JAC Advance Access published online on August 5, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn299
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Original research |
Accumulation of CVIET Pfcrt allele of Plasmodium falciparum in placenta of pregnant women living in an urban area of Dakar, Senegal
1 Unité d’Immunologie, Institut Pasteur de Dakar, Sénégal 2 Unité Immunologie Moléculaire des Parasites, CNRS URA 2581, Institut Pasteur de Paris, France 3 Hôpital Roi Baudouin de Guediawaye, Dakar, Sénégal 4 Unité mère-enfant, Institut Recherche et Développement, Dakar, Sénégal
* Corresponding author. Present address. Vascular Immunology Unit, Faculty of Medicine, University of Sydney, Medical Foundation Building (K25), 92-94 Parramatta Rd, Camperdown, NSW 2042, Australia. Tel: +61-2-9036-3222; Fax: +61-2-9036-3177; E-mail: rjambou{at}med.usyd.edu.au
Received 22 November 2007; returned 26 February 2008; revised 10 June 2008; accepted 29 June 2008
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Abstract |
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Objectives: Chemoprophylaxis is recommended during pregnancy to reduce the risk of placental infection. However, in areas with increasing drug resistance, it can trigger selection of resistant parasites in the placenta and increase the frequency of placental malaria. The objective of this study was to analyse the selection of drug-resistant parasites in the placenta in an area where chloroquine was still recommended as prophylaxis.
Patients and methods: We analysed the polymorphism of parasites from matched placental and venous blood samples at the time of delivery from women in Dakar. Polymorphism of the isolates was studied using nested PCR typing of MSA1 and MSA2 genes, and full sequence of PfCRT exon 2.
Results: Of 692 women recruited at delivery, 72 had placental malaria. Two Pfcrt exon 2 genotypes were found, and 86% of the placentas had monoallelelic CVIET infection compared with 39% that had peripheral blood infection. Mixed parasite populations of CVIET/CVMNK occurred in 53% of the peripheral blood samples but only in 7% of the infected placentas. This selection of CVIET in placenta was not related to a decreased polymorphism of the parasites, as a large diversity of MSA1 and MSA2 was found in both placenta and venous blood. This diversity confirms that a multiplicity of circulation isolates can occur at low parasite transmission. msp1 and msp2 genotyping revealed mostly distinct populations of parasites in venous and placental blood.
Conclusions: These data suggest that, even in low transmission areas, diverse parasite populations can accumulate in the placenta during pregnancy despite strong selection at the PfCRT locus due to chemoprophylaxis with chloroquine.
Key Words: pregnancy , malaria , P. falciparum , chloroquine resistance
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Introduction |
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In endemic regions, pregnant women are subjected to an increased risk of Plasmodium falciparum malaria.1,2 A transient depression of immunity during pregnancy and a specific adherence of infected red blood cells to placenta-specific receptors account for the development of a long-lasting infection of the placenta, which can trigger premature delivery and result in reduced birth weight and stillbirth.1–3 This accumulation of parasites in the placenta is partially explained by the binding of the parasite to syncitio-trophoblasts via the adhesin varCSA, a variant of the PfEMP1 family that binds to CSA on trophoblastic cells. However, antibodies against varCSA can be acquired after the first malaria attack during pregnancy,4 which can explain the decrease of the placental parasite load with the number of pregnancies. Despite this mechanism of accumulation, in Sahelian areas where the rainy season is short and the transmission rather low, the risk of infection and the incidence of placental malaria should also be low, especially for women using chemoprophylaxis. However, this was not observed,5 especially in the Dakar area.3 To explain this unexpectedly high prevalence of placental malaria, several hypotheses can be proposed. Previous studies reported high diversity of parasites during pregnancy and this was poorly related to the level of transmission, which can allow parasites to avoid both immunity and intra-host selection of subpopulations of parasites. The use of chemoprophylaxis by pregnant women could also trigger the selection of drug-resistant parasites in the placenta.
To test these hypotheses, our study analysed the polymorphism of parasites from matched placenta and venous blood samples collected at delivery in women from the urban areas of Dakar (Senegal), where transmission of malaria only occurs during the rainy season. Antigenic diversity of the parasites was addressed using standard msp1 block 2 and msp2 nested PCR.6 Selection of resistant isolates in placenta was addressed by sequencing analysis of the mutations in exon 2 of PfCRT7 in parasites from venous blood and placental samples.
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Study area and patients
Samples were collected, as described elsewhere,3 during 2000 and 2001, at the maternity ward of Guediawaye, a suburb of Dakar subject to a seasonal and hypo-endemic malaria transmission. Women registered for this study were residents of the town and declared no travel outside the area during the previous 2 months. For all women giving informed consent, placental blood was tested after delivery using an immuno-chromatography test (ICT) (P. falciparum HRPII, Sorachim Ltd). All the women with an ICT-positive placenta (positive group) were sampled for venous blood. Term of delivery was defined using uterus measurement.
During the time of the study, a 600 mg/week chloroquine uptake was still recommended by the national health policy in Senegal for chemoprophylaxis in pregnancy. However, during the same period, we conducted in vivo (n = 200) and in vitro drug sensitivity studies (n = 300) among adults and children (M. N. and R. J., unpublished data), which identified 25% chloroquine treatment failure and 40% in vitro resistant strains. The use of prophylaxis and bednet was determined for each woman through oral interrogation by members of the team, using a standard questionnaire in Wolof (a local dialect). Precise answers on (i) the drug and the dose used, (ii) the weekly time of uptake and (iii) the price of the dose were considered evidence for a high probability of chloroquine use.
All aspects of the study were approved by the National Ethics Committee (NEC) of the Ministry of Health. All the enrolled women gave their informed consent for the protocol. In accordance with the NEC, the HIV status of the participants was not established, but previous studies showed that the seroprevalence of HIV in pregnant women in Dakar was very low (1.2%).8
For each woman with a positive ICT, 5 mL of peripheral venous blood was collected in EDTA. Anaemia was defined as <10 g/100 mL of haemoglobin on full blood count. Placentas were collected at delivery, maintained at 4°C and transported to the laboratory for processing within 4 h. Pieces of the placentas were frozen with and without previous fixation (paraformaldehyde 4%). Peripheral and placental parasitaemia were determined by microscopic examination of Giemsa stained blood smears or tissue sections and expressed as the number of parasites counted per 100 leucocytes. Placental infections were classified on cryosection, as described previously,9,10 into (i) acute infection (AI), when only infected erythrocytes (IEs) were detected in placental intervillous spaces; (ii) chronic infection (CI), when IEs and haemozoin were both detected; (iii) past infection (PI), when only haemozoin was detected in placental tissue; and (iv) non-parasitized, when neither IEs nor haemozoin were detected in the placental sections, but with an HRPII+ test.
DNA extraction from blood samples was carried out as previously described.6 For placenta, 200 mg of frozen tissues was ground to a fine powder in a mortar with liquid nitrogen, and homogenized at 50°C for 12 h in an extraction buffer.11 DNA was extracted using standard phenol–chloroform procedures, precipitated with ethanol and resuspended in water.
Pfcrt exon 2 was sequenced as previously described12 and chromatographs were analysed using Seqscape software (Applied Biosystems) to allow detection of multiple infections. msp1 block 2 and msp2 diversity were identified by family-specific nested PCR,6,13 as recommended for drug sensitivity studies. These two loci, extensively used as molecular markers for epidemiology studies, are not linked but submitted to immune pressure. The size of the amplicon was determined on a 2% agarose gel in Tris Acetate EDTA buffer, using standard molecular weight markers and polynomial regression (Taxolab® package, Grimont, Institut Pasteur). Placental and peripheral blood samples were simultaneously processed in single blind. For graphic representation, PCR products were grouped into ‘alleles’ according to their size, using a 10 bp class range for msp1 block 2 and a 32 bp class range for msp2, as described by Eisen et al.14 Comparison of parasite populations in the paired placental and peripheral blood was performed based on the size of the PCR products. For PCR products amplified with the same primer pair, a difference in size of <1% was considered non-significant.
Differences between the two groups were compared with a Mann–Whitney U-test (MW), or a 
2 test. Differences between more than two groups were compared by the Kruskal–Wallis test (KW). Correlation was calculated using the Spearman R test. Alpha risk was set at 0.05.
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Clinical and parasitological data
During the rainy season, 692 pregnant women were registered in our study of whom 72 had HRPII-positive placenta (Table 1). Epidemiological data are discussed in detail in Sarr et al.3 In brief, 35% were primigravidae, 14% presented with fever and 90% declared having used chloroquine as prophylaxis throughout their pregnancy. However, based on the questionnaire, only 51% showed convincing evidence of adequate chloroquine consumption. This low rate of primigravidae among infected women can be related to the short period of annual malaria transmission in the study area, which decreases the risk of malaria during the time of pregnancy and spreads it across gravidities. There was no correlation between the weight of the newborns and the age of the mother. Sixty percent of the women were anaemic and 25% had more than 10 000 leucocytes/µL. The percentage of women with observable parasites (in placenta and/or in peripheral blood) was maximal between the ages of 20–30 years. For women with HRPII+ placenta, parasites were detected in 84% of the placentas and in 81% of the peripheral blood samples (20% harbouring more than 5000 parasites per µL of blood). The mean parasite count in placenta decreased with the age of the mother and the number of pregnancies (data not shown). CIs were more frequent than AIs: 31 placentas (43%) were classified as having CI and 16 (22%) AI. Prevalence of CI decreased with parity (1–2 versus >2 pregnancies, 2 P = 0.01) and with age (>20 versus <20 years, 2 P = 0.03). Parasitaemia was significantly higher in AI than in CI (MW P = 0.001) in all the samples (peripheral and placental tissue) (Table 1). On sections of the AI placenta, it was found that 89% of the parasites were at late stages, 17% of which were stacked onto the cytotrophoblast. Haemozoin alone was detected in 12 placentas (PI 17%). No mature gametocytes were found in either placenta or in blood at the time of delivery. However, this is not surprising, as detection of gametocytes stage 1–4 is quite difficult on histological sections without a specific antibody.
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Pfcrt alleles
The full sequence of Pfcrt exon 2 was obtained for 141 samples (72 peripheral blood samples and 69 placentas). Codons 74, 75 and 76 were I or M, E or N and T or K, respectively, and all other exon 2 codons were identical to strain 3D7. Resistance mutations (74I, 75E and 76T) were the most prevalent in all samples. In samples with a single allele (from placenta and venous blood, n = 98), only two haplotypes were found, i.e. CVMNK (n = 11) and CVIET (n = 87). In 43 sequences, there was ambiguity at the 74–76 positions, with both allelic forms detected at each of the positions, consistent with the presence of a mix of CVMNK and CVIET haplotypes (Figure 1a). Partly due to this mixed infection in venous blood, no significant difference was found in the frequency of parasites carrying the resistant threonine codon at position 76 of the pfcrt sequence between venous or placental blood (92% and 93%, respectively) (Figure 1a). However, the proportion of infections harbouring wild-type lysine (K) codon at position 76 was significantly higher in the periphery than in the placenta (42 of 69 individuals harboured 76K in the periphery, compared with 10 of 59 in the placenta; odds ratio of 9.18, 95% CI 3.78–23.3, P < 0.001; Table 2). This underlines a significantly less common wild-type allele in placental isolates.
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Mixed genotypes in venous blood were more prevalent in multiparous than in primigravidae women (
2 P < 0.01) and were associated with anaemia (haemoglobin <10 g/dL, 2 P < 0.01). Women with mixed genotype had a higher rate of late or CI of the placenta than others (44% versus 16%, respectively, 2 P < 0.001). Chemoprophylaxis compliance was clearly associated with a decrease of CVMNK haplotype in the placenta (frequency 7% versus 21%), but not in venous blood (frequency 65% versus 63%).
Comparison of the exon 2 Pfcrt sequence in 69 paired peripheral/placental blood samples (Table 2) showed a perfect match between the single allele observed in both tissues in only 26 cases (38%). Totally discordant peripheral and placental Pfcrt genotypes were observed in only three (4.3%) of the paired samples. The other 39 paired samples (57%) showed a partial overlap, with a single allele found in one sample and both alleles detected in the paired one. In 34 of 39 cases, a single mutated allele was detected in the placenta, and a mix of mutated and wild-type was found in peripheral blood.
Comparison of the PCR products was based on the size of the product, which underestimates the polymorphism of the sequences (Table 3). The three K1, Mad20 and R033 family types were observed, with 14, 8 and 1 distinct allele, respectively, and an average of 1.9 alleles per woman. The two FC27 and 3D7 msp2 families were present, with six and seven distinct alleles, respectively, and 1.5 msp2 alleles per sample. Thus, despite the low transmission rate, the polymorphism of the populations of parasites was high (Figure 1a and b).
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The multiplicity of infection (MOI, Figure 1c and d) detected by msp1 PCR was higher than by msp2 PCR (MW P = 0.01), and was higher in placental than in peripheral blood (MW non-significant). For msp1 the MOI was positively correlated with the parasite density in peripheral or placental blood (KW P < 0.001). The mean number of alleles per sample for msp1 and msp2 was not related to drug prophylaxis, fever, term, age of the mother, parity or the type of placental infection. Thus, difference in MOI between venous blood and placenta cannot explain the decrease of polymorphism of PfCRT in the placenta.
In the same way, genetic differences were found for MSA1 subfamilies (2 P = 0.04) and within each subfamily, between sequestered and venous isolates (Figure 2). Two K1-type alleles accounted for 60% of all msp1 PCR products in the venous blood samples, whereas two dominant MAD20-type alleles were found in the placental samples. For MSA2 3D7-type, only one allele was prevalent in the peripheral blood. Moreover, if we compare matched placental and venous blood (Table 3) from the same woman (n = 54 pairs), 61% presented the same PCR profile without any difference on size and subtype of the PCR products for msp1, but only 28% had the same profile for msp2. On the whole, only one-third of the parasites were shared by placental and venous samples, without relation to the age, parasitaemia or parity (KW test). These data confirm that, for the same woman, populations of parasites are different for the placental and venous blood compartments, as previously suggested,13 supporting the hypothesis of selection of parasites in the placenta.
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Discussion |
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According to UNESCO, in the next 15 years, half of the African population will live in urban areas, which will modify the clinical expression of numerous diseases like tuberculosis, HIV and malaria. This change of setting will induce changes both in the immune response of the patients and in the physiopathology.15 However, in West Africa, this estimation hides a great heterogeneity. In Senegal, 30% of the population is concentrated in only 3.6% of the territory16 (regions of Thiès and Dakar). These regions are highly urbanized,17 and a low transmission of malaria occurs during the rainy season, affecting a non-immune population. This population can easily access medical facilities, but adults usually prefer to purchase low quality drugs at street markets, which sustains propagation of drug resistance. In this context, pregnant women are briefly exposed to the risk of malaria and can easily attend dispensaries that provide good quality follow-up of the pregnancy. So why does placental malaria remain so prevalent? In our study, we found that more than 10% of the women enrolled were infected. This shows that placental malaria is still a major problem for the Malaria National Control Programme policy, with more than 5000 pregnant women infected each year in the area of Dakar. At the time of the study, weekly uptake of chloroquine was still recommended during pregnancy. However, despite the low price of this drug, our estimation was that no more than 51% of the women had used it properly. Detection of the drug in blood, which was not possible during this study, would have most likely lowered this score.
This study shows that a very large fraction (93%) of the placental parasites harboured a Pfcrt K76T mutation and that most of them (88%) had a single CVIET Pfcrt allele. In the venous blood samples, half of the women had mixed infection, with both wild and mutated parasites, but 39% had a single mutated population of parasites. Sequencing is not the most sensitive method to detect a mixed infection, but is as sensitive as PCR_RFLP (parasites occurring with a frequency of <10% of the total are usually not detected). However, our data clearly support the selection of chloroquine resistance genotypes in the placenta. When taking into account the actual MOI (e.g. at least two clonal types per placenta as detected by msp1/msp2 typing), the adjusted prevalence of the resistance mutation becomes 89.1% in the placenta and only 65.3% in the peripheral blood.
Only two haplotypes were clearly detected: CVMNK (the wild-type, chloroquine-sensitive allele) and CVIET (the South-east Asian chloroquine-resistant allele).7,18 This is also consistent with the observation that most alleles with a key 76T mutation observed so far in Africa harbour this specific South-east Asian CVIET haplotype.18,19 However, other haplotypes were recently described in the Central African Republic,20 in Madagascar (CVIDT),21 in the Congo (SVIET)22 and in Tanzania (SVMNT).23 None of these (particularly neither 75D nor 72S) were observed in our samples or in an additional 96 samples sequenced from the same area (data not shown). Thus, our data indicate efficient invasion of the local parasite population by the resistant allele of South-east Asian origin, which has spread across Africa.18,19
Previous observations in another Senegalese setting also pointed to the selection of chloroquine resistance genotypes in pregnant women under chloroquine prophylaxis.24 This study on venous blood, performed during pregnancy, showed a higher prevalence of Pfcrt 76T mutants in chloroquine-treated women than in asymptomatic untreated controls.24 Our data demonstrated an accumulation of the mutated parasites in placenta, which suggests that previous results were an underestimation.
msp1 and msp2 genotyping showed that placental and venous blood samples had mostly distinct populations of parasites. This partial overlapping has already been observed in a group of pregnant women recruited in the same setting 3 years before this study.25 Recently, Jafari-Guemouri et al.,26 using a different technique, reported a much higher MOI in placental and peripheral blood. However, msp genotyping is considered (especially by WHO) as the international standard and clearly confirms that dominant parasite populations differ between placental and venous blood, thus supporting cryptic infection of placenta by cytoadherence parasites.28
Based only on the polymorphism of size, 23 and 13 distinct msp1 and msp2 types were identified, which suggests a large diversity of the parasites at the molecular level.27 Each pregnant woman harboured an average of two alleles for each locus, a slightly lower figure than that observed a few years before in the same area.25 This polymorphism can be due to the input of isolates from the countryside,29,30 subsequently maintained by local transmission31–33 in urban farming areas.
Several hypotheses can be formulated to explain the accumulation of parasites with a chloroquine resistance genotype in the placenta. This could be due to the history of infection of the women: several infections could have occurred during pregnancy, but only resistant parasites could have survived the chemoprophylaxis. On the other hand, parasites detected at the time of delivery in the peripheral venous blood could be related to the latest infection experienced by the women. Absence of linkage disequilibrium of the Pfcrt resistance allele with the msp1 block 2 and/or the msp2 locus does not support accumulation in the placenta of one or more chloroquine-resistant strains circulating in this area of Dakar. Placental infection seems due to colonization by numerous diverse strains sharing a mutated Pfcrt locus. This observation contrasts with one of the reports,34 but is consistent with a multitude of data supporting chloroquine-driven genetic sweep of the Pfcrt locus.18,19 Finally, selection of resistant parasites in the placenta could be due to a higher concentration, or slower decay, of chloroquine in this organ (as suggested by pharmacokinetic data35) or in the foetus.36–39
In conclusion, our results argue that chloroquine prophylaxis in pregnant women is associated with preferential selection of chloroquine-resistant parasites in the placenta. As a consequence, drug-resistant infections in pregnant women may remain unnoticed and the efficacy of chemoprophylaxis misinterpreted if only studies on peripheral blood are conducted. Molecular typing of drug target genes in placental parasites at the time of delivery should be a sensitive indicator for drug policy and a useful additional tool for monitoring the efficacy of new strategies as preventive intermittent treatment.40
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This programme was supported by the Louis D. Foundation of the National Academy of Sciences, Paris, the UE ‘RESMAL-Chip’ programme and the FSP-RAI programme of the French Foreign Office. D. S. was supported by a grant from FSP-RAI.
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None to declare.
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Footnotes |
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Present address. School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551 Singapore, Singapore. 
Present address. Institut Pasteur de guadeloupe B.P. 484, 97165 Pointe à Pitre Cedex, France.
¶ Present address. Unité d’Immunologie Moléculaire des Parasites, Institut Pasteur Paris, 25 Rue du Dr Roux, 75015 Paris, France.
Present address. Dispensaire Roi Baudouin de Guédiawaye, Dakar, Sénégal.
|| Present address. Unité d’Epidémiologie, Institut Pasteur de Dakar, B.P. 220, Dakar, Sénégal.
# Present address. Unité de Recherche Santé de la Mère et de l’Enfant, Institut de Recherche Développement, Dakar, Sénégal.
** Present address. Department of Infectious Diseases and CTEGD, University of Georgia, 500 DW Brooks Drive, N326 Paul Coverdell Center, Athens, GA 30602, USA.
Present address. Laboratoire Anatomo-pathlogie, Institut Pasteur de Dakar, B.P. 220, Dakar, Sénégal.
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Acknowledgements |
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We thank all the medical staff of the Nursery, Roi Baudouin, for their technical support.
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References |
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