1 Institut de Recherche pour le Développement (IRD), UR010, Mother and Child Health in the Tropics, Faculté de Pharmacie, 75006 Paris, France; 2 IRD UR010, BP1386 CP 18524 Dakar; 3 Hôpital de Thiadiaye, Thiadiaye, Senegal
Received 7 July 2004; returned 5 November 2004; revised 26 November 2004; accepted 15 February 2005
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Abstract |
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Methods: The study was conducted in the rural maternity hospital of Thiadiaye, Senegal, where malaria is seasonal. Sixty-nine P. falciparum isolates from infected women were collected at delivery. These women were part of a cohort study; they were followed from their first antenatal visit and advised to take chloroquine prophylaxis. For each woman, the earliest P. falciparum-infected blood sample was also used. A control group of 49 non-pregnant individuals with asymptomatic P. falciparum infection was enrolled.
Results: During pregnancy, prevalence of T76 mutant parasites was higher than in the 49 non-pregnant controls (P <0.001). Among pregnant women, this rate was highest at delivery (P=0.06), and tended to be higher in women who had taken chloroquine prophylaxis, as assessed in urine samples (P=0.08).
Conclusions: Chloroquine prophylaxis is responsible for increased drug consumption and increased drug pressure that may lead to the selection of drug-resistant parasites. This is the first report showing that P. falciparum-infected pregnant women harbour pfcrt T76 mutant parasites more often than non-pregnant individuals, and that the prevalence of this mutation is higher at term than earlier during pregnancy.
Keywords: malaria , pregnancy , drug resistance
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Introduction |
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For a long time, WHO advocated the use of chloroquine prophylaxis in pregnant women to prevent the adverse consequences of malaria during pregnancy. Although WHO is currently modifying its policy, now advocating the use of intermittent preventive treatment, the Ministries of Health in many African countries, including Senegal, are still currently recommending that pregnant women receive chloroquine chemoprophylaxis throughout pregnancy. However, the recent increase of chloroquine-resistant P. falciparum has become a major problem in Senegal, reaching rates of 50%,2 and the Ministry of Health is considering a modification of its recommendations.
The spread of drug resistance owing to the use of malaria prevention drugs in pregnant women has never been assessed. However, the drug pressure occurring in pregnant women given chloroquine prophylaxis might induce a positive selection of chloroquine-resistant P. falciparum parasites. The effects of such prophylaxis may be even stronger given the high frequency of poor compliance.3 Most notably, an allele of the P. falciparum chloroquine-resistance transporter gene (pfcrt) encoding threonine at position 76 (pfcrt T76) has been demonstrated to be strongly linked to chloroquine resistance.4 Multiple studies in areas where falciparum malaria is endemic have demonstrated that mutant parasites presenting with the T76 point mutation in the P. falciparum chloroquine resistance transporter, encoded by the pfcrt gene, are linked to chloroquine resistance in vitro and in vivo.5,6 The aim of this study was to compare the prevalence rate of the pfcrt T76 mutation in P. falciparum-infected pregnant and non-pregnant individuals from Senegal.
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Materials and methods |
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This study was conducted in the rural maternity hospital of Thiadiaye, Senegal. Malaria is seasonally transmitted in this area, during the rainy season from August through December. Women delivering at the maternity ward between October 2001 and June 2002 were enrolled in the study, providing they presented with P. falciparum infection. Placental and peripheral blood were collected for thick smear preparation and filter paper blotting.
These women were part of a cohort study, and had been followed up from their first antenatal visit (mean pregnancy term 19.3 ± 4.2 weeks) to delivery. During each antenatal visit, women were recommended to take prophylaxis with 300 mg of chloroquine twice weekly, and blood was collected for thick smear and filter paper blot, as well as urine samples for chloroquine detection,7 to assess compliance with the prophylaxis regimen. For each woman, the first P. falciparum-infected blood sample (the earliest in pregnancy; mean pregnancy term 27.8 ± 6.4 weeks) was used for this study. Women were enrolled after informed consent was obtained. A control group of 49 non-pregnant individuals presenting with symptomless P. falciparum infection were also enrolled in the same area. Informed consent was obtained from all patients. Human experimentation guidelines of both French and Senegalese governments were followed in the conduct of clinical research. The study was approved by the ethics committee of the Ministry of Health, Senegal.
DNA extraction, PCRrestriction fragment length polymorphism (RFLP) of the pfcrt gene
Blood collected on filter papers was dried and conserved at room temperature until DNA extraction using chelex.8 The oligonucleotides primers were designed from published sequences. The lower primer was 5'-AATAAAgTTgTgAgTTTCggA-3', hybridizing from positions 280 to 300.9 The upper primer was 5'-TgTgCTCATgTgTTTAAACTT-3', hybridizing from positions 130 to 150 in the pfcrt sequence (Genbank accession number AF495378).10 The PCR components, in a final volume of 25 µL, were 1.6 mM MgCl2, 640 µM deoxynucleotide triphosphate, buffer 1x, 0.3 µM of each primer, 0.5 U of Ampli Taq polymerase (Ampli Taq Gold; Applied Biosystems, Foster City, CA, USA) and 4 µL of DNA samples. The cycling protocol was: 95°C for 7 min for initial denaturation; 40 cycles of 94°C for 30 s, 57°C for 30 s and 72°C for 30 s; and a final extension of 72°C for 10 min.
After amplification, a volume of 20 µL of PCR product was incubated overnight at 55°C with the mutation-specific restriction enzyme ApoI to detect the pfcrt K76T mutation. In the PCR products, the DNA sequence was cleaved at the wild-type codon site (if present) into two fragments (98 and 72 bp), while the mutant allele was not cut (170 bp). The digested products were separated by electrophoresis in a 2% agarose gel containing ethidium bromide, and DNA was visualized by ultraviolet transillumination. DNA fragments were compared by size and with the PCR products generated from genomic DNA of the 3D7 and W2 strains (used as references for susceptible and resistant genotypes, respectively).
Statistical analysis
The 2-test for unpaired samples (or Fisher's exact test when required) was used to test for significant differences between categorical variables. When paired analysis was required, the Bowker test, an extension of the McNemar test, was used. A P value <0.05 was considered statistically significant.
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Results |
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Genotyping of the pfcrt gene of all infected samples was achieved by PCRRFLP. During pregnancy, 47 of the 62 infected pregnant women harboured mutant parasites, 11 women wild-type parasites, while four presented with a mixed infection with both wild-type and mutant parasites (Figure 1). In the non-pregnant control group, infection with mutant, wild-type and mixed genotypes was observed in 26, six and 17 patients, respectively. Therefore, the prevalence of mutant parasites was higher in pregnant women than in controls, while that of mixed infections was lower (P <0.001).
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Discussion |
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Numerous studies have demonstrated that pregnant women are at a higher risk of suffering from clinical malaria, and drug prevention has been advocated for years, using either weekly chloroquine prophylaxis or, more recently in several countries, intermittent presumptive treatment.1 The Senegalese Ministry of Health recommends a weekly chloroquine prophylaxis for each pregnant woman from the beginning of pregnancy to delivery. One possible drawback of such increased drug consumption is an increased drug pressure that may lead to the selection of drug-resistant parasites. There is some evidence in favour of this hypothesis. Women from Thiadiaye presented a lower prevalence level of pfcrt T76 mutant parasites at an early stage of pregnancy than at delivery. Moreover, women who were assumed, by urine sample, to have taken their prophylaxis correctly presented with a higher prevalence rate of pfcrt K76T mutant parasites. This is the first report showing that P. falciparum-infected pregnant women harbour pfcrt T76 mutant parasites more often than non-pregnant individuals, and that the prevalence rate of this mutation is higher at term than earlier during pregnancy.
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Acknowledgements |
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References |
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