Skip to main content
PMC home page
Malaria Journal logoLink to Malaria Journal
. 2012 Jan 5;11:7. doi: 10.1186/1475-2875-11-7

Polymorphisms of the pfmdr1 but not the pfnhe-1 gene is associated with in vitro quinine sensitivity in Thai isolates of Plasmodium falciparum

Teera Poyomtip 1, Nantana Suwandittakul 2, Narumon Sitthichot 2, Rommanee Khositnithikul 2, Peerapan Tan-ariya 3, Mathirut Mungthin 2,✉
PMCID: PMC3287963  PMID: 22221394

Abstract

Background

The emergence of Plasmodium falciparum resistance to most currently used anti-malarial drugs is a major problem in malaria control along the Thai-Myanmar and Thai-Cambodia borders. Quinine (QN) with tetracycline/doxycycline has been used as the second-line treatment for uncomplicated falciparum malaria. In addition, QN monotherapy has been the first-line treatment for falciparum malaria in pregnant women. However, reduced in vitro and in vivo responses to QN have been reported. To date, a few genetic markers for QN resistance have been proposed including Plasmodium falciparum chloroquine resistance transporter (pfcrt), P. falciparum multidrug resistance 1 (pfmdr1), and P. falciparum Na+/H+ exchanger (pfnhe-1). This study was to investigate the role of the pfmdr1 and pfnhe-1 gene on in vitro QN sensitivity in Thai isolates of P. falciparum.

Methods

Eighty-five Thai isolates of P. falciparum from the Thai-Myanmar and Thai-Cambodia borders from 2003-2008 were determined for in vitro QN sensitivity using radioisotopic assay. Polymorphisms of the pfmdr1 and pfnhe-1 gene were determined by PCR-RFLP and sequence analysis. Associations between the in vitro QN sensitivity and the polymorphisms of the pfmdr1 and pfnhe-1 gene were evaluated.

Results

The mean QN IC50 was 202.8 nM (range 25.7-654.4 nM). Only four isolates were QN resistant when the IC50 of >500 nM was used as the cut-off point. Significant associations were found between the pfmdr1 mutations at codons N86Y and N1042D and in vitro QN sensitivity. However, no associations with the number of DNNND, DDNNNDNHNDD, and NHNDNHNNDDD repeats in the microsatellite ms4760 of the pfnhe-1 gene were identified.

Conclusion

Data from the present study put doubt regarding the pfnhe-1 gene as to whether it could be used as the suitable marker for QN resistance in Thailand. In contrast, it confirms the influence of the pfmdr1 gene on in vitro QN sensitivity.

Background

The emergence of anti-malarial resistance in Plasmodium falciparum is a major public health threat worldwide, especially in tropical developing countries. The situation of multidrug-resistant falciparum malaria is most serious along the Thai-Myanmar and Thai-Cambodia borders [1]. To handle this situation, WHO recommends artemisinin derivative-based combination treatment (ACT) for the treatment of uncomplicated falciparum malaria [2]. Artesunate-mefloquine combination has been used as the first-line treatment in Thailand for more than 15 years [3]. Quinine (QN)-tetracycline/doxycycline has been used as the second-line treatment for uncomplicated falciparum malaria in Thailand. In addition, QN monotherapy is the first-line treatment for pregnancy [3,4]. Unfortunately reduced in vitro and in vivo response to QN has been reported in Southeast Asia [5,6]. Investigations have been carried out to identify the mechanisms of QN resistance. At least three candidate genes including Plasmodium falciparum chloroquine resistance transporter (pfcrt), Plasmodium falciparum multidrug resistance 1 (pfmdr1), and Plasmodium falciparum Na+/H+ exchanger (pfnhe-1) have been linked to reduced QN sensitivity [7-12]. Cooper et al. (2002) showed the association between the pfcrt mutation at codon 76 and QN sensitivity [7]. In addition, recent studies have shown that parasites containing a novel mutation in the pfcrt gene, Q352K/R, C350R, altered QN sensitivity [8,9]. Concerning the pfmdr1 gene, it has been shown that both mutations and copy number influenced in vitro QN sensitivity [10-12]. Using quantitative trait loci on the genetic cross of HB3 and Dd2 strains, an additional candidate gene, pfnhe-1, for QN resistance was identified [13]. This gene encodes a 226 kDa parasite plasma membrane protein containing 12 transmembrane domains, and 3 microsatellite regions, msR1, ms3580 and ms4760. Variations in QN susceptibilities between different parasite strains have also been linked to repeat polymorphisms in the microsatellite locus ms4760 of pfnhe-1 [13]. A few studies showed that the number of DNNND, DDNNNDNHNDD, and NHNDNHNNDDD repeats in the microsatellite ms4760 influenced in vitro QN sensitivity [14-19]. However, there is a lack of consensus regarding the specific nature of these associations [20-25]. For instance, while some studies report an association between reduced susceptibility to QN and an increase in the number of 'DNNND' repeats in ms4760 [15,18,19], others could not verify this association [22-24], or found that two DNNND repeats was the optimal number for conferring a reduction in QN sensitivity [17]. Moreover, amplification of a second ms4760 repeat 'NHNDNHNNDDD' has been linked to increases in the parasite's susceptibility to QN [15,18,19]. However, the reverse association has also been reported [22], and several studies have failed to confirm either of these findings [23,24]. Considering the situation of multidrug-resistant P. falciparum in Thailand, determination of the molecular basis of QN resistance is crucial to be able to monitor parasite resistance. This study was to investigate the influence of the pfmdr1 and pfnhe-1 genes on in vitro QN sensitivity of Thai isolates of P. falciparum from both the Thai-Myanmar and Thai-Cambodia borders.

Methods

Plasmodium falciparum strains and cultivation

The 85 isolates of P. falciparum used in this study were collected from patients with uncomplicated falciparum malaria, who attended malaria clinics and hospitals in malaria endemic areas along the Thai-Myanmar (Kanchanaburi and Ranong) and Thai-Cambodia (Chantaburi and Srisaket) borders from 2003 to 2008. The research protocol was reviewed and approved by the Ethics Committee of the Royal Thai Army Medical Department. Parasites were maintained in continuous cultures using a modification of the method of Trager and Jensen [26].

In vitro sensitivity assays

QN sensitivity of P. falciparum isolates was determined by measurement of [3H] hypoxanthine incorporation into parasite nucleic acids as previously described [27]. Drug IC50 (i.e. 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 pfmdr1 and pfnhe-1 genes

Parasite DNA was extracted using the Chelex-resin method [28]. Five microliters of DNA preparation was used for a 25 μl PCR reaction. Mutations in the pfmdr1 gene were determined by the nested PCR and restriction endonuclease digestion method developed by Duraisingh et al. for detection of the mutations at codons 86, 184, 1034, 1042 and 1246. K1 and 7G8 strain were used as positive controls [29]. The pfmdr1 gene copy number was determined by TaqMan real-time PCR (ABI sequence detector 7000; Applied Biosystems) as developed by Price et al. [30]. The K1 and Dd2 clone containing 1 and 4 pfmdr1 copies, respectively was used as the reference DNA sample. The pfmdr1 and β-tubulin amplification reactions were run in duplicate. Relative pfmdr1 copy number was assessed as previously described. PCR amplification for the pfnhe-1 ms4760 microsatellite was performed as previously described [14]. DNA purification and DNA sequencing were conducted by Bioservice Unit, Bangkok, Thailand. Sequences were analyzed for the number of DNNND, DDNNNDNHNDD, and NHNDNHNNDDD repeats in the pfnhe-1 ms4760 microsatellite by BioEdit sequence alignment editor (version 7.0.9.0).

Statistical analysis

Data were analysed by SPSS for Windows version 18 (SPSS Inc., Chicago, IL). The SPSS license number is ID5071846. The QN IC50 of each isolate was the mean IC50 of three independent experiments. Each experiment was carried out in triplicate. Normally distributed IC50 data were assessed by the Kolmogorov Smirnov test. Differences among parasites with different genotypes was analysed by Chi square and Fisher's exact test. Correlations between QN IC50 and the pfmdr1 copy number and the number of DNNND, DDNNNDNHNDD, and NHNDNHNNDDD repeats in the pfnhe-1 ms4760 microsatellite were assessed by Pearson's correlation. Differences of the mean QN IC50 among parasites from different groups were analyzed by Independent t test or One-way ANOVA. Post Hoc test (Scheffe) for multiple comparisons was used to test for differences among groups. The level of significance was set at a p value of <0.05.

Results

In vitro QN sensitivity

Characteristics of parasite isolates are presented in Table 1. The mean IC50 (± SD) for QN was 202.8 ± 123.3 nM (range 25.7-654.4 nM). QN IC50s in this population of isolates were normally distributed. No significant differences were found between QN IC50 of parasites isolated from the Thai-Cambodia and Thai-Myanmar borders (p = 0.641, Independent t test). Of 85 isolates, only 4 (4.7%) isolates exhibited QN IC50 of >500 nM. Characterization of the four QN-resistant P. falciparum isolates is shown in Table 2.

Table 1.

In vitro sensitivity to QN and distribution of pfmdr1 polymorphisms of the 85 adapted parasites from Thai-Myanmar and Thai-Cambodia areas

Area No. Mean QN Mean pfmdr1 mutations
IC50 (nM) pfmdr1 copy number 86Y 184F 1034C 1042D 1246Y
Thai- Myanmar 37 209.9 ± 117.1 2.9 ± 1.4* 6 (16.2%) 15 (40.5%) 4 (10.8%) 4 (10.8%) -
Thai- Cambodia 48 197.2 ± 128.9 1.2 ± 0.7 9 (18.8%) 38 (79.2%)** 10 (20.8%) 13 (24.1%) -
Total 85 202.8 ± 123.3 2.0 ± 1.3 15 (17.6%) 53 (61.4%) 14 (16.5%) 17 (20.0%) -
Open in a new tab

* Significant difference between two areas determined by Independent t test (p < 0.001)

** Significant difference between two areas determined by Chi square test (p < 0.001)

Table 2.

Characterization of the four QN-resistant isolates of Plasmodium falciparum

SK20 SK22 MR2 KB12
Origin Srisaket Srisaket Chantaburi Kanchanaburi
QN IC50 (nM) 521.8 503.0 530.6 654.4
Pfmdr1 mutations
 N86Y N N N N
 Y184F F F F F
 S1034C S S S C
 N1042D N N N D
 D1246Y D D D D
Pfmdr1 copy number 0.8 0.9 1.2 2.0
Pfnhe-1
 DNNND repeats 3 4 1 3
 DDNNNDNHNDD repeats 1 2 1 1
 NHNDNHNNDDD repeats 1 1 0 1
Open in a new tab

Characterization of the pfmdr1 and pfnhe-1 genes

Characterization of the pfmdr1 gene of 85 isolates is shown in Table 1. Approximately 60% of the parasite isolates contained the pfmdr1 184F allele. Determination of the pfmdr1 gene copy number showed that these isolates contained pfmdr1 copy numbers with a mean of 2.0 (range 0.7-5.6). The pfmdr1 184F allele was more common in the parasites isolated from the Thai-Cambodia border compared with those from the Thai-Myanmar border. In contrast, the parasites isolated from the Thai-Myanmar border had significantly higher copy numbers. A total of 81 isolates were characterized for polymorphisms in the number of DNNND, DDNNNDNHNDD, and NHNDNHNNDDD repeats in the pfnhe-1 ms4760 microsatellite (Table 3). The most common number of DNNND, DDNNNDNHNDD, and NHNDNHNNDDD repeats was 4 (48.2%), 2 (79%) and 1 (86.4%). Genotypic characterization of the four QN-resistant isolates is shown in Table 2.

Table 3.

Comparison of in vitro QN sensitivity among Plasmodium falciparum with different pfmdr1and pfnhe-1 genotypes

Parasite genotypes No. (%) Mean QN IC50 p value
(nM)
pfmdr1
86 N86 70 (82.4) 216.5 ± 127.5 0.025*
86Y 15 (17.6) 138.3 ± 76.1
184 Y184 32 (37.7) 160.4 ± 93.6 0.013*
184F 53 (62.3) 228.3 ± 132.6
1034 S1034 71 (83.5) 194.1 ± 114.0 0.144
1034C 14 (16.5) 246.9 ± 160.5
1042 N1042 68 (80) 185.8 ± 113.8 0.010*
1042D 17 (20) 270.5 ± 139.9
Copy no. <3 69 (81.2) 195.8 ± 132.2 0.284
≥ 3 16 (18.8) 232.7 ± 68.8
pfnhe-1
DNNND repeats 0 1 (1.2) 183.6 0.958
1 3 (3.7) 246.1 ± 147.1
2 5 (6.2) 163.7 ± 44.2
3 26 (32.1) 203.0 ± 155.3
4 39 (48.2) 213.0 ± 108.6
5 7 (8.6) 195.9 ± 92.0
DDNNNDNHNDD repeats 0 3 (3.7) 115.7 ± 71.3 0.049**
1 14 (17.3) 272.6 ± 193.1
2 64 (79.0) 195.8 ± 102.4
NHNDNHNNDDD repeats 0 9 (11.1) 196.4 ± 137.6 0.378
1 70 (86.4) 210.8 ± 124.2
2 2 (2.5) 87.0 ± 47.4
Open in a new tab

* Significant difference determined by Independent t test

* Significant difference determined by One-way ANOVA

The association between in vitro quinine sensitivity and the pfmdr1 and pfnhe-1 genes

No correlation was found between QN IC50 and the pfmdr1 copy number (r = 0.099, p = 0.367) and the number of DNNND (r = 0.017, p = 0.880), DDNNNDNHNDD (r = -0.76, p = 0.503), and NHNDNHNNDDD (r = -0.420, p = 0.711) repeats in the pfnhe-1 ms4760 microsatellite. Table 3 shows the in vitro QN sensitivities of these adapted Thai isolates containing different pfmdr1 genotypes and different number of DNNND, DDNNNDNHNDD, and NHNDNHNNDDD repeats in the pfnhe-1 ms4760 microsatellite. Parasite isolates with pfmdr1 184F and 1042D showed significantly higher QN IC50 than those containing pfmdr1 184Y and 1042N, respectively. In contrast, parasites having pfmdr1 86Y exhibited significantly lower QN IC50 than those having pfmdr1 86N. Parasites containing different pfmdr1 copy number and the number of DNNND and NHNDNHNNDDD repeats in the pfnhe-1 ms4760 microsatellite showed no significant difference in the mean QN IC50. When the parasites were categorized into subgroups according to their copy number of the pfmdr1gene, using 3 copies as the cut-off point gave the greatest difference of the QN IC50 between 2 groups compared to other figures. However no significant difference was detected. These parasites were also classified into subgroups with a different number of DNNND repeats. No significant difference were found between QN IC50 of parasites containing ≥2 and less repeats (204.9 ± 121.5 nM & 230.5 ± 204.1 nM, p = 0.692), ≥3 and less repeats (207.7 ± 124.7 nM & 207.7 ± 124.7 nM, p = 0.748) or ≥4 and less repeats (210.4 ± 105.5 nM & 200.5 ± 148.1 nM, p = 0.728). Although analysis by One-way ANOVA showed a significant difference of QN IC50 in the parasites with a different number of DDNNNDNHNDD repeats, multiple comparison showed no significant difference between groups.

According to their pfmdr1 haplotypes, parasites were classified into five groups (Table 4), i.e., the isolates containing the pfmdr1 86Y, 1042D, 184F with copy number <3, 184Y with copy number <3 and 184Y with copy number ≥3. Significant differences of QN IC50 were found among these groups (p = 0.019, One-way ANOVA). Multiple comparison indicated that only parasites containing pfmdr1 1042D were significantly less sensitive to QN than parasites containing pfmdr1 86Y (p = 0.048).

Table 4.

Comparison of in vitro QN sensitivity among Plasmodium falciparum with different pfmdr1 haplotypes

Group Pfmdr1 haplotypes No. (%) Mean QN IC50 (nM) ± SD p value
N86Y Y184F N1042D Copy No.
1 Y Y/F N <3/≥3 15 (17.6) 138.3 ± 76.1 0.019
2 N F D <3/≥3 17 (20.0) 270.5 ± 139.9
3 N F N < 3 30 (35.3) 200.4 ± 133.3
4 N Y N < 3 12 (14.1) 162.1 ± 113.0
5 N Y N ≥3 11 (12.9) 236.7 ± 74.0
Total 85 (100) 202.8 ± 123.3
Open in a new tab

Significant difference among groups determined by One-way ANOVA

Discussion

In the present study, using the IC50 of >500 nM as the cut-off point for in vitro QN resistance, only 4.7% (4/85) exhibited QN resistance. Although a higher cut-off point at 800 nM has been proposed, no parasite isolate was found showing QN IC50 of >800 nM. This result is similar to those previous reports showing that most Thai isolates of P. falciparum were QN sensitive [6,31,32]. However data from these studies might not be comparable since different methods including schizont maturation inhibition, isotopic and SYBR green 1 based fluorescence assays were used for the determination of QN IC50. In addition, since culture-adapted isolates were used in some studies including the present study, specific phenotypes might be selected during the adaptation process. The situation of QN resistance in Thailand is less serious than those found in mefloquine and chloroquine. This may be due to a lower drug pressure of QN since it has a shorter half life. However, a decline in QN sensitivity of P. falciparum isolated from the Thai-Myanmar border has been indicated in recent study [6].

All parasite isolates in this study contained chloroquine-resistant haplotype, CVIET of the pfcrt. The association between in vitro QN sensitivity and polymorphisms of the pfmdr1 gene, but not the pfnhe-1 gene, was identified. Although a genetic cross study indicated that QN sensitivity can be modulated by the pfnhe-1 gene [13], the role of the pfnhe-1 gene as a molecular marker for QN resistance is still controversial. Some but not all in vitro and in vivo studies identified the association between DNNND, DDNNNDNHNDD, and NHNDNHNNDDD repeats of the pfnhe-1 ms4760 microsatellite and QN sensitivity and treatment outcome, respectively [14-19,23-25]. Nearly half of Thai isolates in the present study contained 4 DNNND repeats while most parasites from other areas in Southeast Asia including Vietnam and the China-Myanmar border contained 3 DNNND repeats [18,19]. Similar to these 2 studies, most isolates in the present study contained 1 repeat of NHNDNHNNDDD. The studies with positive association usually showed that parasites with 2 or more than 2 DNNND repeats had a significantly reduced QN sensitivity compared with those with 1 repeat. This association has been found in the studies from Vietnam and the China-Myanmar border as well [18,19]. In contrast, no significant difference of QN IC50 between parasites containing ≥2 and less DNNND repeats was identified. Inconsistent findings of the association between the response to QN and pfnhe-1 gene might be due to its interaction with other genes such as pfcrt and pfmdr1. This postulation has been provided to explain the result of a knockdown pfnhe-1 expression resulting in increased QN sensitivity in 2 of 3 parasite lines [16]. In addition, a recent study in Kenya found no significant difference of QN IC50 among parasites with different DNNND repeats [17]. However parasites containing 2 DNNND repeats with 86Y pfmdr1 showed a decrease in QN sensitivity. When the QN IC50 of the parasites containing a similar number of DNNND repeats in the pfnhe-1 ms4760 microsatellite with different 86 alleles in the pfmdr1 gene was compared, no significant difference of QN IC50 among these parasites was identified (data not shown). In addition, no significant correlations between the number of DDNNNDNHNDD, and NHNDNHNNDDD repeats of the pfnhe-1 ms4760 microsatellite and in vitro QN sensitivity were found. Since there were only four isolates exhibiting reduced QN susceptibility, this may be restrictive for identification or validation of these new markers.

In contrast to the pfnhe-1 gene, in vitro QN sensitivity was significantly associated with the mutations in the pfmdr1 gene in these Thai isolates. The parasites containing the pfmdr1 184F and 1042D allele showed less sensitivity to QN while those with the pfmdr1 86Y exhibited increased QN sensitivity. However, when the parasites were categorized according to their haplotypes of the pfmdr1 gene, the pfmdr1 86Y and 1042D allele influenced QN sensitivity. Compelling evidence for a significant role of N1042D mutation on in vitro QN sensitivity has been shown in a few studies using allelic exchange strategies [10,11]. Reed et al. [10] showed that insertion of the pfmdr1 gene containing the 1034C, 1042D and 1246Y alleles made a QN - sensitive parasite become more resistant to QN [10]. Conversely, a QN -resistant line became more sensitive to QN after these alleles were removed. More recently, a study by Sidhu et al. (2005) identified that a single mutation, the N1042D, could modulate the parasites become less sensitive to QN [11]. Unlike the N1042D mutation, the functional role of the N86Y mutation on QN sensitivity has been explored by expression of the pfmdr1 gene in a heterologous system, Xenopus oocytes [33]. Substituting the asparagines (N) at position 86 for tyrosine (Y) resulted in a loss of QN transport ability. Since the site of QN action is in the food vacuole, [34,35] transport ability of the wild-type Pgh1 reduces drug concentration in the food vacuole, and consequently results in decreased QN susceptibility. This finding is compatible to that found in a few studies using parasites isolated from Southeast Asia, including the present study showing that the parasite isolates containing the pfmdr1 86Y allele showed more sensitive to QN [20,32]. However, a contrary result was shown in the study of parasite isolates from Kenya [17]. The decrease in QN susceptibility was associated with the pfmdr1 86Y allele in parasites harbouring the two DNNND repeats in the pfnhe-1 ms4760 microsatellite. Since reduced susceptibility to QN appear to be governed by a number of proteins whose contributions vary between strains. The contrasting findings might be explained by different variations in genetic background of parasites from different geographical areas. The influence of the pfmdr1 copy number on QN sensitivity has been confirmed by the study of Sidhu et al. using the knockdown strategy [12]. A few studies of parasite isolates from the Thai-Myanmar border also showed the influence of pfmdr1 copy number on in vitro QN sensitivity. However, this association has not been identified in some studies [21,22]. In this study, no association between the pfmdr1 copy number and in vitro QN sensitivity neither in parasites from the Thai-Myanmar nor the Thai-Cambodia border was identified.

In conclusion, the present study confirms the involvement of the pfmdr1 gene in QN sensitivity. Both N86Y and N1042D mutations significantly modulate in vitro response to QN. Although previous studies from Southeast Asia including Vietnam and China-Myanmar border have demonstrated that the pfnhe-1 gene is involved in QN sensitivity, the present study showed no association. This has raised doubt regarding the pfnhe-1 gene as to whether it could be used as the suitable marker for QN resistance in Thailand.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

TP and MM contributed to the conception and design of the study. NSu, NSi and RK performed in vitro susceptibility test and genotyping. TP, PT and MM analyzed the data and wrote the manuscript. All authors read and approved the final version that was submitted for publication.

Contributor Information

Teera Poyomtip, Email: teera_075@hotmail.com.

Nantana Suwandittakul, Email: suwanna_b@hotmail.com.

Narumon Sitthichot, Email: mude_143@hotmail.com.

Rommanee Khositnithikul, Email: kik_kuru@yahoo.com.

Peerapan Tan-ariya, Email: scptn@mahidol.ac.th.

Mathirut Mungthin, Email: mathirut@pmk.ac.th.

Acknowledgements

This study was financially supported by the Office of Research Development, The Ministry of Defence, Thailand and Phramongkutklao Research Fund.

References

  1. World Health Organization. Global report on antimalarial drug efficacy and drug resistance 2000-2010. Geneva: WHO; 2010. [Google Scholar]
  2. World Health Organization. Guidelines for the treatment of malaria. Geneva: WHO; 2006. [Google Scholar]
  3. White NJ. The treament of malaria. N Engl J Med. 1996;335:800–806. doi: 10.1056/NEJM199609123351107. [DOI] [PubMed] [Google Scholar]
  4. Looareesuwan S, Wilairatana P, Vanijanonta S, Kyle D, Webster K. Efficacy of quinine-tetracycline for acute uncomplicated falciparum malaria in Thailand. Lancet. 1992;i:367–370. doi: 10.1016/0140-6736(92)91690-a. [DOI] [PubMed] [Google Scholar]
  5. Pukrittayakamee S, Supanaranond W, Looareesuwan S, Vanijanonta S, White NJ. Quinine in severe falciparum malaria: evidence of declining efficacy in Thailand. Trans R Soc Trop Med Hyg. 1994;88:324–327. doi: 10.1016/0035-9203(94)90102-3. [DOI] [PubMed] [Google Scholar]
  6. Hüttinger F, Satimai W, Wernsdorfer G, Wiedermann U, Congpuong K, Wernsdorfer WH. Sensitivity to artemisinin, mefloquine and quinine of Plasmodium falciparum in northwestern Thailand. Wien Klin Wochenschr. 2010;122(Suppl 3):52–56. doi: 10.1007/s00508-010-1438-6. [DOI] [PubMed] [Google Scholar]
  7. Cooper RA, Ferdig MT, Su XZ, Ursos LM, Mu J, Nomura T, Fujioka H, Fidock DA, Roepe PD, Wellems TE. Alternative mutations at position 76 of the vacuolar transmembrane protein PfCRT are associated with chloroquine resistance and unique stereospecific quinine and quinidine responses in Plasmodium falciparum. Mol Pharmacol. 2002;61:35–42. doi: 10.1124/mol.61.1.35. [DOI] [PubMed] [Google Scholar]
  8. Cooper RA, Lane KD, Deng B, Mu J, Patel JJ, Wellems TE, Su X, Ferdig MT. Mutations in transmembrane domains 1, 4 and 9 of the Plasmodium falciparum chloroquine resistance transporter alter susceptibility to chloroquine, quinine and quinidine. Mol Microbiol. 2007;63:270–282. doi: 10.1111/j.1365-2958.2006.05511.x. [DOI] [PubMed] [Google Scholar]
  9. Valderramos SG, Valderramos JC, Musset L, Purcell LA, Mercereau-Puijalon O, Legrand E, Fidock DA. Identification of a mutant PfCRT-mediated chloroquine tolerance phenotype in Plasmodium falciparum. PLoS Pathog. 2010;6:e1000887. doi: 10.1371/journal.ppat.1000887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Reed MB, Saliba KJ, Caruana SR, Kirk K, Cowman AF. Pgh1 modulates sensitivity and resistance to multiple antimalarials in Plasmodium falciparum. Nature. 2000;403:906–909. doi: 10.1038/35002615. [DOI] [PubMed] [Google Scholar]
  11. Sidhu AB, Valderramos SG, Fidock DA. pfmdr1 mutations contribute to quinine resistance and enhance mefloquine and artemisinin sensitivity in Plasmodium falciparum. Mol Microbiol. 2005;57:913–926. doi: 10.1111/j.1365-2958.2005.04729.x. [DOI] [PubMed] [Google Scholar]
  12. Sidhu AB, Uhlemann AC, Valderramos SG, Valderramos JC, Krishna S, Fidock DA. Decreasing pfmdr1 copy number in Plasmodium falciparum malaria heightens susceptibility to mefloquine, lumefantrine, halofantrine, quinine, and artemisinin. J Infect Dis. 2006;194:528–535. doi: 10.1086/507115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ferdig MT, Cooper RA, Mu J, Deng B, Joy DA, Su XZ, Wellems TE. Dissecting the loci of low-level quinine resistance in malaria parasites. Mol Microbiol. 2004;52:985–997. doi: 10.1111/j.1365-2958.2004.04035.x. [DOI] [PubMed] [Google Scholar]
  14. Vinayak S, Alam MT, Upadhyay M, Das MK, Dev V, Singh N, Dash AP, Sharma YD. Extensive genetic diversity in the Plasmodium falciparum Na+/H + exchanger 1 transporter protein implicated in quinine resistance. Antimicrob Agents Chemother. 2007;51:4508–4511. doi: 10.1128/AAC.00317-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Henry M, Briolant S, Zettor A, Pelleau S, Baragatti M, Baret E, Mosnier J, Amalvict R, Fusai T, Rogier C, Pradines B. Plasmodium falciparum Na+/H + exchanger 1 transporter is involved in reduced susceptibility to quinine. Antimicrob Agents Chemother. 2009;53:1926–1930. doi: 10.1128/AAC.01243-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Nkrumah LJ, Riegelhaupt PM, Moura P, Johnson DJ, Patel J, Hayton K, Ferdig MT, Wellems TE, Akabas MH, Fidock DA. Probing the multifactorial basis of Plasmodium falciparum quinine resistance: evidence for a strain-specific contribution of the sodium-proton exchanger PfNHE. Mol Biochem Parasitol. 2009;165:122–131. doi: 10.1016/j.molbiopara.2009.01.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Okombo J, Kiara SM, Rono J, Mwai L, Pole L, Ohuma E, Borrmann S, Ochola LI, Nzila A. In vitro activities of quinine and other antimalarials and pfnhe polymorphisms in Plasmodium isolates from Kenya. Antimicrob Agents Chemother. 2010;54:3302–3307. doi: 10.1128/AAC.00325-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Meng H, Zhang R, Yang H, Fan Q, Su X, Miao J, Cui L, Yang Z. In vitro sensitivity of Plasmodium falciparum clinical isolates from the China-Myanmar border area to quinine and association with polymorphism in the Na+/H + exchanger. Antimicrob Agents Chemother. 2010;54:4306–4313. doi: 10.1128/AAC.00321-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sinou V, Quang le H, Pelleau S, Huong VN, Huong NT, Tai le M, Bertaux L, Desbordes M, Latour C, Long LQ, Thanh NX, Parzy D. Polymorphism of Plasmodium falciparum Na(+)/H(+) exchanger is indicative of a low in vitro quinine susceptibility in isolates from Viet Nam. Malar J. 2011;10:164. doi: 10.1186/1475-2875-10-164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Pickard AL, Wongsrichanalai C, Purfield A, Kamwendo D, Emery K, Zalewski C, Kawamoto F, Miller RS, Meshnick SR. Resistance to antimalarials in Southeast Asia and genetic polymorphisms in pfmdr1. Antimicrob Agents Chemother. 2003;47:2418–2423. doi: 10.1128/AAC.47.8.2418-2423.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Price RN, Cassar C, Brockman A, Duraisingh M, van Vugt M, White NJ, Nosten F, Krishna S. The pfmdr1 gene is associated with a multidrug-resistant phenotype in Plasmodium falciparum from the western border of Thailand. Antimicrob Agents Chemother. 1999;43:2943–2949. doi: 10.1128/aac.43.12.2943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lim P, Alker AP, Khim N, Shah NK, Incardona S, Doung S, Yi P, Bouth DM, Bouchier C, Puijalon OM, Meshnick SR, Wongsrichanalai C, Fandeur T, Le Bras J, Ringwald P, Ariey F. Pfmdr1 copy number and arteminisin derivatives combination therapy failure in falciparum malaria in Cambodia. Malar J. 2009;8:11. doi: 10.1186/1475-2875-8-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Andriantsoanirina V, Ménard D, Rabearimanana S, Hubert V, Bouchier C, Tichit M, Bras JL, Durand R. Association of microsatellite variations of Plasmodium falciparum Na+/H + exchanger (Pfnhe-1) gene with reduced in vitro susceptibility to quinine: lack of confirmation in clinical isolates from Africa. Am J Trop Med Hyg. 2010;82:782–787. doi: 10.4269/ajtmh.2010.09-0327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Briolant S, Pelleau S, Bogreau H, Hovette P, Zettor A, Castello J, Baret E, Amalvict R, Rogier C, Pradines B. In vitro susceptibility to quinine and microsatellite variations of the Plasmodium falciparum Na+/H + exchanger (Pfnhe-1) gene: the absence of association in clinical isolates from the Republic of Congo. Malar J. 2011;10:37. doi: 10.1186/1475-2875-10-37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Baliraine FN, Nsobya SL, Achan J, Tibenderana JK, Talisuna AO, Greenhouse B, Rosenthal PJ. Limited ability of Plasmodium falciparum pfcrt, pfmdr1, and pfnhe1 polymorphisms to predict quinine in vitro sensitivity or clinical effectiveness in Uganda. Antimicrob Agents Chemother. 2011;55:615–622. doi: 10.1128/AAC.00954-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Trager W, Jensen JB. Human malaria parasites in continuous culture. Science. 1976;193:673–675. doi: 10.1126/science.781840. [DOI] [PubMed] [Google Scholar]
  27. Desjardins RE, Canfield J, Haynes D, Chulay JD. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob Agents Chemother. 1979;16:710–718. doi: 10.1128/aac.16.6.710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wooden J, Gould EE, Paull AT, Sibley CH. Plasmodium falciparum: A simple polymerase chain reaction method for differentiating strains. Exp Parasitol. 1992;75:207–212. doi: 10.1016/0014-4894(92)90180-I. [DOI] [PubMed] [Google Scholar]
  29. Duraisingh MT, Roper C, Walliker D, Warhurst DC. Increased sensitivity to the antimalarials mefloquine and artemisinin is conferred by mutations in the pfmdr1 gene of Plasmodium falciparum. Mol Microbiol. 2000;36:955–961. doi: 10.1046/j.1365-2958.2000.01914.x. [DOI] [PubMed] [Google Scholar]
  30. Price RN, Uhlemann AC, Brockman A, McGready R, Ashley E, Phaipun L, Patel R, Laing K, Looareesuwan S, White NJ, Nosten F, Krishna S. Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number. Lancet. 2004;364:438–447. doi: 10.1016/S0140-6736(04)16767-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Chaijaroenkul W, Wisedpanichkij R, Na-Bangchang K. Monitoring of in vitro susceptibilities and molecular markers of resistance of Plasmodium falciparum isolates from Thai-Myanmar border to chloroquine, quinine, mefloquine and artesunate. Acta Trop. 2010;113:190–194. doi: 10.1016/j.actatropica.2009.10.016. [DOI] [PubMed] [Google Scholar]
  32. Phompradit P, Wisedpanichkij R, Muhamad P, Chaijaroenkul W, Na-Bangchang K. Molecular analysis of pfatp6 and pfmdr1 polymorphisms and their association with in vitro sensitivity in Plasmodium falciparum isolates from the Thai-Myanmar border. Acta Trop. 2011;120:130–135. doi: 10.1016/j.actatropica.2011.07.003. [DOI] [PubMed] [Google Scholar]
  33. Sanchez CP, Rotmann A, Stein WD, Lanzer M. Polymorphisms within PfMDR1 alter the substrate specificity for anti-malarial drugs in Plasmodium falciparum. Mol Microbiol. 2008;70:786–798. doi: 10.1111/j.1365-2958.2008.06413.x. [DOI] [PubMed] [Google Scholar]
  34. Hawley SR, Bray PG, Mungthin M, Atkinson JD, O'Neill PM, Ward SA. Relationship between anti-malarial drug activity, accumulation, and inhibition of heme polymerization in Plasmodium falciparum in vitro. Antimicrob Agents Chemother. 1998;42:682–686. doi: 10.1128/aac.42.3.682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Mungthin M, Bray PG, Ridley RG, Ward SA. Central role of hemoglobin degradation in mechanisms of action of 4-aminoquinolines, quinoline methanols, and phenanthrene methanols. Antimicrob Agents Chemother. 1998;42:2973–2977. doi: 10.1128/aac.42.11.2973. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Malaria Journal are provided here courtesy of BMC

RESOURCES