PFCRT and DHFR-TS Sequences for Monitoring Drug Resistance in Adzopé Area of Côte d’Ivoire After the Withdrawal of Chloroquine and Pyrimethamine

Purpose: Drug resistance is probably the greatest challenge to most malaria-control programmes. The goal of this study was to evaluate polymorphisms in parasite resistance gene markers, pfcft and dhfr, from falciparum malaria isolates collected in Adzopé City, Côte d’Ivoire in 2007. Methods: Blood samples were collected in filter paper from 72 children infected with Plasmodium falciparum living in Adzopé area. Plasmodium falciparum DNA was extracted and nested PCRs were performed using specific primers of pfcrt and dhfr-ts. During the study, chloroquine and sulphadoxinepyrimethamine (which previously were the firstand second-line treatments, respectively, for malaria in Côte d’Ivoire) were not given to the enrolled children, having been withdrawn in 2004. Results: The results revealed the presence of the mutant-type pfcrt and dhfr-ts in 51 (62.2 %) and 29 (35.4 %) samples, respectively. The mutant-type pfcrt alleles consisted of four single mutations (Met74/Asn75/Thr76) and 47 triple mutations (Ile74/Glu75/Thr76). However, the frequency distribution of mutations in dhfr-ts was 35.4 % for dhfr-Asn108, 17 % for dhfr-Ile51 and 21 % for dhfr-Arg59. Conclusion: The results of this study show high presence of chloroquine (CQ) resistance markers while for sulphadoxine-pyrimethamine (SP), a much lower prevalence was detected for the markers under study. Chloroquine remains an inadequate drug for malaria therapy in the study region. Furthermore, in spite of the official withdrawal of CQ and SP in favour of the arteminisin-based combinations (ACTs), it appears the population of this area continues to use the drugs via self-medication.


INTRODUCTION
Malaria remains a major cause of morbidity and mortality in sub-Saharan Africa [1].Worldwide malaria morbidity is estimated to be 300-500 million cases with about 1 million deaths each year, of which 90% occur in sub-Saharan Africa [2].The vast majority of deaths occur among children below five years of age and pregnant women, especially in remote rural areas with poor access to health services [3].
Malaria control is hampered by increasing spread of drug resistance.Although sulphadoxine-pyrimethamine (SP) has been abandoned as first-or second-line treatments, respectively, by most African malaria endemic countries in favour of artemisinin-based combination treatments (ACT), it is still used as intermittent preventive treatment during pregnancy (IPTp) [4].However, SP resistance has been on the rise in the past few years and, although the link between molecular markers and treatment failure has not been firmly established, at least for pregnant women, these markers should be monitored.Pyrimethamine inhibits dihydrofolate reductase (DHFR), whereas sulfadoxine inhibits dihydropteroate synthetase (DHPS) [5].
Resistance to these drugs has been associated with single nucleotide polymorphisms (SNP) in the genes encoding DHFR and DHPS [6].The key amino acid substitutions that confer falciparum malaria resistance to sulfadoxine and pyrimethamine are Ala437Gly and Lys540Glu or Ala437Gly in dihydropteroate synthase (DHPS), and Ser108Asn or Ser108Thr in dihydrofolate reductase (DHFR).In addition, dhfr mutations from Asn-51 to Ile-51 and Cys-59 to Arg-59 in association with Asn-108 were found to be associated with greater resistance to pyrimethamine [7].
In most countries, chloroquine (CQ) was, for many years, the first-line drug for malaria treatment.Unfortunately, resistance to this drug is nowadays widespread.However, in areas where drug pressure has been removed, the molecular markers linked to CQ drug resistance seem to disappear, indicating that local parasites are again sensitive to this drug [8,9].
It is, therefore, important to use molecular markers for monitoring drug resistance in a country and understand its spread as this may be important in designing strategies to guide the selection of drug resistant parasites.The spread of point mutations linked to drug resistance could be monitored by carrying out regular cross sectional surveys and then genotyping the samples from infected people.Molecular tools for the detection and identification of these SNPs, such as restriction fragment length polymorphism PCR (RFLP-PCR), are available [10].CQ resistance is associated with a mutation in the Plasmodium falciparum chloroquine resistance transporter gene (K76T) [11].This mutation is generally not isolated but is associated, depending on the geographical setting, with mutations at other codons, Cys72Ser, Met74Ile, Asn75Glu, Ala220Ser, Gln271Glu, Asn326Ser, Ile356Thr and Arg371Ile, the role of which is not well defined.
In Côte d'Ivoire, until 2003, CQ and SP were used as first-and second-line drugs, respectively, for the treatment of uncomplicated malaria.ACT replaced CQ as the recommended first-line drug.SP remains recommended for IPT among pregnant women.A few years after, it was considered important to measure the prevalence of molecular markers of chloroquine and pyrimethamine resistance.Indeed, in Malawi, a sub-Saharan country, from 1992 to 2000, the prevalence of the CQ marker gradually decreased, disappearing completely by 2001 after the withdrawal of CQ [12].
The study aimed to evaluate SP and CQ resistance in falciparum malaria isolates, following withdrawal of these drugs, in Adzopé, Côte d'Ivoire, using molecular characterization of the dhfr-ts and pfcrt genes, respectively.This city is considered as one of the sentinel sites of the national programme for malaria control.

Blood collection area
This study is a part of the cross-sectional surveys carried out in 2007 in Adzopé (lat.06°06´N and long.03°51´W), situated in the south of Côte d'Ivoire, 108 km from Abidjan, the economic capital city.The rainy season occurs from March to July (average rainfall: 1,789 mm/yr).The population was estimated at 43,821 inhabitants in 1998.Malaria is hyperendemic with no seasonal transmission.The most common vectors are Anopheles gambiae and Culex Quinquefasciatus.Plasmodium falciparum is the predominant malaria parasite.
Blood samples used in this study were collected at random from 72 children aged between 6 months and 14 years, during a cross sectional survey in Adzopé.Fingerprick samples of capillary blood were collected from the selected children onto a filter paper strip (Isocode Stix®; Schleichler and Schuell, Ecquevilly, France).The samples were dried and stored at room temperature in an individual zip-lock polyethylene bags prior to DNA extraction and PCR studies.

DNA extraction and amplification
DNA was extracted from the dried blood on each filter-paper strip by rinsing each strip with 500 µL distilled water, immersing it in 75 µL distilled water in a 0.5mL microtube and then incubating at 99 °C for 30 min [13].
However, in spite of the use of these enzymes, all the PCR products or DNA fragments from agarose gel (mixed alleles) were purified using the High Pure Kit purification product (Roche Diagnosis) and then sequenced in order to investigate additional mutations.

Quality control and Data analysis
A known DNA of P. falciparum was used as positive control in the PCR evaluation.The electropherograms of the purification PCR products sequenced by MWG-biotech (MWG-Eurofin Operon) were analyzed by using 4 peaks, Chromas Lite version 2.01 and ApE-A plasmid Editor V1.12 software.
According to the haplotypes 74, 75 and 76 of pfcrt gene investigated, the wild-type pfcrt isolate was defined by the following combination: Met74/Asn75/Lys76, while for dhfr-ts, the allelic combination Ala16/Asn51/Cys59/Ser108/I164 defined the wild-type isolates.However, for each gene, the key amino-acid mutations that confer the resistance are Lys76 for pfcrt and Ser108 for dhfr-ts, respectively.

Ethical review
The study protocol was approved by the ethics committee of the Ivorian Ministry of Public Health and all legal parents or guardians of enrolled children gave their consent for blood sampling.
Of the 82 samples of P. falciparum isolates investigated, 53 (64.6 %) had wild-type dhfrts, while 29 (35.4 %) were mutant-type dhfrts.They consisted of four single mutant-type samples (Asn51/Cys59/Asn108), nineteen double mutations distributed as follows: Ile51/Cys59/Asn108 (eight) and Asn51/Arg59/Asn108 (eleven).Six samples were triple mutant-type dhfr-ts Ile51/Arg59/Asn108 mutation (Table 2).Haplotypes 16 and 164 of the dhfr-ts in all DNA of the wild type.Any Thr108 allele in DHFR-TS was not detected in DNA fragments analyzed.The frequency distribution of the point mutations in dhfr among the mutant samples detected in the present study is shown in Figure 1.
Among the 82 samples analysed in the study, 59 (72 %) were affected by at least one point mutation in pfcrt and/or dhfr-ts genes (Table 2).Although the frequency of mutation of dhfr-ts (Asn108, 35.4%) is relatively less than that seen with CQ in the same area (62.2 %), it is still alarming considering that the use of sulphadoxine-pyrimethamine as intermittent preventive treatment in pregnancy is unavoidable because suitable alternatives are not available yet.However, the prevalence of molecular markers of malarial organisms resistant to both chloroquine and pyrimethamine represented 48.8 % of the total samples.One of the reasons for this high level of the molecular markers of CQ and SP resistance is probably the wide usage of these drugs in Côte d'Ivoire in spite of their official replacement with ACT for the treatment of uncomplicated falciparum malaria.Maybe it is too early to evaluate the sensitivity of these drugs by monitoring the molecular markers for falciparum malaria resistance.In that case, it would be necessary to wait for some years before embarking on another study of this type.

CONCLUSION
The study was the first molecular study carried out in this geographical area which is considered as one of the sentinel sites for malaria control programme of Côte d'ivoire.
The results of the epidemiological study on the prevalence of genotypes associated with drug resistance, carried out in Adzopé, showed a high presence of chloroquine resistance markers, while for SP a lower prevalence was detected.Since 2003, following the withdrawal of these antimalarials as first-and second-line drugs, respectively, the status of chloroquine and pyriméthamine-resistant parasite has not changed much in Adzopé with regard to the mutations affecting dhfr-ts and pfcrt genes.

Table 1 :
Distribution of DNA samples according to genetic profile

Table 2 :
Amino-acid residues obtained in DNA fragments in PFCRT and DHFR-TS of Plasmodium falciparum samples *The amino-acids residues transferred at different positions in PFCRT and DHFR-TS alleles are highlighted.For DHFR-TS, the haplotypes 16 and 164 were wild-type in all samples