Introduction
The development and spread of Plasmodium falciparum resistant to chloroquine has been a major factor in the resurgence of life-threatening P. falciparum malaria worldwide.1 Chloroquine resistance is associated with reduced cellular drug accumulation at a high-affinity target site within the parasite food vacuole.2 A genetic cross experiment identified a gene on chromosome 7, Plasmodium falciparum chloroquine resistance transporter (pfcrt), as the main determinant of chloroquine resistance.3–5 A point mutation on the pfcrt gene resulting in replacement of lysine by threonine in the PfCRT at codon 76 has been proven to be critical for chloroquine resistance by transfection experiment.6,7 The K76T mutation has been linked to chloroquine resistance in parasite isolates collected worldwide.1,8–12 In addition, more than 15 mutations in the pfcrt gene resulting in amino acid changes have been reported.13
To date, a number of pfcrt haplotypes have been identified based on amino acids 72–76. These haplotypes have been linked to the geographic origin of the isolates and their chloroquine susceptibility status. Two common haplotypes, CVIET and SVMNT, were identified in chloroquine-resistant isolates from Asia/Africa and South America, respectively, and a CVMNK haplotype is universally identified in chloroquine-sensitive parasites.6 Recent studies indicate the important role of amino acid polymorphisms in the PfCRT on the pattern and level of antimalarial drug resistance. A genetic cross experiment showed that parasites with CVIET and SVMNT haplotypes exhibited different patterns of chloroquine and monodesethylamodiaquine resistance.14 Moreover, additional amino acid substitutes in the PfCRT altered the level of chloroquine resistance.15–17
There is compelling evidence that P. falciparum multidrug resistance 1 (pfmdr1), a gene on chromosome 5 encoding a P-glycoprotein homolog 1 (Pgh1), also contributes to chloroquine resistance.18 A study by Reed and others using an allelic exchange experiment has definitively confirmed the involvement of pfmdr1 polymorphisms in high-level chloroquine resistance.19 In addition, a few studies of resistant parasites from in vitro drug selection indicate that alteration of the pfmdr1 gene copy number contributes to changes in the level of chloroquine resistance.20–22
Thus, it has been postulated that the pfmdr1 gene is working in concert with the pfcrt gene to control the level of chloroquine resistance.23 However, some but not all studied of field isolates could identify this association.24–26 Because different patterns of pfcrt and pfmdr1 polymorphisms respond to different patterns of drug resistance, it would be useful to identify the distribution of these polymorphisms. In this study, pfcrt and pfmdr1 polymorphisms in P. falciparum isolated along the Thailand-Myanmar and Thailand-Cambodia borders were determined. In addition, the role of these polymorphisms in controlling the level of chloroquine resistance was examined.
Materials and Methods
P. falciparum strains and cultivation.
Eighty-nine isolates of P. falciparum used in this study were collected from malaria-endemic areas along the Thailand-Myanmar and Thailand-Cambodia borders during 2003–2005. Parasites were maintained in continuous cultures by using a modification of the method of Trager and Jensen.27
In vitro sensitivity assays.
Chloroquine sensitivity of P. falciparum isolates was determined by measurement of [3H] hypoxanthine incorporation into parasite nucleic acids as described.28 Drug IC50 (i.e., concentration of a drug that inhibits parasite growth by 50%) was determined from the log dose/response relationship as fitted by GRAFIT (Erithacus Software, Kent, United Kingdom).
Genotypic characterization of pfcrt and pfmdr1 genes.
Parasite DNA was extracted by using the Chelex-resin method.29 Five microliters of DNA preparation was used for a 25-μL polymerase chain reaction (PCR). Nested PCR and allele-restricted PCR and/or restriction endonuclease digestion as described by Fidock and others6 and Djimde and others30 were used for detection of pfcrt mutations encoded amino acids at positions 76, 220, 271, 326, 356 and 371.
A primer pair, i.e., E1-2F (5′-CgACATTCCgATATATTTTAgAC-3′) and E1-2R (5′-TATATgTgTAATgTTTTATATTgg-3′) was used to amplify exon1 and 2 and produced the expected amplicon of 732 basepairs. The reaction mixture was composed of 1× colorless Go Taq® Flexi buffer, 1.5 mM MgCl2, 0.2 mM each of dNTP, 2 units of Taq DNA polymerase, and 20 pmol of each primer. The mixture was processed in a programmable DNA thermal cycler (PTC-200 Peltier Thermal Cycler; MJ Research Inc., Waltham, MA). The program consisted of one cycle at 94°C for 3 minutes to denature the genomic DNA; then 35 cycles of denaturation at 92°C for 30 secons, annealing at 55°C for 30 seconds, and extension at 62°C for 1.5 minutes. For the last cycle, the extension step at 62°C was performed for 5 minutes to complete partial polymerization. The PCR amplicons were then stored at –20°C until used.
DNA purification and DNA sequencing were conducted by Bioservice Unit (Bangkok, Thailand). DNA sequences of the pfcrt gene and amino acid sequences of the PfCRT from 89 P. falciparum isolates were aligned against available sequences in GenBank (Dd2 and HB3) by using the Lasergene MegAlign Program (DNASTAR Inc., Madison, WI) with the method of ClustalV. The nucleotide sequences were translated to amino acid sequences. Each polymorphism position was identified as to whether located inside or outside the transmembrane protein of PfCRT by comparing with the predicted peptides from the TMMHM Program (CBS, Lyngby, Denmark).
Mutations in the pfmdr1 gene were determined by nested PCR and the restriction endonuclease digestion method developed by Duraisingh and others31 for detection of the mutations at codons 86, 184, 1034, 1042, and 1246. Strains K1 and 7G8 were used as positive controls. 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.32 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 described.32
Statistical analysis.
Data were analyzed by using SPSS for Windows (SPSS Inc., Chicago, IL). The chloroquine IC50 of each isolate was the mean IC50 of three independent experiments. Differences in mean IC50 and copy number of the pfmdr1 gene among parasites from different areas were analyzed by using the independent t-test. Difference in median and interquartile range of chloroquine IC50 for parasites with different genotypes was analyzed by using the Mann-Whitney U test or the Kruskal-Wallis test. A chloroquine IC50 value > 25 nM was considered as in vitro chloroquine resistance, and an IC50 value > 100 nM was the cut-off point for in vivo chloroquine resistance.33 Association between genotypes and in vitro chloroquine resistance of P. falciparum was analyzed by using the chi-square test and Fisher's exact test.
Results
In vitro chloroquine sensitivities.
Characteristics of parasite isolates are shown in Table 1. The mean ± SD IC50 for chloroquine was 87.0 ± 43.8 nM (range = 13.9–190.5 nM). The parasites isolated along the Thailand-Cambodia border exhibited significantly more resistance to chloroquine than those from along the Thailand-Myanmar border. Of 89 isolates, 2 (2.2%), 55 (61.8%), and 32 (36.0%) were had IC50 values ≤ 25 nM, 26–100 nM, and > 100 nM, respectively.
In vitro sensitivities to chloroquine and distribution of pfcrt and pfmdr1 polymorphisms of 89 recently adapted Plasmodium falciparum isolates from Thailand-Myanmar and Thailand-Cambodia areas*
| Area | No. of isolates | Mean ± SD chloroquine IC50 (nM) | pfcrt mutation | Mean ± SD pfmdr1 copy number | pfmdr1 mutations | ||||
|---|---|---|---|---|---|---|---|---|---|
| 76T | 86Y | 184F | 1034C | 1042D | 1246Y | ||||
| Thailand-Myanmar | 68 | 76.9 ± 39.6 | 67 (98.5%) | 3.0 ± 1.4 | 5 (7.4%) | 19 (27.9%) | 2 (2.9%) | 2 (2.9%) | – |
| Thailand-Cambodia | 21 | 119.7 ± 40.9† | 20 (95.2%) | 1.6 ± 0.9† | 2 (9.5%) | 17 (81.0%)‡ | 1 (4.8%) | 1 (4.8%) | – |
| Total | 89 | 87.0 ± 43.8 | 87 (97.8%) | 2.6 ± 1.4 | 7 (7.9%) | 36 (40.4%) | 3 (3.4%) | 3 (3.4%) | – |
pfcrt = Plasmodium falciparum chloroquine resistance transporter; pfmdr1 = P. falciparum multidrug resistance 1; IC50 = 50% inhibitory concentration.
Significant difference between 2 areas determined by independent t test (P < 0.001).
Significant difference between 2 areas determined by Fisher's exact test (P < 0.001).
Characterization of pfcrt and pfmdr1 genes.
Detection of the mutations in the pfcrt gene using nested PCR and allele-restricted PCR and/or restriction endonuclease digestion showed that all isolates contained the 220S, 271E, 326S, and 371I mutations. Two (2.2%) and four (4.5%) isolates contained the 76K and 356I mutations, respectively. Sequences of pfcrt codons 72–76 confirmed that two chloroquine-sensitive isolates contained the CVIEK haplotype and 87 chloroquine-resistant isolates contained the CVIET haplotype. In addition, sequencing of exons 1 and 2 of the pfcrt gene showed four additional points of nucleotide polymorphisms, i.e., 12, 16, 198, and 290. All additional mutations were found only in parasites containing the CVIET haplotype. Only nucleotide polymorphisms at positions 16 and 290 resulted in amino acid changes. A substitution of C for A at position 16 resulting in amino acid change from lysine to glutamine at position 6 (K6Q) was found in three chloroquine-resistant isolates. An amino acid change from histidine to leucine at position 97, caused by a replacement of T for A at position 290, was found in four chloroquine-resistant isolates. The replacement at positions 6 and 97 occurred in cytosol peptide 1 and transmembrane domain 2, respectively.
Of these 89 parasite isolates, 7 (7.9%), 36 (40.4%), 3 (3.4%), 3 (3.4%), and 0 (0%) isolates contained pfmdr1 86Y, 184F, 1034C, 1042D, and 1246Y mutations, respectively. These isolates had mean pfmdr1 gene copy numbers of 2.6 (range = 0.8–5.6). The pfmdr1 184F allele was more common in parasites isolated along the Thailand-Cambodia than in those isolated along the Thailand-Myanmar border. In contrast, parasites isolated along the Thailand-Myanmar border had significantly higher copy numbers.
Association between in vitro chloroquine sensitivity and pfcrt and pfmdr1 genes.
Two parasite isolates containing pfcrt 76K exhibited absolute chloroquine sensitivity, and those harboring pfcrt 76T had a chloroquine IC50 > 25 nM. The in vitro chloroquine sensitivities of 87 isolates containing chloroquine-resistant haplotype CVIET with different additional pfcrt mutations and different pfmdr1 genotypes are shown in Table 2. Of these 87 chloroquine-resistant isolates, 4 isolates containing additional PfCRT 97L exhibited significantly higher chloroquine IC50, and isolates with PfCRT 6Q showed slightly lower chloroquine IC50. Parasite isolates with pfmdr1 184F showed a higher level of chloroquine resistance, and those with a pfmdr1 copy number ≥ 4 exhibited significantly lower chloroquine IC50. The association between parasite genotypes and level of chloroquine resistance in 87 parasite isolates are shown in Table 3. Parasites with a chloroquine IC50 > 100 nM were significantly associated with PfCRT 97L and pfmdr1 184F, and a pfmdr1 copy number ≥ 4 was more common in those with a chloroquine IC50 ≤ 100 nM.
Comparison of in vitro chloroquine sensitivity among Plasmodium falciparum isolates with different pfcrt and pfmdr1 genotypes*
| Parasite genotypes | No. (%) | Median CQ IC50 (IQR), nM | P | |
|---|---|---|---|---|
| pfcrt | ||||
| K6Q | 6K | 84 (96.6) | 76.8 (57.8–119.8) | 0.043† |
| 6Q | 3 (3.4) | 47.2 (ND) | ||
| H97L | 97H | 83 (95.4) | 74.9 (55.4–115.8) | 0.006† |
| 97L | 4 (4.6) | 158.6 (124.7–184.8) | ||
| I356T | 356I | 4 (4.6) | 99.3 (58.9–173.8) | 0.325 |
| 356T | 83 (95.4) | 75.8 (55.5–116.2) | ||
| pfmdr1 | ||||
| 86 | 86N | 81 (93.1) | 75.0 (54.6–117.9) | 0.873 |
| 86Y | 6 (6.9) | 89.7 (69.9–125.1) | ||
| 184 | 184Y | 51 (58.6) | 67.5 (51.2–113.9) | 0.008† |
| 184F | 36 (41.4) | 104.7 (63.9–120.8) | ||
| 1034 | 1034S | 84 (96.6) | 75.4 (55.8–116.2) | 0.237 |
| 1034C | 3 (3.4) | 170.7 (ND) | ||
| 1042 | 1042N | 84 (96.6) | 75.4 (55.8–116.2) | 0.237 |
| 1042D | 3 (3.4) | 170.7 (ND) | ||
| Copy no. | < 4 | 71 (81.6) | 80.4 (60.4–122.3) | 0.002† |
| ≥ 4 | 16 (18.4) | 56.1 (42.1–66.3) | ||
| Total | 87 (100) | 75.8 (55.5–119.6) | ||
pfcrt = Plasmodium falciparum chloroquine resistance transporter; pfmdr1 = P. falciparum multidrug resistance 1; CQ = chloroquine; IC50 = 50% inhibitory concentration; IQR = interquartile range; ND = not determined.
Significant difference determined by Mann-Whitney U test.
Association of parasite genotypes and the level of in vitro chloroquine sensitivity in Plasmodium falciparum isolates containing the pfcrt 76T allele*
| Parasite genotypes | Chloroquine IC50, no. (%) | P | ||
|---|---|---|---|---|
| ≤ 100 nM | > 100 nM | |||
| pfcrt K6Q | K6 | 51 (60.7) | 33 (39.3) | 0.285 |
| 6Q | 3 (100) | 0 (0) | ||
| pfcrt H97L | H97 | 54 (65.1) | 29 (34.9) | 0.018† |
| 97L | 0 (0) | 4 (100) | ||
| pfmdr1 Y184F | Y184 | 37 (72.5) | 14 (27.5) | 0.016‡ |
| 184F | 17 (47.2) | 19 (52.8) | ||
| pfmdr1 copy number | < 4 | 39 (54.9) | 32 (45.1) | 0.004† |
| ≥ 4 | 15 (93.8) | 1 (6.3) | ||
| Total | 54 (62.1) | 33 (37.9) | ||
pfcrt = Plasmodium falciparum chloroquine resistance transporter; pfmdr1 = P. falciparum multidrug resistance 1; IC50 = 50% inhibitory concentration.
Significant difference determined by Fisher's exact test.
Significant difference determined by chi-square test.
According to their pfcrt and pfmdr1 haplotypes, chloroquine-resistant parasites were classified into four groups (Table 4): i.e., isolates containing pfcrt 97H and pfmdr1 184Y with a copy number < 4; pfcrt 97L; pfcrt 97H and pfmdr1 184F with a copy number < 4; and pfcrt 97H and pfmdr1 184Y with a copy number ≥ 4. A total of 84 isolates were included in these four groups, and the other three isolates contained pfmdr1184Y with a copy number ≥ 4. To clearly determine the effect of the Y184F mutation and the copy number of the pfmdr1 gene, we excluded these three isolates from the analysis. There were significant differences in chloroquine IC50s among these groups (P < 0.001, by Kruskal-Wallis test). The parasites containing pfmdr1 184Y with a copy number ≥ 4 were significantly less resistant to chloroquine than the parasites containing pfmdr1 184Y with a copy number < 4 (P = 0.023) and pfmdr1 184F with a copy number < 4 (P = 0.001). In contrast, parasites containing pfcrt 97L were significantly more resistant than parasites containing pfmdr1 184Y with a copy number < 4 (P = 0.004), pfmdr1 184F with a copy number < 4 (P = 0.016), and pfmdr1 184Y with a copy number ≥ 4 (P = 0.003).
Comparison of in vitro chloroquine sensitivity among Plasmodium falciparum isolates with different pfcrt and pfmdr1 haplotypes*
| Group | Parasite haplotypes | No. (%) | Median CQ IC50 (nM) | IQR | |||
|---|---|---|---|---|---|---|---|
| pfcrt | Pfmdr1 | ||||||
| K76T | H97L | Y184F | Copy no. | ||||
| 1 | 76T | H97 | Y184 | < 4 | 36 (42.9) | 72.1 | 54.8–121.3 |
| 2 | 76T | 97L | Y184/184F | < 4 | 4 (4.8) | 158.6 | 124.7–184.8 |
| 3 | 76T | H97 | 184F | < 4 | 31 (36.9) | 105.9 | 34.4–119.9 |
| 4 | 76T | H97 | Y184 | ≥ 4 | 13 (15.5) | 56.7 | 42.4–65.1 |
| Total | 84 (100) | 76.3 | 57.0–119.8 | ||||
pfcrt = Plasmodium falciparum chloroquine resistance transporter; pfmdr1 = P. falciparum multidrug resistance 1; CQ = chloroquine; IC50 = 50% inhibitory concentration; IQR = interquartile range. Significant differences (P < 0.001) observed among groups were determined by Kruskal-Wallis test.
Discussion
In the present study, nearly all (87/89) isolates exhibited chloroquine-resistant haplotypes. The CVIET haplotype was identified in all 87 chloroquine-resistant isolates. This finding confirms the results of Hatabu and others, who showed that chloroquine-resistant isolates from Thailand contained CVIET.34 In contrast, chloroquine-resistant isolates from other counties in Southeast Asia showed a different distribution of the pfcrt haplotypes. Three additional haplotypes, CVIDT, CVMNT, and CVTNT, were identified in P. falciparum isolates from Cambodia.12,35 The SVMNT haplotype, was also detected in approximately 30% of parasites isolated from northern Lao PDRs.36 In this study, only two chloroquine-sensitive haplotypes were identified. Both isolates contained CVIEK, a less common chloroquine-sensitive haplotype. This haplotype has only been reported in a few isolates from Sudan, India, and The Philippines.6,26,37
Sequencing of exons 1 and 2 of the pfcrt gene identified two other nucleotide polymorphisms that caused the changing of amino acid at positions 6 and 97. The K6Q mutation located in cytosol peptide 1 is reported in this study. Only two previous studies have reported polymorphisms at position 97 of the pfcrt gene. Interestingly, different patterns of amino acid changes at this position were identified according to the geographic areas where the parasite isolates were found. Two parasites isolated from Colombia contained 97Q, and the 97L allele was identified in an isolate from Thailand.6,10 Our study identified one and three isolates from the Thailand-Myanmar and Thailand-Cambodia borders, respectively, that contained the 97L allele.
Although an IC50 value > 100 nM is generally used for defining in vivo chloroquine resistance,33 the chloroquine sensitive/resistant status might not be correlated with the pfcrt haplotypes in some studies.8,35 Because this cut-off value was estimated from the correlation between in vivo and in vitro test results, this value reflects not only parasite's susceptibility but also host factors such as immune status. Thus, some studies, particularly those using laboratory isolates used a cut-off value of 25 nM.38,39 Using this value, we found that in vitro parasite susceptibility was clearly correlated with pfcrt haplotypes in the present study. Two chloroquine-sensitive isolates containing the CVIEK haplotype showed an IC50 < 25 nM, and the chloroquine IC50 of those with the CVIET haplotype ranged from 25.2 to 190.5 nM. However, the parasite isolates containing the pfcrt 76T allele displayed such a broad range of chloroquine IC50s, indicating that the level of chloroquine resistance must be under multilocus/multigenic control.
A few studies using field and laboratory isolates selected for drug resistance showed the influence of amino acid changes in PfCRT transmembrane domains on the chloroquine susceptibility of the parasite.15–17 These amino acid substitutions, which resulted in changes in charge, are thought to affect the interaction of PfCRT and positively charged chloroquine, thus altering chloroquine accumulation in the food vacuole of the parasite and modulating chloroquine susceptibility. PfCRT is a member of the drug/metabolite transporter superfamily, which contains 10 transmembrane domains. On the basis of information from other members of the drug/metabolite transporter superfamily, each transmembrane domain has its specific function.40 In the present study, chloroquine-resistant isolates with an amino acid substitution at position 97 in transmembrane domain 2 but not at position 356 in transmembrane domain 9 of the PfCRT exhibited significantly higher chloroquine resistance. Functions of transmembrane domain 2 are similar to those of transmembrane domain 1, where the K76T mutation is located, i.e., recognition and discrimination of substrate. Transmembrane domain 9 is also important for binding and translocation of the substrate. Our finding might be explained by replacement of positively charged histidine by neutral leucine at amino acid position 97; no alteration of charge for the I356T substitution was found. Alteration of charge in the transmembrane domain might affect the drug susceptibility of the parasite more than that occurring in the cytosol peptide. A slight difference in the chloroquine IC50 was noted between the parasite isolates harboring a positively charged lysine and a neutral glutamine at the PfCRT position 6.
Our study identified some known point mutations by PCR–restriction fragment length polymorphism and additional mutations in exons 1 and 2 by sequencing. To determine the influence of pfcrt polymorphisms on the level of chloroquine resistance, all mutations should be identified by full-length sequencing of the pfcrt gene. In addition, because all available evidence indicates the involvement of other gene(s) in in vitro chloroquine susceptibility, alteration of chloroquine IC50 in these parasites may not be completely modulated by pfcrt polymorphisms. The effect of these charge-loss mutations should be further investigated by suitable techniques such as the transfection technique.
A few studies of field isolates have reported significantly interactive roles of two genes, usually pfcrt 76T and pfmdr1 86Y, in chloroquine resistance.24,41 In the present study, among the parasite isolates containing the pfcrt 76T allele, both copy number of the pfmdr1 gene and the changed amino acid at position 184 in the Pgh1 influenced the level of chloroquine resistance. Different predominant pfmdr1 haplotypes, that might confer different levels of chloroquine resistance were found in parasites isolated along the Thailand-Myanmar and Thailand-Cambodia borders. Parasites from the Thailand-Myanmar areas contained higher copy numbers of the pfmdr1 gene, and most parasites from the Thailand-Cambodia border contained the pfmdr1 184F allele. Selection for different pfmdr1 haplotypes in these two areas may have been caused by different drug policies in the past. The combination of mefloquine and artesunate has been strictly used for the treatment of uncomplicated P. falciparum malaria in the Thailand-Cambodia area for more than 15 years. In contrast, mefloquine with or without artesunate was used, depending on the level of mefloquine resistance, along the Thailand-Myanmar border. Until 2003, the combination of mefloquine and artesunate had been used as the first-line drug in this area.
In conclusion, our results indicated that an additional charge-loss mutation in the pfcrt gene and copy number and mutation of the pfmdr1 gene influenced the level of chloroquine resistance. In addition, molecular markers for chloroquine resistance could be different in each geographic area.
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