene could affect in vitro piperaquine sensitivity.27 However, a few studies using standard laboratory and adapted strains from different geographical areas showed no association between in vitro piperaquine sensitivity and the K76T mutation in the pfcrt gene.2426 Recently, a few studies have focused on the association between SNPs and CNV of the pfmdr1 gene and in vitro piperaquine sensitivity.50,5557 Using a drug pressure experiment, de-amplification of a region in chromosome 5 encompassing the pfmdr1 gene was identified in piperaquine-resistant clones compared with the parent strains. This finding suggests a possible linkage between pfmdr1 copy number and piperaquine sensitivity.55 A study of parasites isolated from the Thai–Myanmar border showed that parasites with one pfmdr1 copy number exhibited significantly reduced piperaquine sensitivity compared with the parasites containing more than one pfmdr1 copy number.56 However, the result from our study shows no association between the pfmdr1 copy number and in vitro piperaquine sensitivity. The contrasting findings might be explained by differences in genetic background of parasites from different geographical areas. For example, a study of parasites isolated from three provinces of Cambodia showed influence of the pfmdr1 copy number on in vitro piperaquine sensitivity only in the parasites isolated from Pursat Province in the western Cambodia, but neither Preah Vihear nor Ratanakiri provinces in the eastern Cambodia.50 Recently, a nonsynonymous SNP encoding a Glu415Gly mutation in a putative exonuclease (exo-E415G), and amplification of plasmepsin II and plasmepsin III genes have been identified as genetic markers of piperaquine resistance in Cambodia.58,59

On the other hand, the parasites in this study containing the pfmdr1 86Y allele showed significantly higher piperaquine IC50s compared with those containing the pfmdr1 N86 allele. Multivariate analysis also identified the pfmdr1 86Y allele as an associated factor of reduced piperaquine sensitivity. The important role of SNPs in the pfmdr1 gene on in vitro piperaquine sensitivity has been confirmed by a recent study using genetically modified P. falciparum lines. The N86Y and Y184F mutations modulated piperaquine sensitivity in strains containing an Asian/African variant of the PfCRT, CVIET.57 In Thailand, P. falciparum isolates from different areas contained different patterns of pfmdr1 polymorphisms.41 The majority of parasites from the Thai–Cambodian border contained the pfmdr1 184F allele with a lower copy number, whereas parasites collected from the Thai–Myanmar border usually contained either the 184Y or 184F allele with a higher copy number of the pfmdr1 gene. In contrast, the parasites from the southernmost provinces of Thailand predominantly contained the pfmdr1 86Y allele.41 Since the nationwide implementation of fixed-dose ACT, that is, dihydroartemisinin/piperaquine would be started in Thailand, parasites with reduced piperaquine sensitivity might be selected in such areas. Thus, in vitro and in vivo monitoring should be regularly performed.

In conclusion, this study determined the baseline in vitro piperaquine sensitivity in Thai isolates of P. falciparum. The parasites containing the pfmdr1 86Y allele exhibited reduced in vitro piperaquine sensitivity. Thus, dihydroartemisinin–piperaquine should be carefully evaluated especially where the parasites with this particular genotype are predominant before it can be considered as the first-line treatment in Thailand.

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Author Notes

* Address correspondence to Mathirut Mungthin, Department of Parasitology, Phramongkutklao College of Medicine, Ratchawithi Road, 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) and the Phramongkutklao Research Fund.

Authors' addresses: Mathirut Mungthin, Naruemon Sitthichot, Nantana Suwandittakul, and Rommanee Khositnithikul, Department of Parasitology, Phramongkutklao College of Medicine, Bangkok, Thailand, E-mails: mathirut@hotmail.com, mude_143@hotmail.com, suwanna_b@hotmail.com, and kik_kuru@yahoo.com. Ekularn Watanatanasup, Mahidol University International College, Nakhon Pathom, Thailand, E-mail: ekularn.wat@mahidol.ac.th. Stephen A. Ward, Division of Molecular and Biochemical Parasitology, Liverpool School of Tropical Medicine, Liverpool, United Kingdom, E-mail: steve.ward@lstmed.ac.uk.

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