Open Access

Distribution of pfmdr1 polymorphisms in Plasmodium falciparum isolated from Southern Thailand

  • Mathirut Mungthin1Email author,
  • Somchai Intanakom2,
  • Nantana Suwandittakul1,
  • Preeyaporn Suida3,
  • Sakultip Amsakul4,
  • Naruemon Sitthichot1,
  • Suwich Thammapalo2 and
  • Saovanee Leelayoova1
Malaria Journal201413:117

https://doi.org/10.1186/1475-2875-13-117

Received: 25 December 2013

Accepted: 22 March 2014

Published: 27 March 2014

Abstract

Background

Drug resistance in Plasmodium falciparum is a major problem in malaria control especially along the Thai-Myanmar and Thai-Cambodia borders. To date, a few molecular markers have been identified for anti-malarial resistance in P. falciparum, including the P. falciparum chloroquine resistance transporter (pfcrt) and the P. falciparum multidrug resistance 1 (pfmdr1). However no information is available regarding the distribution pattern of these gene polymorphisms in the parasites from the Thai-Malaysia border. This study was conducted to compare the distribution pattern of the pfcrt and pfmdr1 polymorphisms in the parasites from the lower southern provinces, Thai-Malaysia border and the upper southern provinces, Thai-Myanmar border. In addition, in vitro sensitivities of anti-malarial drugs including chloroquine, mefloquine, quinine, and artesunate were determined.

Methods

In all, 492 P. falciparum-positive blood samples were collected from the lower southern provinces: Songkhla, Yala and Narathiwas. From the upper southern part of Thailand, Ranong and Chumphon, 66 samples were also collected. Polymorphisms of the pfcrt and the pfmdr1 gene were determined using PCR techniques. In vitro sensitivities of anti-malarial drugs were determined using radioisotopic method.

Results

All parasites from both areas contained the pfcrt 76 T allele. The pfmdr 1 86Y allele was significantly more common in the parasites isolated from the lower southern areas. In contrast, the pfmdr1 184F allele was predominant among the parasites from the upper southern areas especially Ranong. In addition, the parasites from Ranong contained higher copy numbers than the parasites from other provinces. All adapted parasite isolates exhibited CQ-resistant phenotype. Neither QN nor MQ resistance was detected in these isolates.

Conclusion

The parasites from Thai-Malaysia border exhibited different resistant patterns compared to other areas along the international border of Thailand. This information will be useful for anti-malarial drug policy in Thailand.

Background

Multidrug resistance in Plasmodium falciparum has been a major problem in malaria control along the international borders of Thailand especially, Thai-Myanmar and Thai-Cambodia border [1]. Artemisinin-based combination therapy (ACT), using a combination of artesunate (ART) and mefloquine (MQ), has been introduced for the treatment of uncomplicated falciparum malaria to address this problem [2]. In the past few years, emergence of artemisinin resistance in these areas is a matter of concern [3, 4]. Prolonged parasite clearance has now been used as the indicator of artemisinin resistance [4].

A few molecular markers have been identified for anti-malarial resistance in P. falciparum. The P. falciparum chloroquine resistance transporter (pfcrt) has been identified as the main determinant of chloroquine (CQ) resistance [5]. A point mutation on the pfcrt gene resulting in replacement of lysine by threonine in the PfCRT at codon 76 has been linked to CQ resistance in parasite isolates collected worldwide [6]. The P. falciparum multidrug resistance 1 (pfmdr1), a gene on chromosome 5 encoding a P-glycoprotein homologue 1 (pgh1) also contributes to CQ resistance [710]. Several studies have shown that single nucleotide polymorphisms and amplification of the pfmdr1 gene is also associated with in vitro response and clinical efficacy of MQ, an arylaminoalcohol [1114]. Evidence suggests that the pfmdr1 gene plays a role in the in vitro response to other quinolines such as quinine (QN) and lumefantrine (LF) and artemisinin derivatives [1518].

To date, the distribution of the pfcrt and pfmdr1 polymorphisms were only reported in the parasites collected from the Thai-Myanmar and Thai-Cambodia borders, but not the Thai-Malaysia border [10, 13, 14, 17, 18]. Since different patterns of pfcrt and pfmdr1 polymorphisms in P. falciparum exhibit varied anti-malarial drug susceptibilities [10, 17, 18], knowing the distribution of these polymorphisms in these areas would be useful. In this study, the pfcrt and pfmdr1 polymorphisms in P. falciparum isolated from the Thai-Malaysia border were determined compared with the parasites isolated from upper southern provinces, Thai-Myanmar border. In addition, the in vitro sensitivities of anti-malarial drugs including CQ, MQ, QN and ART were determined in recently adapted P. falciparum isolated from this area.

Methods

Blood sample collection

Finger-prick blood (approximately 200 μl) samples from patients who visited malaria clinics of the Office of Disease Prevention and Control 12 (the lower southern provinces including Songkhla, Yala and Narathiwas) and the Office of Disease Prevention and Control 11 (the upper southern provinces including Ranong and Chumphon) were collected onto Whatman No 3 filter paper. At these clinics, Giemsa-stained thick film was performed for the diagnosis of malaria. The dried filter paper samples were kept in plastic zipper bags and sent to the Department of Parasitology, Phramongkutklao College of Medicine, for molecular analysis. A total of 492 microscopically positive P. falciparum blood samples were collected from three lower southern provinces in 2009. Of these 492 samples, 43, 215 and 234 samples were collected from Songkhla, Yala and Narathiwas, respectively. Sixty-six samples were also collected from the upper southern part of Thailand, i e, 42 samples from Ranong and 24 samples from Chumphon. All these samples were confirmed to be P. falciparum positive by PCR technique as described by Snounou et al. [19]. The research protocol was reviewed and approved by the Ethics Committee of the Royal Thai Army Medical Department.

Plasmodium falciparum cultivation and in vitro sensitivity assays

Fifteen isolates of P. falciparum collected from Yala, a province along the Thai-Malaysia border in 2010 were adapted. Parasites were maintained in continuous cultures using a modification of the method of Trager and Jensen [20]. These isolates were tested against commonly used anti-malarial drugs including CQ, MQ, QN and ART using radioisotopic assay as previously described [21]. 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, UK). Each in vitro sensitivity experiment was carried out in triplicate. The IC50 of each isolate was the mean IC50 of at least three independent experiments.

Genotypic characterization for pfcrt and pfmdr1 genes

Parasite DNA was extracted using the Chelex-resin method [22]. Five μl of DNA preparation was used for a 25 μl PCR reaction. PCR and allele-specific restriction analysis were performed for the detection of the pfcrt mutations encoded amino acids at position 76 [23]. Mutations in the pfmdr1 gene were determined by the nested PCR and restriction endonuclease digestion method developed by Duraisingh et al. [15] for detection of the mutations at codons 86, 184, 1034, 1042 and 1246 [24]. K1, a laboratory strain containing the 86Y allele was used as a control for the detection of the N86Y mutation. For the positive control of the 184 F, 1034C, 1042D and 1246Y alleles, 7G8 strain was used. The 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) as developed by Price et al. [25]. 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 copy number of the pfmdr 1 gene was determined as previously described [25].

Statistical analysis

Data were analysed by STATA/MP, Version 12. Differences of the mean copy number of the pfmdr1 gene among the parasites from different areas were analysed by Independent t test and One-way ANOVA. Post hoc test (Scheffe) for multiple comparisons was used to test for differences among groups. Association between genotypes and P. falciparum from different areas was analysed by Chi square test. The level of significance was set at a p value of < 0.05.

Results

Characterization of the pfcrt and pfmdr1 genes

Genotypic characterization of the parasite isolates from upper and lower southern Thailand is shown in Table 1 and Figure 1. All parasites from both areas contained the pfcrt 76T allele. Of the 492 parasite isolates from lower southern Thailand, 474 (96.3%), 16 (3.3%), and two (0.4%) isolates contained the pfmdr 1 86Y, 184F and 1034C mutations, respectively. The distribution of the pfmdr 1 polymorphisms of the parasites isolated among three lower southern provinces, i e Yala, Narathiwas and Songkhla, was similar. The pfmdr 1 86Y allele was significantly more common in the parasites isolated from lower southern areas than those isolated from upper southern areas (Chi square, p < 0.001). In contrast, the pfmdr1 184F allele was more common in the parasites from upper southern areas (Chi square, p < 0.001). In this area, the pfmdr 1 184F allele was significantly more common in the parasites isolated from Ranong compared to those from Chumphon (Chi square, p < 0.001). In contrast, the pfmdr1 86Y allele was significantly predominant in the parasites from Chumphon (Chi square, p < 0.001). Parasites containing mixed alleles of the pfmdr1 gene were not detected in all samples.
Table 1

Distribution of the pfcrt and pfmdr 1 polymorphisms of the parasites isolated from upper and lower southern areas

Area

No. of isolates

pfcrt 76T

Mean pfmdr 1 copy number

pfmdr 1 mutations

86Y

184F

1034C

1042D

1246Y

Upper southern

66

66 (100%)

2.3 ± 1.0*

24 (36.4%)**

42 (63.6%)**

0 (0%)

0 (0%)

0 (0%)

Ranong

42

42 (100%)

2.6 ± 0.8

6 (14.3%)

36 (85.7%)

0 (0%)

0 (0%)

0 (0%)

Chumphon

24

24 (100%)

1.7 ± 0.9

18 (75.0%)

6 (25.0%)

0 (0%)

0 (0%)

0 (0%)

Lower southern

492

492 (100%)

1.2 ± 0.4

474 (96.3%)

16 (3.3%)

2 (0.4%)

0 (0%)

0 (0%)

Yala

215

215 (100%)

1.3 ± 0.5

204 (94.9%)

10 (4.7%)

2 (0.9%)

0 (0%)

0 (0%)

Narathiwas

234

234 (100%)

1.2 ± 0.4

228 (97.4%)

5 (2.1%)

0 (0%)

0 (0%)

0 (0%)

Songkhla

43

43 (100%)

1.2 ± 0.4

42 (97.7%)

1 (2.3%)

0 (0%)

0 (0%)

0 (0%)

*Significant difference between parasites isolated from upper and lower southern area determined by Independent t test (p < 0.001).

**Significant difference between parasites isolated upper and lower southern area determined by Chi square test (p < 0.001).

Figure 1

Predominant pfmdr1 genotypes in different areas of Thailand. The present study locations including two provinces in the upper south, (1) Chumphon and (2) Ranong and three provinces in the lower south, (3) Songkhla, (4) Yala and (5) Narathiwas and previously reported areas including (6) Tak and (7) Kanchanaburi [17, 18, 31], (8) Chantaburi and (9) Trat [17, 18, 29].

Determination of the pfmdr1 gene copy number showed that the isolates from upper southern areas contained the pfmdr1 copy numbers with the mean of 2.3 (range 1 to 4) which was significantly higher than those found in the parasite from lower southern areas (mean = 1.2, range 1 to 3). Significant differences of the pfmdr1 copy numbers were found among the parasites from different provinces (One-way ANOVA, p < 0.001). Multiple comparison indicated that the parasites from Ranong contain more copy number than the parasites from Chumphon, Yala, Narathiwas and Songkhla (all p < 0.001). The parasites from Chumphon also had more copy number than the parasites from lower southern areas, i e Yala, Narathiwas and Songkhla (p = 0.007, p = 0.004, and p = 0.012, respectively).

In vitro anti-malarial sensitivities against 15 Plasmodium falciparum isolates from Yala

Table 2 shows in vitro response of parasites isolated from Yala to four commonly used anti-malarial drugs. All isolates exhibited CQ-resistant phenotype [26]. Neither QN resistance (QN IC50 of > 800 nM) [27] nor MQ resistance (MQ IC50 of > 30 nM) [28] was detected in these isolates. All isolates contained the pfcrt 76T and a single copy of the pfmdr1 gene with the 86Y allele.
Table 2

In vitro anti-malarial sensitivities in 15 P. falciparum isolates from Yala

 

Min IC50(nM)

Min IC50(nM)

Mean IC50(nM) ± SD

Chloroquine

63.0

189.7

129.2 ± 45.2

Quinine

102.7

278.2

185.2 ± 61.7

Mefloquine

10.7

24.5

16.6 ± 3.9

Artesunate

3.0

4.4

3.8 ± 0.5

Discussion

Majority of P. falciparum isolates collected from three provinces along the Thai-Malaysia border, i.e. Yala, Narathiwas and Songkhla, contained the pfmdr1 86Y allele (96.3%) with the mean copy number of 1.2. Malaria cases in the southernmost provinces have significantly increased since 2005, after political unrest in this area. Previously, only a small number of P. falciparum isolates collected from this area were genetically characterized. Similar to the present study, Pickard et al. identified the pfmdr1 86Y allele in all eight P. falciparum isolates from Yala, one of the southernmost provinces [13]. This information indicates the different patterns of the pfmdr1 polymorphisms in P. falciparum isolated from different international borders of Thailand. In a few studies of Thai P. falciparum isolates, polymorphisms of the pfmdr1 gene were determined in the parasites mostly collected from either Thai-Myanmar or the Thai-Cambodia borders [10, 13, 14, 17, 18]. The recently collected parasites from the Thai-Cambodia border contained the pfmdr1 184F allele with a lower copy number compared with the parasites from the Thai-Myanmar border [17, 18, 29, 30]. In contrast, parasites collected from the Thai-Myanmar border usually contained either the 184Y or 184F allele with a higher copy number of the pfmdr1 gene [17, 18, 31]. To compare the parasites collected from these three southernmost provinces, the polymorphisms of the pfmdr1 gene in the parasites from Ranong and Chumphon, provinces along the Thai-Myanmar border, were also determined. Similar to previous studies, most of these parasites, especially those collected from Ranong, had the 184F allele and increased copy number of the pfmdr1 gene. In contrast to the pfmdr1 gene, all the study parasites contained the CQ-resistant genotype, the 76T allele.

The different patterns of the pfmdr1 polymorphisms among the parasites from these international borders of Thailand might be due to the response to a different drug pressure in the past. Since 1995, the combination of ART/MQ has been adopted by the Ministry of Public Health for the treatment of uncomplicated falciparum malaria in Thailand [1, 32, 33]. In the beginning, this combination was used only in the highly MQ- resistant areas, including the Thai-Myanmar area, Tak and the Thai-Cambodia area, Trat and Chantaburi. In some areas of the Thai-Myanmar border, including Ranong, ART has been added just a few years later. In addition, dosage of ART and MQ might vary among different areas. For example, in 2002, a single dose of 15 mg/kg of MQ was used in the non- or low MQ-resistant areas of Kanchanaburi while in the moderate-MQ-resistant areas of this province, divided doses of 12 mg/kg ART were added. In contrast, in the high MQ-resistant areas such as Mae Sod, Tak province, a combination of 25 mg/kg of MQ and 12 mg/kg of ART was used. The combination of ART/MQ has been used in the malaria clinics of the Office of Disease Prevention and Control in these southernmost provinces since 2004. However, a few hospitals in the area might use other regimens such as a combination of QN/doxycycline. In addition, in Thailand, vivax malaria shares similar endemic areas with falciparum malaria. Thus, CQ, the first-line treatment for vivax malaria could cause a drug pressure and influence the distribution of the pfmdr1 polymorphisms, especially where vivax malaria was predominant.

The distribution of the pfmdr1 polymorphisms in each area along the international border of Thailand might be influenced by the parasites from neighbouring countries via human movement. Indeed, approximately half of malaria cases in Thailand have been foreign migrant workers [34]. A few studies of the parasites from the Thai-Cambodia border showed that most parasites collected either from Thailand or Cambodia shared a similar genotype, i e, the pfmdr1 184F allele [29, 30]. A recent study showed a high level of genetic diversity in the parasites from the Thai-Myanmar and Thai-Cambodia border where cross-border migrations commonly occurred [35]. This study also identified the parasites with the same genotype in patients who were infected in Thailand and Myanmar [35]. In contrast, an identical haplotype was found in all parasites collected from Yala. Similar to this previous study, nearly all parasites collected from three provinces of the Thai-Malaysia border, including Yala, contained the similar pfmdr1 genotype, the 86Y allele. The distribution of the pfmdr1 polymorphisms in the parasites from these three southernmost provinces of Thailand was different from those from Pahang, Peninsula Malaysia, containing the pfmdr1 N86 allele in most isolates [36]. Low variation of the parasite population in this area could be due to recent expansion of the local parasites with fewer introductions of the parasites with other genotypes in the area. Foreign migrant workers in this area were fewer compared to the Thai-Myanmar area, such as Ranong, because of political unrest.

All adapted P. falciparum isolates from Yala exhibited a CQ-resistant phenotype. These parasites were QN- and MQ- sensitive. In vitro drug susceptibility pattern of the adapted P. falciparum isolates in this study was similar to the results of the study by Rungsihirunrat et al. [37]. Although the pfcrt 76T allele has been proved to be a key determinant for in vitro CQ resistance, the polymorphisms of pfmdr1 gene could modulate the level of CQ resistance [9, 10]. Besides, the 86Y allele was also identified as the predictor of CQ treatment failure [38]. It has been shown that parasites with different resistant phenotypes and genotypes exhibited different levels of fitness which might explain the unique parasite structure of P. falciparum in the Thai-Malaysia border. The influence of parasites’ fitness was indicated when CQ-sensitive strains were re-emerging in some countries where CQ use was discontinued because of widespread CQ resistance [39, 40]. Using allelic replacement technique, insertion of the 7G8, CQ-resistant alleles into CQ-sensitive strain, D10 resulted in the loss of parasites’ fitness [41]. In contrast, a recent study of clinical samples showed that the parasites with the pfmdr1 86Y and D1246 alleles had the fitness advantage over others [42]. Moreover, the parasites with the pfmdr1 86Y allele could produce a higher number of gametocytes [43] which would gain the advantage in term of transmission.

Conclusion

A nationwide implementation of a three-day ART instead of a two-day ART in combination with two-day MQ regimen has been started in Thailand since 2008 to overcome a low cure rate in some areas [1]. However, a new fixed dose ACT will inevitably be considered by the Ministry of Public Health in the near future. The present study showed that P. falciparum isolated from different areas along the international border of Thailand exhibited different resistant phenotypic and genotypic patterns. This information will be useful for anti-malarial drug policy in Thailand. New candidate drugs should be adopted at least based on their activity against these phenotypic and genotypic parasites in different areas of Thailand.

Declarations

Acknowledgements

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

Authors’ Affiliations

(1)
Department of Parasitology, Phramongkutklao College of Medicine
(2)
Office of Disease Prevention and Control 12, Department of Disease Control, Ministry of Public Health
(3)
Vecter Born Disease Control Center 12.1, Ministry of Public Health
(4)
Office of Disease Prevention and Control 11, Ministry of Public Health

References

  1. WHO: Global Report on Antimalarial Drug Efficacy and Drug Resistance 2000–2010. 2010, Geneva: World Health OrganizationGoogle Scholar
  2. WHO: Guidelines for the Treatment of Malaria. 2006, Geneva: World Health OrganizationGoogle Scholar
  3. Noedl H, Se Y, Schaecher K, Smith BL, Socheat D, Fukuda MM, Artemisinin Resistance in Cambodia 1 (ARC1) Study Consortium: Evidence of artemisinin-resistant malaria in western Cambodia. N Engl J Med. 2008, 359: 2619-2620. 10.1056/NEJMc0805011.View ArticlePubMedGoogle Scholar
  4. Dondorp AM, Nosten F, Yi P, Das D, Phyo AP, Tarning J, Lwin KM, Ariey F, Hanpithakpong W, Lee SJ, Ringwald P, Silamut K, Imwong M, Chotivanich K, Lim P, Herdman T, An SS, Yeung S, Singhasivanon P, Day NP, Lindegardh N, Socheat D, White NJ: Artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med. 2009, 361: 455-467. 10.1056/NEJMoa0808859.PubMed CentralView ArticlePubMedGoogle Scholar
  5. Fidock DA, Nomura T, Talley AK, Cooper RA, Dzekunov SM, Ferdig MT, Ursos LM, Sidhu AB, Naudé B, Deitsch KW, Su XZ, Wootton JC, Roepe PD, Wellems TE: Mutations in the P. falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Mol Cell. 2000, 6: 861-871. 10.1016/S1097-2765(05)00077-8.PubMed CentralView ArticlePubMedGoogle Scholar
  6. 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. 10.1124/mol.61.1.35.View ArticlePubMedGoogle Scholar
  7. Foote SJ, Kyle DE, Martin RK, Oduola AM, Forsyth K, Kemp DJ, Cowman AF: Several alleles of the multidrug-resistance gene are closely linked to chloroquine resistance in Plasmodium falciparum. Nature. 1990, 345: 255-258. 10.1038/345255a0.View ArticlePubMedGoogle Scholar
  8. 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. 10.1038/35002615.View ArticlePubMedGoogle Scholar
  9. Babiker HA, Pringle SJ, Abdel-Muhsin A, Mackinnon M, Hunt P, Walliker D: Highl-level chloroquine resistance in Sudanese isolates of Plasmodium falciparum is associated with mutations in the chloroquine resistance transporter gene pfcrt and the multidrug resistance gene pfmdr1. J Infect Dis. 2001, 183: 1535-1538. 10.1086/320195.View ArticlePubMedGoogle Scholar
  10. Setthaudom C, Tan-ariya P, Sitthichot N, Khositnithikul R, Suwandittakul N, Leelayoova S, Mungthin M: 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. 2011, 85: 606-611. 10.4269/ajtmh.2011.11-0108.PubMed CentralView ArticlePubMedGoogle Scholar
  11. Wilson CM, Volkman SK, Thaithong S, Martin RK, Kyle DJ, Milhous WK, Wirth DF: Amplification of pfmdr1 associated with mefloquine and halofantrine resistance in Plasmodium falciparum from Thailand. Mol Biochem Parasitol. 1993, 57: 151-160. 10.1016/0166-6851(93)90252-S.View ArticlePubMedGoogle Scholar
  12. Cowman AF, Galatis D, Thompson JK: 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 U S A. 1994, 91: 1143-1147. 10.1073/pnas.91.3.1143.PubMed CentralView ArticlePubMedGoogle Scholar
  13. 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 Agent Chemother. 2003, 47: 2418-2423. 10.1128/AAC.47.8.2418-2423.2003.View ArticleGoogle Scholar
  14. Nelson AL, Purfield A, McDaniel P, Uthaimongkol N, Buathong N, Sriwichai S, Miller RS, Wongsrichanalai C, Meshnick SR: Pfmdr1 genotyping and in vivo mefloquine resistance on the Thai-Myanmar border. Am J Trop Med Hyg. 2005, 72: 586-592.PubMedGoogle Scholar
  15. Duraisingh MT, Cowman AF: Contribution of the pfmdr1 gene to antimalarial drug-resistance. Acta Trop. 2005, 94: 181-190. 10.1016/j.actatropica.2005.04.008.View ArticlePubMedGoogle Scholar
  16. Duraisingh MT, Jones P, Sambou I, von Seidlein L, Pinder M, Warhurst DC: The tyrosine-86 allele of the pfmdr 1 gene of Plasmodium falciparum is associated with increased sensitivity to the anti-malarials mefloquine and artemisinin. Mol Biochem Parasitol. 2000, 108: 12-23.View ArticleGoogle Scholar
  17. Poyomtip T, Suwandittakul N, Sitthichot N, Khositnithikul R, Tan-ariya P, Mungthin M: Polymorphisms of the pfmdr1 but not the pfnhe-1 gene is associated with in vitro quinine sensitivity in Thai isolates of Plasmodium falciparum. Malar J. 2012, 11: 7-10.1186/1475-2875-11-7.PubMed CentralView ArticlePubMedGoogle Scholar
  18. Mungthin M, Khositnithikul R, Sitthichot N, Suwandittakul N, Wattanaveeradej V, Ward SA, Na-Bangchang K: Association between the pfmdr1 gene and in vitro artemether and lumefantrine sensitivity in Thai isolates of Plasmodium falciparum. Am J Trop Med Hyg. 2010, 83: 1005-1009. 10.4269/ajtmh.2010.10-0339.PubMed CentralView ArticlePubMedGoogle Scholar
  19. Snounou G, Viriyakosol S, Jarra W, Thaithong S, Brown KN: Identification of the four human malaria parasite species in field samples by the polymerase chain reaction and detection of a high prevalence of mixed infections. Mol Biochem Parasitol. 1993, 58: 283-292. 10.1016/0166-6851(93)90050-8.View ArticlePubMedGoogle Scholar
  20. Trager W, Jensen JB: Human malaria parasites in continuous culture. Science. 1976, 193: 673-675. 10.1126/science.781840.View ArticlePubMedGoogle Scholar
  21. 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. 10.1128/AAC.16.6.710.PubMed CentralView ArticlePubMedGoogle Scholar
  22. 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. 10.1016/0014-4894(92)90180-I.View ArticlePubMedGoogle Scholar
  23. Djimdé A, Doumbo OK, Cortese JF, Kayentao K, Doumbo S, Diourté Y, Dicko A, Su XZ, Nomura T, Fidock DA, Wellems TE, Plowe CV, Coulibaly D: A molecular marker for chloroquine-resistant falciparum malaria. N Engl J Med. 2001, 344: 257-263. 10.1056/NEJM200101253440403.View ArticlePubMedGoogle Scholar
  24. Duraisingh MT, Roper C, Walliker D, Warhurst DC: Increased sensitivity to the antimalarials mefloquine and artemisinin is conferred by mutations in the pfmdr 1 gene of Plasmodium falciparum. Mol Microbiol. 2000, 36: 955-961. 10.1046/j.1365-2958.2000.01914.x.View ArticlePubMedGoogle Scholar
  25. 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 pfmdr 1 gene copy number. Lancet. 2004, 364: 438-447. 10.1016/S0140-6736(04)16767-6.PubMed CentralView ArticlePubMedGoogle Scholar
  26. Bickii J, Basco LK, Ringwald P: Assessment of three in vitro tests and an in vivo test for chloroquine resistance in Plasmodium falciparum clinical isolates. J Clin Microbiol. 1998, 36: 243-247.PubMed CentralPubMedGoogle Scholar
  27. Basco LK, Le Bras J: In vitro susceptibility of Cambodian isolates of Plasmodium falciparum to halofantrine, pyronaridine and artemisinin derivatives. Ann Trop Med Parasitol. 1994, 88: 137-144.PubMedGoogle Scholar
  28. Hatin I, Trape JF, Legros F, Bauchet J, Le Bras J: Susceptibility of Plasmodium falciparum strains to mefloquine in an urban area in Senegal. Bull World Health Org. 1992, 70: 363-367.PubMed CentralPubMedGoogle Scholar
  29. Mungthin M, Suwandittakul N, Chaijaroenkul W, Rungsrihirunrat K, Harnyuttanakorn P, Seugorn A, Na Bangchang K: The patterns of mutation and amplification of Plasmodium falciparum pfcrt and pfmdr1 genes in Thailand during the year 1988 to 2003. Parasitol Res. 2010, 107: 539-545. 10.1007/s00436-010-1887-x.View ArticlePubMedGoogle Scholar
  30. Vinayak S, Alam MT, Sem R, Shah NK, Susanti AI, Lim P, Muth S, Maguire JD, Rogers WO, Fandeur T, Barnwell JW, Escalante AA, Wongsrichanalai C, Ariey F, Meshnick SR, Udhayakumar V: Multiple genetic backgrounds of the amplified Plasmodium falciparum multidrug resistance (pfmdr1) gene and selective sweep of 184 F mutation in Cambodia. J Infect Dis. 2010, 201: 1551-1560. 10.1086/651949.PubMed CentralView ArticlePubMedGoogle Scholar
  31. 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. 10.1016/j.actatropica.2011.07.003.View ArticlePubMedGoogle Scholar
  32. Rojanawatsirivej C, Vijaykadga S, Amklad I, Wilairatna P, Looareesuwan S: Monitoring the therapeutic efficacy of antimalarials against uncomplicated falciparum malaria in Thailand. Southeast Asian J Trop Med Public Health. 2003, 34: 536-541.PubMedGoogle Scholar
  33. Vijaykadga S, Rojanawatsirivej C, Cholpol S, Phoungmanee D, Nakavej A, Wongsrichanalai C: 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. 2006, 11: 211-219. 10.1111/j.1365-3156.2005.01557.x.View ArticlePubMedGoogle Scholar
  34. WHO: Malaria in the Greater Mekong Subregion: Regional and Country Profiles. 2010, Geneva: World Health OrganizationGoogle Scholar
  35. Pumpaibool T, Arnathau C, Durand P, Kanchanakhan N, Siripoon N, Suegorn A, Sitthi-Amorn C, Renaud F, Harnyuttanakorn P: Genetic diversity and population structure of Plasmodium falciparum in Thailand, a low transmission country. Malar J. 2009, 8: 155-10.1186/1475-2875-8-155.PubMed CentralView ArticlePubMedGoogle Scholar
  36. Atroosh WM, Al-Mekhlafi HM, Mahdy MA, Surin J: The detection of pfcrt and pfmdr1 point mutations as molecular markers of chloroquine drug resistance, Pahang, Malaysia. Malar J. 2012, 11: 251-10.1186/1475-2875-11-251.PubMed CentralView ArticlePubMedGoogle Scholar
  37. Rungsihirunrat K, Chaijareonkul W, Seugorn A, Na-Bangchang K, Thaithong S: Association between chloroquine resistance phenotypes and point mutations in pfcrt and pfmdr1 in Plasmodium falciparum isolates from Thailand. Acta Trop. 2009, 109: 37-40. 10.1016/j.actatropica.2008.09.011.View ArticlePubMedGoogle Scholar
  38. Andriantsoanirina V, Ratsimbasoa A, Bouchier C, Tichit M, Jahevitra M, Rabearimanana S, Raherinjafy R, Mercereau-Puijalon O, Durand R, Ménard D: Chloroquine clinical failures in P. falciparum malaria are associated with mutant Pfmdr-1, not Pfcrt in Madagascar. PLoS One. 2010, 5: e13281-10.1371/journal.pone.0013281.PubMed CentralView ArticlePubMedGoogle Scholar
  39. Kublin JG, Cortese JF, Njunju EM, Mukadam RA, Wirima JJ, Kazembe PN, Djimdé AA, Kouriba B, Taylor TE, Plowe CV: Reemergence of chloroquine-sensitive Plasmodium falciparum malaria after cessation of chloroquine use in Malawi. J Infect Dis. 2003, 187: 1870-1875. 10.1086/375419.View ArticlePubMedGoogle Scholar
  40. Wang X, Mu J, Li G, Chen P, Guo X, Fu L, Chen L, Su X, Wellems TE: Decreased prevalence of the Plasmodium falciparum chloroquine resistance transporter 7 6 T marker associated with cessation of chloroquine use against P. falciparum malaria in Hainan, People's Republic of China. Am J Trop Med Hyg. 2005, 72: 410-414.PubMedGoogle Scholar
  41. Hayward R, Saliba KJ, Kirk K: Pfmdr1 mutations associated with chloroquine resistance incur a fitness cost in Plasmodium falciparum. Mol Microbiol. 2005, 55: 1285-1295. 10.1111/j.1365-2958.2004.04470.x.View ArticlePubMedGoogle Scholar
  42. Ochong E, Tumwebaze PK, Byaruhanga O, Greenhouse B, Rosenthal PJ: Fitness consequences of Plasmodium falciparum pfmdr1 polymorphisms inferred from ex vivo culture of Ugandan parasites. Antimicrob Agents Chemother. 2013, [Epub ahead of print]Google Scholar
  43. Osman ME, Mockenhaupt FP, Bienzle U, Elbashir MI, Giha HA: Field-based evidence for linkage of mutations associated with chloroquine (pfcrt/pfmdr1) and sulfadoxine-pyrimethamine (pfdhfr/pfdhps) resistance and for the fitness cost of multiple mutations in P. falciparum. Infect Genet Evol. 2007, 7: 52-59. 10.1016/j.meegid.2006.03.008.View ArticlePubMedGoogle Scholar

Copyright

© Mungthin et al.; licensee BioMed Central Ltd. 2014

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Advertisement