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Am. J. Trop. Med. Hyg., 75(3), 2006, pp. 381-387
Copyright © 2006 by The American Society of Tropical Medicine and Hygiene

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POLYMORPHISMS IN PFCRT, PFMDR1, DHFR GENES AND IN VITRO RESPONSES TO ANTIMALARIALS IN PLASMODIUM FALCIPARUM ISOLATES FROM BANGUI, CENTRAL AFRICAN REPUBLIC

DIDIER MENARD*, FERDINAND YAPOU, ALEXANDRE MANIRAKIZA, DJIBRINE DJALLE, MARCELLE DIANE MATSIKA-CLAQUIN, AND ANTOINE TALARMIN
Pasteur Institute of Bangui, Bangui, Central African Republic


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Drug resistance is probably the greatest challenge to most malaria-control programs. Given the limited resources for other malarial-control measures, rational drug used is crucial. Molecular markers for parasite resistance such as pfcrt, pfmdr-1, and dhfr have the potential to be used in an integrated fashion to provide timely information that is useful to policy makers. Therefore, we evaluated polymorphisms in these genes from Plasmodium falciparum and their association with in vitro antimalarial drug resistance to 135 parasites samples collected in Bangui in 2004. For the dhfr gene, we found a strong association between the dhfr genotype and chemosensitivity to pyrimethamine. For the pfcrt gene, we found that haplotypes with mutant-type alleles led to significant changes in the IC50 values for chloroquine, monodesethylamodiaquine, and quinine. We found no correlations for the pfmdr1 gene. These findings suggest that a regular monitoring and screening for resistance markers for antifolates and for chloroquine could act as an adjunct to in vivo trials.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The resistance to antimalarial drugs, especially chloroquine (CQ), of Plasmodium falciparum is one of the principal factors contributing to the worldwide increase in morbidity and mortality due to malaria.1,2 Different approaches have been developed to monitor the extent of antimalarial drug resistance and to determine the biologic mechanisms by which the parasite has evaded the action of the drug.

The susceptibility of P. falciparum to antimalarial drugs is usually assessed by therapeutic responses (in vivo test). This method has allowed the thresholds of treatment failure that are crucial for adjusting antimalarial drug policies to be determined.3 However, the risk of loss of patients during long follow-up periods and the logistical demands of clinical response studies in endemic areas have led to the development of laboratory strategies for identifying anti-malaria drug resistance. Laboratory strategies include in vitro drug sensitivity tests and evaluation of molecular markers associated with drug resistance.4,5 Antimalarial drug sensitivity tested in vitro provides information on the frequency of the resistant phenotype among the populations of parasites being transmitted and the possible cross-resistance patterns of anti-malaria drugs.6 In vitro testing is also useful for evaluating the efficacy of new drugs and for investigating the biologic mechanisms of drug action and resistance.7,8 Although this method is useful, its application is limited because samples must be immediately cultured or cryopreserved for transport, parasites must be adapted to culture (which is laborious), and the tests use expensive and radioactive materials.9 Although more rigorous in vitro tests of culture-adapted isolates are more reproducible, the processes of freezing, thawing, and adaptation to culture also introduce the possibility of selecting sub-populations of parasites, so that the parasites ultimately assayed may be genetically and phenotypically unrepresentative of the original parasite population.

These limitations of in vivo and in vitro methods have led to the search for genetic markers of resistance. Potential molecular markers for parasite resistance to quinoline antimalarials are being identified. The association of CQ resistance and single nucleotide polymorphisms (SNPs) in the pfcrt (CQR transporter) and pfmdr1 (multidrug resistance 1) genes are presently well established.10–13 For example, the K76T mutation in pfcrt gene was perfectly associated with in vitro resistance in all progeny of a genetic cross between chloroquine-sensitive and chloroquine-resistant parental clones and among a set of geographically diverse parasite isolates.14 Polymorphisms in pfmdr1, which encodes the P. falciparum P glycoprotein homologue 1, modulate chloroquine resistance in mutant pfcrt-harboring parasites in vitro,15 although their role in vivo has yet to be substantiated.10 The mutations most often cited as potential contributors to chloroquine resistance are pfmdr1 N86Y and D1246Y. In the same way, several point mutations in P. falciparum dhfr and dhps genes have been associated with sulfadoxine-pyrimethamine (SP) treatment failure in Africa.16–22 Univariate analysis have shown that the dhfr triple mutant S108N/N51I/C59R or the dhps double mutant A437G/K540E or the dhfr and dhps quintuple mutant carrying all these 5 mutations are all associated with SP treatment failure in patients.19,21,22

In the Central African Republic (CAR), Plasmodium falciparum resistance to CQ has been documented since 198323 and to SP since 1987.24 Since 2003, when the latest in vivo study was carried out,25 amodiaquine + SP combination has been used as interim first-line treatment, until the best alternative treatments, such as Artemisinin-based Combination Therapies (ACT), become available at low prices in the CAR. We have previously determined, in samples collected in 2004, the in vitro susceptibility of P. falciparum strains to anti-malarials currently being used in the CAR (chloroquine, amodiaquine, quinine, and pyrimethamine), to antimalarials that will be available for future use in this region (artemisinin, halofantrine), and to prophylactic antimalarials (mefloquine, atovaquone, doxycycline).26 We have also previously determined the baseline frequency distribution of the mutant alleles of genes associated with antimalarial drug resistance (pfcrt, pfmdr1, dhfr, and dhps).27

Here, we aim to complete these studies by determining the utility of the molecular markers such as pfcrt, pfmdr1, and dhfr in monitoring antimalarial drug resistance. We have estimated the association of these molecular makers with in vitro responses to antimalarial drugs (chloroquine, mono-desethylamodiaquine, quinine, dihydroartemisinin, mefloquine, halofantrine, and pyrimethamine) in Plasmodium falciparum isolates from Bangui, Central African Republic.


METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
P. falciparum isolates. This study was conducted in Bangui, the capital of the CAR, between March and July 2004. Bangui is located beside the Oubangui river in the middle of central Africa, north of the Democratic Republic of the Congo (Geographic coordinates: 7 00 N, 21 00 E). The climate is tropical and rainfall is at its highest from April to November. The average temperature varies from 19–32°C. Malaria transmission occurs throughout the year, with peak transmission at the beginning and the end of the rainy season. Malaria is hyperendemic in this region and Plasmodium falciparum is the principal parasite. The prevalence of this parasite in children less than 5 years of age is 31.8%.28

We obtained 381 P. falciparum clinical isolates from symptomatic patients from the CAR attending several health centers in Bangui before the patients were treated. Venous blood samples from patients giving informed consent were collected in a tube coated with EDTA (Vaicutainer® tubes, Becton Dickinson, Rutherford, NJ, USA). Giemsa-stained thin and thick blood smears were examined to check for mono-infection with P. falciparum and to determine parasite density. Blood samples with a parasite density > 0.1% were assayed in vitro within 8 hours of blood extraction. The patients were treated with amodiaquine-sulfadoxine-pyrimethamine combination or quinine, as recommended by the National Malaria Control Program in the CAR.

Ethical approval. As there is no National Ethics Committee in the CAR, study protocols were approved by the expert committee for antimalarial drug policy and the CAR Ministry of Health. Blood samples were obtained from patients after obtaining informed consent during routine malaria diagnosis.

In vitro drug sensitivity assay. Stock solutions of chloroquine, monodesethylamodiaquine, dihydroartemisinin, quinine, mefloquine, and halofantrine were prepared in methanol. The stock solution of pyrimethamine was prepared in ethanol. The final concentration in methanol and ethanol did not exceed 0.05%. Twofold (4-fold for pyrimethamine) serial dilutions of the stock solutions were prepared in distilled water. The solutions tested had concentrations ranging from 12.5–3200 nM for chloroquine, 25–3200 nM for quinine, 7.5–1920 nM for monodesethylamodiaquine, 0.25–64 nM for dihydroartemisinin, 0.25–32 nM for halofantrine, 1.5–400 nM for mefloquine, and 50–40,000 nM for pyrimethamine. Aliquots (20 µL) of each solution (at all concentrations tested) were transferred to 96-well tissue culture plates in triplicate.

The venous blood samples were washed 3 times in RPMI 1640 medium. The white blood cell interface was removed after each wash. The erythrocytes were then resuspended in a volume of complete RPMI 1640 medium (RPMI 1640 medium supplemented with 10% human serum (Serum AbCys®, France, lot no S02909S4190), 25 mM HEPES buffer, and 25 mM sodium bicarbonate) giving a hematocrit of 1.5% and an initial parasitemia of 0.1–0.5%. Para-aminobenzoic acid-free and folic acid-free RPMI 1640 medium was used to assess the in vitro sensitivity to pyrimethamine. If the blood sample had a parasitemia greater than 0.5%, fresh uninfected erythrocytes were added to adjust the parasitemia to 0.3%. The technical procedure for the in vitro isotopic microtest and calculation of 50% inhibitory concentrations (IC50) has been previously described.26

Extraction of DNA. The DNA template for PCR and the detection of mutant alleles was prepared from the whole blood sample. The blood was centrifuged and the erythrocytes were frozen at –20°C until extraction. Parasite DNA was extracted from 100 µL of thawed red blood cell pellets by treatment with 0.1M NaOH for 3 minutes at 100°C. The supernatant was collected and treated with 250 µL of lysing solution (0.1 M Tris HCl, Triton 100X, 1 M NaCl, SDS 10X, 0.5 M EDTA) and 20 mg/ml proteinase K for 1 hour at 37°C, and then extracted twice with phenol/chloroform (1:1). The DNA was then precipitated with ethanol, resuspended in 100 µL of distilled water, and stored at –20°C.

Amplification by polymerase chain reaction and detection of mutant alleles. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis were carried out for 3 genes (dihydrofolate reductase, dhfr gene at codons 16, 50, 51, 59, 108, 164; chloroquine resistance transporter, pfcrt gene at codons 72, 74, 75, 76 and in multi-drug resistance 1, pfmdr1 gene at codons 86 and 1246) to identify the presence of mutant alleles.

A detailed description of these methods is available on the Web at http://medschool.umaryland.edu/CVD/plowe.html.

All PCR amplifications were performed with Gene Amp PCR system 9700® thermocycler (Applied Biosystems®, Courtaboeuf, France) and Taq DNA Polymerase Promega® (Promega France, Charbonnieres, France). Five microliters of PCR products were incubated with restriction enzymes (New England Biolabs®, Beverly, MA, USA) according to manufacturer’s protocols in a 15-µL final reaction. Laboratory strains of P. falciparum served as controls (positive and negative) and were included in all PCR and enzyme digests procedures (purified genomic DNA from W2, HB3, and 3D7 reference strains from the Malaria Research Reference Reagent Resource Center, MR4/ATCC, Manassas, Virginia). A subset of 45 samples also was sequenced in Genopole at Institut Pasteur (Paris, France). Briefly, 50 µL of PCR products were purified using polyacrylamide P-100 Gel (Bio-Gel P-100, BioRad®, Marnes-la-Coquette, France) on 96- well plate filtration (Millipore®, St. Quentin en Yvelines, France). Evaluation of template quantity and quality was performed using agarose 1.2% gel and buffer TBE 0.5% (Invitrogen®, Cergy Pontoise, France) on Electrofast micro gel system 96 wells (Fischer Scientific Labosis®, Ottawa, Canada) with DNA quantification kit (Molecular weight, Abgene®, Courtaboeuf, France). Sequencing reactions were performed on a 96-well format, with individual caps (MicroAmp Optical 96-well reaction plate) using BigDye Terminator chemistry (BigDye v 3.1 terminator, Applied BioSystem®, Courtaboeuf, France) on GeneAmp-9700 (Applied BioSystem®, Courtaboeuf, France). Sequencing was realized with 10 µL of EDTA 0.3 mM par well on automatic DNA analyser ABI 3700 (Applied BioSystems®, Courtaboeuf, France). For data analysis, we used SeqScape® software (Applied BioSystem®, Courtaboeuf, France).

msp-1 and msp-2 genotyping. Polymerase chain reaction genotyping was performed as described by Snounou,29 using repetitive regions found in 2 polymorphic genetic makers, namely msp-1 (block 2) and msp-2 (block 3). Allelic variants of msp-1 (MAD20, K1 and RO33) and msp-2 (3D7 and FC27) were detected by allelic family-specific nested PCR. All PCR amplifications (Gene Amp PCR system 9700®, Applied Bio-systems®, Courtaboeuf, France) contained a positive control (purified genomic DNA from W2, HB3, and 3D7 reference strains from the Malaria Research Reference Reagent Resource Center, MR4/ATCC, Manassas, Virginia for msp-1 and msp-2 allelic families) and a negative control (containing no target DNA).

Statistical analysis. The prevalence of each mutant allele was determined without prior knowledge of the in vitro susceptibility status of the isolates. We used ANOVA or the non-parametric Mann-Whitney U test to compare the geometric means of the IC50 values with respect to the pfcrt, pfmdr1, and dhfr genotypes. We used Fisher’s exact test to assess the statistical associations between point mutations and the in vitro resistance phenotypes. We also determined the positive predictive value (PPV) for molecular markers as the probability that a resistant phenotype isolate was restricted to isolates carrying a mutant-type allele. All tests were 2-sided, and P < 0.05 were considered statistically significant.


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Among 381 blood samples collected from P. falciparum-infected individuals, only monoclonal samples (1 genotype found with msp-1 and msp-2 genotyping) with most prevalent genotypes and data from the in vitro isotopic drug sensitivity assay were analyzed: chloroquine (N = 134), monodesethylamodiaquine (N = 134), quinine (N = 135), dihydroartemisinin (N = 51), halofantrine (N = 24), mefloquine (N = 49), and pyrimethamine (N = 39). The mean age of the falciparum-infected individuals was 25 years (range, 1–80 years) and 36% were male. The geometric mean of parasitemia was 15,442 parasites/µl of blood (range 78–443,071 parasites/µl of blood).

Data from the in vitro isotopic drug sensitivity assay, the baseline frequency distribution of the mutant alleles of pfcrt, pfmdr1, dhfr, and dhps genes and clonality of infections by msp-1 and msp-2 genotyping in P. falciparum isolates have been previously described.26,27,30 Briefly, mutant alleles of the pfcrt gene were found in two thirds of samples and the frequencies of the deduced haplotype were (amino-acid residues at positions 72, 74, 75, and 76): CIET 45%, CMNK 33.3%, CMNT 12.3%, CIEK 4.7%, SIET 2.3%, SIEK 1.2%, SMET 0.6%, and CINT 0.6%. (CIET, CIEK, SIET SIEK, SMET, CINT haplotypes were confirmed by sequencing.) The most common allele of the pfmdr1 gene among the field isolates of P. falciparum was 86Y (21.9%). The 1246Y allele was also common (18.0%). Frequencies of deduced haplotype profiles for the dhfr gene of Plasmodium falciparum isolates were (amino-acid residues at positions 16, 50, 51, 59, 108, and 164): ACIRNI 57.5%, ACIRSI 9.6%, ACNCNI 8.4%, ACNRNI 7.8%, ACNCSI 7.2%, ACNRSI 3.0%, ACICNI 3.0%, ACICSI 2.4%, ACNCTI 0.6%, ACIRSL 0.6%, and ACIRNL 0.6%. (ACIRNI, ACIRSI, ACNRNI, ACICNI, ACNCTI, ACIRSL, and ACIRNL haplotypes were confirmed by sequencing.)

Correlation between polymorphisms in the pfcrt and pfmdr1 genes and in vitro chloroquine susceptibility. For polymorphisms in the pfmdr1 gene, we found no significant differences for the geometric means of the IC50 in positions 86 and 1246 (P > 0.05, data not shown).

For the pfcrt gene, we compared geometric means of IC50 values for deduced haplotypes in the pfcrt gene and observed significant differences (P = 0.001) between haplotype with wild-type alleles (CMNK, N = 55, mean IC50 = 50 nM, CI 95% 32–68) and haplotypes with mutant-type alleles: CIET, 3 times higher (N = 63, mean IC50 = 151 nM, CI 95% 120–181); CMNT, 2.9 times higher (N = 10, mean IC50 = 143 nM, CI 95% 43–242) and CIEK, 2.7 times higher (N = 6, mean IC50 = 135 nM, CI 95% 18–280). Figure 1Go shows scatter plots of the data.


Figure 1
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  		    FIGURE 1. In vitro susceptibility to chloroquine (A), monodesethylamodiaquine (B), and quinine (C) in isolates of Plasmodium falciparum from the Central African Republic with deduced haplotypes in the pfcrt gene.

 
Haplotypes with mutant-type alleles CIET, CMNT, CIEK were present respectively in 76%, in 75%, and in 55% chloroquine-resistant isolates (P = 0.005) and haplotype with wild-type allele CMNK in 85% chloroquine-sensitive isolates (P = 0.002). We estimated that deduced haplotypes CIET, CMNT, CIEK were 3.2 times, 2.9 times, and 1.2 times more frequent, respectively, in the chloroquine-resistant phenotype.

The positive predictive value (PPV) of mutant-type I74, E75, and T76 alleles for chloroquine-resistance were estimated to 54%, 20%, and 74%, respectively.

Correlation between polymorphisms in the pfcrt and pfmdr1 genes and in vitro monodesethylamodiaquine susceptibility. For polymorphisms in the pfmdr1 gene, we found no significant differences for the geometric means of the IC50 values in positions 86 and 1246 (P > 0.05, data not shown).

For the pfcrt gene, we found significant increases for geometric mean of IC50 values for in vitro monodesethylamodiaquine susceptibility (P = 0.002): 2 times higher for the CIET haplotype (N = 62, mean IC50 = 52 nM, CI 95% 39–64) than the CMNK haplotypes (N = 56, mean IC50 = 26 nM, CI 95% 21–32). Figure 1Go shows scatter plots of the data.

The deduced haplotypes CIET was present in 85% monodesethylamodiaquine-resistant isolates (P = 0.02) and CMNK haplotype in 95% monodesethylamodiaquine-sensitive isolates (P = 0.006). We estimated that deduced haplotypes CIET was 1.6 times more frequent in the monodesethylamodiaquine-resistant phenotype. We estimated the PPV of mutant-type I74, E75, and T76 alleles for monodesethylamodiaquine resistance as 22%, 22%, and 24%, respectively.

Correlation between polymorphisms in the pfcrt and pfmdr1 genes and in vitro quinine susceptibility. For polymorphisms in the pfmdr1 gene, we found no significant differences for the geometric means of the IC50 values in positions 86 and 1246 (P > 0.05, data not shown).

For the pfcrt gene, we also found a significant difference (P = 0.03) between the CIET (N = 64, mean IC50 = 213 nM, CI 95% 183–244 nM) and CMNK (N = 55, mean IC50 = 163 nM, CI 95% 128–159 nM) haplotypes (1.3 times higher for CIET). Figure 1Go shows scatter plots of the data.

Correlation between polymorphisms in the pfcrt and pfmdr1 genes and in vitro susceptibilities of others antimalarial drugs tested. For polymorphisms in the pfmdr1 and pfcrt genes, we found no significant differences (P > 0.05, data not shown) for the geometric means of the IC50 values for in vitro dihydroartemisinin, halofantrine, and mefloquine susceptibility.

Correlation between polymorphisms in dhfr and in vitro pyrimethamine susceptibility. We found significant increases in the geometric means of the IC50 values for in vitro pyrimethamine susceptibility (P = 0.0002): 10 times higher for the mutant-type I51 allele (N = 27, mean IC50 = 5288 nM, CI 95% 3614–6961 nM) than the wild-type N51 allele (N = 12, mean IC50 = 502 nM, CI 95% 118–886 nM); 21 times higher for the mutant-type R59 allele (N = 31, mean IC50 = 4742 nM, CI 95% 3201–6283 nM) than the wild-type C59 allele (N = 8, mean IC50 = 225 nM, CI 95% 85–489 nM); 21 times higher for the mutant-type N108 allele (N = 30, mean IC50 = 4888 nM, CI 95% 3324–6453 nM) than the wild-type S108 allele (N = 9, mean IC50 = 238 nM, CI 95% 16–484 nM). We found 100% of wild-type alleles for codons 16 and 50, one sample with mutant-type 164L and one sample with mixed type 164I/L (not analyzed in this study).

For the deduced haplotypes in dhfr gene, the geometric means of the IC50 values for in vitro pyrimethamine susceptibility increased regularly with the number of mutation (P = 0.002): 22 times higher between ACNCNI (N = 4, mean IC50 = 299 nM, CI 95% 123–1210) and ACNCSI haplotypes (N =5, mean IC50 =13.6 nM, CI 95% 2–88), 53 times higher between ACIRSI (N = 4, mean IC50 = 720 nM, CI 95% 253–1420) and ACNCSI haplotypes, 80 times higher between ACNRNI (N =3, mean IC50 =1081 nM, CI 95% 524–2229) and ACNCSI haplotypes, and 332 times higher for ACIRNI haplotype (N = 23, mean IC50 = 4323 nM, CI 95% 3140–5951) and ACNCSI haplotypes. Figure 2Go shows scatter plots of the data.


Figure 2
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  		    FIGURE 2. In vitro susceptibility to pyrimethamine in isolates of Plasmodium falciparum from the Central African Republic with the number of mutant allele in the dhfr gene.

 
The deduced haplotypes ACIRNI, ACIRSI, ACNRNI, and ACNCNI were only present in pyrimethamine-resistant isolates (P = 10–5) and the deduced haplotypes ACNCSI in the pyrimethamine-sensitive isolates (P = 10–4).

We estimated that the mutant-type I51, R59, and N108 alleles were 27 times, 30 times, 30 times more frequent, respectively, in the pyrimethamine-resistant phenotype. We estimated the PPV of mutant-type I51 and N108 alleles for pyrimethamine-resistant phenotype as 100% for each allele and as 96.8% for the mutant-type R59 allele.


DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Drug resistance is probably the greatest challenge to most malaria-control programs. Given the limited resources for other malarial-control measures, rational drug used is crucial, although economic constraints and the scarcity of drug choice hampers this. Close monitoring of the epidemiology and dynamics of drug resistance are necessary to implement measures to circumvent the problem and to allow the National Malaria Control Program to recommend the best available management of malaria.

We have previously shown a close relationship between in vivo and in vitro studies in Bangui. We used the standard 14-day WHO 2001 protocol to compare therapeutic responses in children less than 5 years of age with acute uncomplicated Plasmodium falciparum malaria in Bangui between February 2002 and March 2004. We found that the overall treatment failure rates with chloroquine, amodiaquine sulfadoxine-pyrimethamine, chloroquine + sulfadoxine-pyrimethamine combination, and amodiaquine + sulfadoxine-pyrimethamine combination were 40.9%, 20.0%, 22.8%, 7.2%, and 0%, respectively.25 Consistent with this were in vitro isotopic drug sensitivity assays carried out in Bangui between March and July 2004. They showed that the proportion of resistant isolates was 37% for chloroquine, 15.9% for amodiaquine, 0% for quinine, 0% for dihydroartemisinin, 1.6% for mefloquine, 3.8% for halofantrine, 4.0% for atovaquone, and 83% for pyrimethamine.26 We also determined with the same samples, the baseline frequency distribution of the mutant alleles of genes associated with resistance to chloroquine and sulfadoxine-pyrimethamine. We found mutant alleles of the pfcrt gene in two thirds of the samples and a high (45%) frequency of the deduced CIET pfcrt haplotype. The most common alleles of the pfmdr1 gene among the field isolates of P. falciparum were the 86Y (21.9%) and 1246Y (18.0%) alleles. Analysis of dhfr gene showed that only 12 isolates carried the wild-type allele (7.2%), whereas many (57.5%) were triple mutants (51I, 59R, 108N).27 More interesting, was to find, for the first time in Central African Republic, the quadruple mutation (51:59/108/164) in 2 samples, using the PCR-RLFP approach, this finding being confirmed by sequencing, subsequently. Unfortunately, IC50 values for in vitro pyrimethamine susceptibility were not available for these samples. According to Nzila and colleagues,31 this result indicated that the quadruple mutant DHFR exists in Central African Republic where SP is widely used, implying that the useful therapeutic life of the new combination such as chlorproguanil plus dapsone (LapdapTM) could be very short.

In this present study, we have shown a strong association between the dhfr genotype and chemosensitivity to pyrimethamine. Isolates with single or combined dhfr I51, R59, or N108 mutations were between 10 and 21 times less susceptible to pyrimethamine. For pyrimethamine, we found a regular increase in IC50 values with the number of dhfr mutations, from a 22-fold increase for a single mutant-type allele to a 332-fold increase for a triple mutant. This is consistent with previous studies correlating dhfr mutations and chemosensitivity in field samples from Brazil32 and Africa.33–35 We also observed a strong relationship between phenotype and genotype for pyrimethamine (all except one isolate containing a mutant allele was found to be resistant), the PPVs of mutant-type I51 and N108 alleles for pyrimethamine-resistant phenotype being 100% and the PPV of mutant-type R59 being 96.8%. Therefore, we believe that PCR-RFLP analysis in the dhfr gene for codons 51, 59, and 108 may be a quick and reliable method being able to replace the in vitro techniques providing particularly poor results for pyrimethamine. However, associations between theses molecular markers and clinical SP treatment outcomes seem to be less straightforward. Even if several studies such as Kublin and associates20 showed that the presence together of all 5 dhfr and dhps mutations that are found in Africa were strongly associated with SP failure, recent study conducted by Francis and colleagues36 strongly suggest that geographic differences in response to antimalarial therapy in Uganda are primarily mediated by acquired immunity associated with malaria transmission intensity, rather than by parasite factors such as molecular markers of antimalarial drug resistance.

We have evaluated the correlation between pfcrt and pfmdr1 mutations and in vitro responses to chloroquine, monodesethylamodiaquine, quinine, dihydroartemisinin, mefloquine, and halofantrine. We found that for haplotypes with mutant-type alleles in the pfcrt gene led to significant changes in the IC50 values of chloroquine, monodesethylamodiaquine, and quinine. This is consistent with our previous in vitro study26 in which we found significant differences in the IC50 values for monodesethylamodiaquine and quinine in chloroquine-sensitive and chloroquine-resistant isolates, and in which there was a significant correlation between the in vitro responses to chloroquine and monodesethylamodiaquine (r = 0.61) and to chloroquine and quinine (r = 0.33). The cross-resistance between these drugs can be partly explained by similarities in their chemical structures.37 Among the 4 haplotypes (CMNK, CIEK, CMNT, and CIET) found in pfcrt gene, CMNK haplotype was clearly associated with chloroquine-sensitive and monodesethylamodiaquine-sensitive isolates, whereas the CIEK, CMNT, and CIET haplotypes were associated with higher IC50 values. We observed significant differences in the geometric means of the IC50 values between the CMNK and CIET haplotypes for chloroquine (3-fold), monodesethylamodiaquine (2-fold), and quinine (1.3-fold) and between CMNK and CMNT (2.9-fold), and CMNK and CIEK (2.7-fold) for only chloroquine. According to Djimde and colleagues10 or Holmgren and associates,38 examination of 76T mutation in the pfcrt gene sequence can be used with reasonable reliability to predict resistance to chloroquine. Most likely this can be applied in the CAR since PPV for chloroquine resistance of 74%. In the same way, associations of mutations in the pfcrt gene with clinical treatment outcomes of chloroquine are less straightforward. This is due in part to the complexity of analyzing relationships between multiple mutations on different genes and the multinomial treatment outcomes and in part because treatment outcomes are affected by factors other than parasite resistance, including the host immune response,39 micronutrient levels,40 and bioavailability and pharmacokinetics.41

Much confusion has surrounded the association of different pfmdr1 alleles to chloroquine resistance, although transfection studies as well as carefully controlled, molecular epidemiologic studies have shown that there are strong associations between polymorphisms and antimalarial resistance.42–45 However, like many other studies,46–48 our present findings have failed to find such associations because the presence of both wild-type and mutant-type alleles in our samples were largely independent of their in vitro responses. This can be for a variety of reasons, including the inability to control for the inoculum effect,49 which can alter the in vitro sensitivity erroneously. In your opinion and according to Duraisingh and associates,13 there may be true epidemiologic differences in resistance resulting from pfmdr1 polymorphisms in different populations and differences in the strength of associations between resistance and pfmdr1 alleles could reflect the different selection histories of these populations. However, correlations between arylaminoalcohols or endoperoxide artemisinin derivatives resistance and amplification of pfmdr1 gene50,51 or new candidate transporter gene52 merit further investigation.

In conclusion, these findings suggest that analysis of the dhfr and pfcrt gene may be a reliable tool for the epidemiologic surveillance of antimalarial drug resistance in the CAR. A regular monitoring and screening for resistance markers for antifolates (such as the dhfr mutations) and for chloroquine (such as the pfcrt mutations) could act as an adjunct to in vivo trials and should be carried out both in Bangui and in the CAR, to allow the National Malaria Control Program to recommend the best available management of malaria (antimalarial drugs used as first-line treatment or intermittent preventive treatment during pregnancy).

Received July 27, 2005. Accepted for publication May 9, 2006.

Acknowledgments: The authors thank the patients for participating in the study.

Financial support: This work was supported by the French Government, via FSP/RAI 2001-168 project (French Ministry of Foreign Affairs). Sequencing was performed by Marie Thérèse Ekala (Genopole, Institut Pasteur à Paris) and supported by the UE (Contract No: QLK2-CT-2002-015) via Development of a malaria resistance DNA chip as public health tool for the management of Plasmodium falciparum malaria drug resistance, RESMALCHIP project.

* Address correspondence to Didier Menard, Institut Pasteur de Madagascar, BP 1274—Antananarivo 101. E-mail: dmenard{at}pasteur.mg Back

Authors’ addresses: Didier Menard, Ferdinand Yapou, Alexandre Manirakiza, Djibrine Djalle, Marcelle Diane Matsika-Claquin, and Antoine Talarmin, Institut Pasteur de Madagascar, BP 1274–Antananarivo 101.


REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Barat LM, Bloland PB, 1997. Drug resistance among malaria and other parasites. Infect Dis Clin North Am 11: 969–987.[ISI][Medline]
  2. Marsh K, 1998. Malaria disaster in Africa. Lancet 352: 924.[ISI][Medline]
  3. WHO, 2003. Assessment and monitoring of antimalarial drug efficacy for the treatment of uncomplicated falciparum malaria. Geneva, Switzerland. (WHO/HTM/RBM/2003.50). Available online: http://mosquito.who.int/cmc_upload/0/000/017/017/ProtocolWHO.pdf.
  4. Bickii J, Basco LK, Ringwald P, 1998. Assessment of three in vitro tests and an in vivo test for chloroquine resistance in Plasmodium falciparum clinical isolates. J Clin Microbiol 36: 243–247.[Abstract/Free Full Text]
  5. Warhurst DC, 2001. A molecular marker for chloroquine-resistant falciparum malaria. N Engl J Med 344: 299–302.[Free Full Text]
  6. Warsame M, Wernsdorfer WH, Payne D, Bjorkman A, 2001. Susceptibility of Plasmodium falciparum in vitro to chloroquine, mefloquine, quinine and sulfadoxine/pyrimethamine in Somalia: relationships between the responses to the different drugs. Trans R Soc Trop Med Hyg 85: 565–569.
  7. Desjardins RE, Canfield CJ, Haynes JD, Chulay JD, 1979. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob Agents Chemother 16: 710–718.[Abstract/Free Full Text]
  8. Foote SJ, Cowman AF, 1994. The mode of action and the mechanism of resistance to antimalarial drugs. Acta Trop 56: 157–171.[ISI][Medline]
  9. Bruce-Chwart LJ, Black RH, Canfield CJ, Clyde DF, Peters W, Wemsdorfer WH, 1981. Chemotherapy of malaria. WHO Monograph Series 27: 211–224.
  10. Djimde A, Doumbo OK, Cortese JF, Kayentao K, Doumbo S, Diourte Y, Dicko A, Su XZ, Nomura T, Fidock DA, Wellems TE, Plowe CV, Coulibaly D, 2001. A molecular marker for chloroquine-resistant falciparum malaria. N Engl J Med 344: 257–263.[Abstract/Free Full Text]
  11. Anderson TJ, Nair S, Sudimack D, Williams JT, Mayxay M, Newton PN, Guthmann JP, Smithuis FM, Tran TH, van den Broek IV, White NJ, Nosten F, 2005. Geographical distribution of selected and putatively neutral SNPs in Southeast Asian malaria parasites. Mol Biol Evol 22: 2362–2374.[Abstract/Free Full Text]
  12. Duraisingh MT, Drakeley CJ, Muller O, Bailey R, Snounou G, Targett GA, Greenwood BM, Warhurst DC, 1997. Evidence for selection for the tyrosine-86 allele of the pfmdr 1 gene of Plasmodium falciparum by chloroquine and amodiaquine. Parasitology 114: 205–211.
  13. Duraisingh MT, Cowman AF, 2005. Contribution of the pfmdr1 gene to antimalarial drug-resistance. Acta Trop 94: 181–190.[ISI][Medline]
  14. Fidock DA, Nomura T, Talley AK, Cooper RA, Dzekunov SM, Ferdig MT, Ursos LM, Sidhu AB, Naude B, Deitsch KW, Su XZ, Wootton JC, Roepe PD, Wellems TE, 2000. Mutations in the P. falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Mol Cell 6: 861–871.[ISI][Medline]
  15. Reed MB, Saliba KJ, Caruana SR, Kirk K, Cowman AF, 2000. Pgh1 modulates sensitivity and resistance to multiple antimalarials in Plasmodium falciparum. Nature 403: 906–909.[Medline]
  16. Plowe CV, Cortese JF, Djimde A, Nwanyanwu OC, Watkins WM, Winstanley PA, Estrada-Franco JG, Mollinedo RE, Avila JC, Cespedes JL, 1997. Mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase and epidemiologic patterns of pyrimethamine-sulfadoxine use and resistance. J Infect Dis 176: 1590–1596.[ISI][Medline]
  17. Jelinek T, Ronn AM, Lemnge MM, Curtis J, Mhina J, Duraisingh MT, Bygbjerg IC, Warhurst DC, 1998. Polymorphisms in the dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) genes of Plasmodium falciparum and in vivo resistance to sulphadoxine/pyrimethamine in isolates from Tanzania. Trop Med Int Health 3: 605–609.[ISI][Medline]
  18. Nzila AM, Mberu EK, Sulo J, Dayo H, Winstanley PA, Sibley CH, Watkins WM, 2000. Towards an understanding of the mechanism of pyrimethamine-sulfadoxine resistance in Plasmodium falciparum: genotyping of dihydrofolate reductase and dihydropteroate synthase of Kenyan parasites. Antimicrob Agents Chemother 44: 991–996.[Abstract/Free Full Text]
  19. Nzila AM, Mberu EK, Nduati E, Ross A, Watkins WM, Sibley CH, 2002. Genetic diversity of Plasmodium falciparum parasites from Kenya is not affected by antifolate drug selection. Int J Parasitol 32: 1469–1476.[ISI][Medline]
  20. Kublin JG, Dzinjalamala FK, Kamwendo DD, Malkin EM, Cortese JF, Martino LM, 2002. Molecular markers for failure of sulfadoxine-pyrimethamine and chlorproguanil-dapsone treatment of Plasmodium falciparum malaria. J Infect Dis 185: 380–388.[ISI][Medline]
  21. Eriksen S, Mwankusye S, Mduma A, Kitua G, Swedberg G, Tomson LL, Gustafsson G, Warsame M, 2005. Patterns of resistance and DHFR/DHPS genotypes of Plasmodium falciparum in rural Tanzania prior to the adoption of sulfadoxine–pyrimethamine as first-line treatment. Trans R Soc Trop Med Hyg 98: 347–353.
  22. Staedke SG, Sendagire H, Lamola S, Kamya MR, Dorsey G, Rosenthal PJ, 2004. Relationship between age, molecular markers, and response to sulphadoxine-pyrimethamine treatment in Kampala, Uganda. Trop Med Int Health 9: 624–629.[ISI][Medline]
  23. Pierce PF, Milhous WK, Campbell CC, 1987. Clinical and laboratory characterization of a chloroquine-resistant Plasmodium falciparum strain acquired in the Central African Republic. Am J Trop Med Hyg 36: 1–2.[Abstract/Free Full Text]
  24. Belec L, Delmont J, Vesters I, Testa J, Christian KS, Georges AJ, 1988. Emergence of multiresistant Plasmodium falciparum malaria in Central African Republic. Press Med 17: 2090–2091.
  25. Menard D, Madji N, Manirakiza A, Djalle D, Koula MR, Talarmin A, 2005. Efficacy of chloroquine, amodiaquine, sulfadoxine-pyrimethamine, chloroquine-sulfadoxine-pyrimethamine combination, and amodiaquine-sulfadoxine-pyrimethamine combination in Central African children with noncomplicated malaria. Am J Trop Med Hyg 72: 581–585.[Abstract/Free Full Text]
  26. Menard D, Yapou F, Siadoua V, Sana S, Matsika-Claquin MD, Madji N, Talarmin A, 2005. Drug-resistant malaria in Bangui (Central African Republic): an in vitro assessment. Am J Trop Med Hyg 73: 239–243.[Abstract/Free Full Text]
  27. Menard D, Djalle D, Yapou F, Manirakiza A, Talarmin A, 2005. Frequency distribution of antimalarial drug-resistant alleles among isolates of Plasmodium falciparum in Bangui, Central African Republic. Am J Trop Med Hyg 74: 205–210.
  28. UNICEF, 2001. Enquête à indicateurs multiples, MICS 2000. Editions B, ed. Rapport Final Bangui Bangui: Ministère du Plan, RCA, 130–131.
  29. Snounou G, Zhu X, Siripoon N, 1999. Biased distribution of msp1 and msp2 allelic variants in Plasmodium falciparum populations in Thailand. Trans R Soc Trop Med Hyg 93: 369–374.[ISI][Medline]
  30. Menard D, Dolmazon V, Matsika-Claquin MD, Manirakiza A, Yapou F, Nambot M, Talarmin A, 2006. Genetic diversity and genotype multiplicity of Plasmodium falciparum infections in symptomatic individuals living in Bangui (CAR). Trans R Soc Trop Med Hyg.
  31. Nzila A, Ochong E, Nduati E, Gilbert K, Winstanley P, Ward S, Marsh K, 2005. Why has the dihydrofolate reductase 164 mutation not consistently been found in Africa yet? Trans R Soc Trop Med Hyg 99: 341–346.[ISI][Medline]
  32. Peterson DS, Di Santi SM, Povoa M, Calvosa VS, Do Rosario VE, Wellems TE, 1991. Prevalence of the dihydrofolate reductase Asn-108 mutation as the basis for pyrimethamine-resistant falciparum malaria in the Brazilian Amazon. Am J Trop Med Hyg 45: 492–497.[Abstract/Free Full Text]
  33. Basco LK, Eldin de Pecoulas P, Wilson CM, Le Bras J, Mazabraud A, 1995. Point mutations in the dihydrofolate reductase-thymidylate synthase gene and pyrimethamine and cycloguanil resistance in Plasmodium falciparum. Mol Biochem Parasitol 69: 135–138.[ISI][Medline]
  34. Nzila-Mounda A, Mberu EK, Sibley CH, Plowe CV, Winstanley PA, Watkins WM, 1998. Kenyan Plasmodium falciparum field isolates: correlation between pyrimethamine and chlorcycloguanil activity in vitro and point mutations in the dihydrofolate reductase domain. Antimicrob Agents Chemother 42: 164–169.[Abstract/Free Full Text]
  35. Khalil I, Ronn AM, Alifrangis M, Gabar HA, Satti GM, Bygbjerg IC, 2003. Dihydrofolate reductase and dihydropteroate synthase genotypes associated with in vitro resistance of Plasmodium falciparum to pyrimethamine, trimethoprim, sulfadoxine, and sulfamethoxazole. Am J Trop Med Hyg 68: 586–589.[Abstract/Free Full Text]
  36. Francis D, Nsobya SL, Talisuna A, Yeka A, Kamya MR, Machekano R, Dokomajilar C, Rosenthal PJ, Dorsey G, 2006. Geographic differences in antimalarial drug efficacy in Uganda are explained by differences in endemicity and not by known molecular markers of drug resistance. J Infect Dis 193: 978–986.[ISI][Medline]
  37. Pradines B, Mabika Mamfoumbi M, Parzy D, Owono Medang M, Lebeau C, Mourou Mbina JR, Doury JC, Kombila M, 1998. In vitro susceptibility of Gabonese wild isolates of Plasmodium falciparum to artemether, and comparison with chloroquine, quinine, halofantrine and amodiaquine. Parasitology 117: 541–545.
  38. Holmgren G, Gil JP, Ferreira PM, Veiga MI, Obonyo CO, Bjorkman A, 2006. Amodiaquine resistant Plasmodium falciparum malaria in vivo is associated with selection of pfcrt 76T and pfmdr186Y. Infect Genet Evol. 6: 309–314.[ISI][Medline]
  39. Wellems TE, Plowe CV, 2001. Chloroquine-resistant malaria. J Infect Dis 184: 770–776.[ISI][Medline]
  40. Van Hensbroek MB, Morris-Jones S, Meisner S, Jaffar S, Bayo L, Dackour R, Phillips C, Greenwood BM, 1995. Iron, but not folic acid, combined with effective antimalarial therapy promotes haematological recovery in African children after acute falciparum malaria. Trans R Soc Trop Med Hyg 89: 672–676.[ISI][Medline]
  41. Watkins WM, Mberu EK, Nevill CG, Ward SA, Breckenridge AM, Koech DK, 1990. Variability in the metabolism of proguanil to the active metabolite cycloguanil in healthy Kenyan adults. Trans R Soc Trop Med Hyg 84: 492–495.[ISI][Medline]
  42. Foote SJ, Kyle DE, Martin RK, Oduola AM, Forsyth K, Kemp DJ, Cowman AF, 1990. Several alleles of the multidrug-resistance gene are closely linked to chloroquine resistance in Plasmodium falciparum. Nature 345: 255–258.[Medline]
  43. Babiker HA, Pringle SJ, Abdel-Muhsin A, Mackinnon M, Hunt P, Walliker D, 2001. High-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 Pfmdr. J Infect Dis 183: 1535–1538.[ISI][Medline]
  44. Nagesha HS, Din-Syafruddin, Casey GJ, Susanti AI, Fryauff DJ, Reeder JC, Cowman AF, 2001. Mutations in the pfmdr1, dhfr and dhps genes of Plasmodium falciparum are associated with in-vivo drug resistance in West Papua, Indonesia. Trans R Soc Trop Med Hyg 95: 43–49.[ISI][Medline]
  45. Von Seidlein L, Duraisingh MT, Drakeley CJ, Bailey R, Greenwodd BM, Pinder M, 1997. Polymorphisms in the pfmdr1 gene and chloroquine resistance in Plasmodium falciparum in the Gambia. Trans R Soc Trop Med Hyg 91: 450–453.[ISI][Medline]
  46. Basco LK, Ringwald P, 1997. pfmdr1 gene mutation and clinical response to chloroquine in Yaounde, Cameroon. Trans R Soc Trop Med Hyg 91: 210–211.[ISI][Medline]
  47. Haruki K, Bray PG, Ward SA, Hommel M, Ritchie GY, Chloroquine resistance of Plasmodium falciparum: further evidence for a lack of association with mutations of the pfmdr1 gene. Trans R Soc Trop Med Hyg 88: 694.
  48. Pillai DR, Labbe AC, Vanisaveth V, Hongvangthong B, Pomphida S, Inkathone S, Zhong K, Kain KC, 2001. Plasmodium falciparum malaria in Laos: chloroquine treatment outcome and predictive value of molecular markers. J Infect Dis 183: 789–795.[ISI][Medline]
  49. Duraisingh MT, Jones P, Sambou I, von Seidlein L, Pinder M, Warhurst DC, 1999. Inoculum effect leads to overestimation of in vitro resistance for artemisinin derivatives and standard antimalarials: a Gambian field study. Parasitology 119: 435–440.
  50. Price RN, Uhlemann AC, Brockman A, McGready R, Ashley E, Phaipun L, Patel R, Laing K, Looareesuwan S, White NJ, Nosten F, Krishna S, 2004. Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number. Lancet 364: 438–447.[ISI][Medline]
  51. Wilson CM, Volkman SK, Thaithong S, Martin RK, Kyle DE, Milhous WK, Wirth DF, 1993. Amplification of pfmdr1 associated with mefloquine and halofantrine resistance in Plasmodium falciparum from Thailand. Mol Biochem Parasitol 57: 151–160.[ISI][Medline]
  52. Mu J, Ferdig MT, Feng X, Joy DA, Duan J, Furuya T, Subramanian G, Aravind L, Cooper RA, Wootton JC, Xiong M, Su XZ, 2003. Multiple transporters associated with malaria parasite responses to chloroquine and quinine. Mol Microbiol 49: 977–989.[ISI][Medline]




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