Background
Malaria in pregnancy (MiP) is a significant public health problem, with substantial adverse effects on both mother and fetus, including maternal anaemia, fetal loss, premature delivery, intrauterine growth retardation, and delivery of low birth-weight infants, which is a risk factor for death [
1‐
4]. MiP current control measures used in most endemic countries, according to WHO recommendations, include the use of insecticide treated nets (ITNs), intermittent preventive treatment with sulfadoxine-pyrimethamine (IPTp-SP) and effective case management of malaria, which, since 2010, includes the use of artemisinine-based combination therapy (ACT) [
5,
6].
Current efforts of malaria control during pregnancy rely mostly on the effectiveness of anti-malarial drugs used for both IPTp and case management. Indeed, although there are other limiting factors, including low attendance rate of antenatal services [
7‐
10], low coverage and compliance to the preventive treatment by pregnant women [
11‐
13] and inadequate protection of fewer than three SP doses where malaria transmission is intense [
12,
14], the major hindrance of the effectiveness of IPTp-SP policy is the spread of
Plasmodium falciparum resistance to SP [
15‐
20]. Given the number of studies reporting the increased resistance of
P. falciparum to SP, there has been several responses: (i) the increase of IPTp-SP doses by WHO in 2012 [
21]; (ii) the evaluation of alternative drugs to SP [
22,
23]; and, (iii) the assessment of alternative or improved strategies to IPTp-SP [
24]. However, none of the alternative drugs or strategies tested has prompted the replacement of the IPTp-SP policy [
25,
26]. Reducing the burden of malaria during pregnancy in high transmission setting remains challenging.
Since 2015, WHO recommends the use of ACT for the treatment of
P. falciparum uncomplicated malaria during the second and third trimester of pregnancy [
27,
28], and such recommendation has already been adopted and implemented by all sub-Saharan African countries [
29]. However, there is limited knowledge on the effect of ACT, treatments such as artemether-lumefantrine (AL), on the selection of
P. falciparum resistance markers during pregnancy that can affect the treatment outcome. Indeed, available information in non-pregnant women have shown that the
P. falciparum multidrug resistance 1 (
pfmdr1) N86 and D1246 alleles might be associated with AL resistance [
30,
31]. In addition, the combination of N86, 184F and D1246, forming the ‘NFD’ haplotype, led to a decreased susceptibility to AL and treatment with AL selects for such a haplotype [
32‐
34]. There is therefore a need for pharmacovigilance studies to monitor any delayed parasite clearance by AL and to assess risk factors associated with the carriage of
P. falciparum resistance markers.
Between 2013 and 2016, a multi-centre, cluster-randomized, controlled trial (COSMIC) was conducted in three West African countries with high (Burkina Faso, Benin) and low (The Gambia) malaria transmission, to assess the protective efficacy of adding community-scheduled screening and treatment of malaria during pregnancy (CSST) to standard IPTp-SP (CSST/IPTp-SP) [
35,
36]. The CSST/IPTp-SP strategy was based on monthly active follow-up by community health workers using rapid diagnostic tests (RDTs). The aim of the combined CSST/IPTp-SP strategy was to provide an opportunity to detect and treat malaria infections during pregnancy with AL and reduce the prevalence of placental malaria [
35]. As part of the COSMIC trial, it has shown a high prevalence of the triple
dhfr mutation with presence of quintuple mutants (triple
dhfr and double
dhps) in Burkina Faso, confirming concerns about the efficacy of IPTp-SP in the near future [
37]. This study aimed to determine the prevalence and factors associated with the carriage of
pfmdr1 polymorphisms (
pfmdr1 N86Y7, Y184F, D1246Y) among pregnant women within the COSMIC trial in Burkina Faso.
Discussion
Despite the widespread implementation of IPTp-SP to prevent MiP, pregnant women in endemic countries often experience peripheral and/or placental malaria infection at delivery [
15,
39,
40]. Although WHO revised IPTp-SP guidelines and increased the SP dose, which has been shown to improve birth outcomes [
41,
42], IPTp-SP strategy is still threatened by increasing
Plasmodium falciparum resistance to SP. Consequently, there is a need to develop new alternative or improved strategies as part of IPTp-SP policy. In line with the latter, CSST of MiP, in addition to standard IPTp-SP (CSST/IPTp-SP), was tested in Burkina Faso, Benin and The Gambia as an intervention to improve maternal health and birth outcomes in areas of different malaria transmission intensity (COSMIC trial, NCT1941264) [
35]. In such a context, the current study was conducted to determine prevalence and factors associated with the carriage of
pfmdr1 polymorphisms among pregnant women participating in the COSMIC trial in Burkina Faso.
Mutations in the gene-encoding
pfmdr1 are known to be associated with aminoquinoline resistance [
43], and therefore represent key
P. falciparum markers for monitoring resistance in both susceptible groups (children under 5 years and pregnant women) and the general population. In this study, the analysis was focused on: (i) mutations in
pfmdr1 N86Y and D1246Y codons, which have been associated with resistance to chloroquine and amodiaquine [
44‐
46], whereas wild-type sequences in these alleles were associated with reduced sensitivity to lumefantrine [
44,
47,
48]; and, (ii) mutation in the
pfmdr1 Y184F codon, which was associated with altered sensitivity to artemisinins and mefloquine [
49]. A prevalence of 13.2 and 12.1% of the
pfmdr1 86Y mutant allele was found at ANC-1 and at delivery, respectively. No mutant allele was observed for
pfmdr1 Y184F and
pfmdr1 D1246Y codons at both ANC-1 and at delivery. The observed prevalence of these mutations at positions 86, 184 and 1246 in this study lack comparable data in the country as previous studies differ with regard to study population (children
vs adults
vs pregnant women),
Plasmodium falciparum isolates (clinical
vs asymptomatic infections), and study periods [
50‐
53]. However, looking at available reports in the study area, these results showed a higher prevalence of the
pfmdr1 86Y mutant allele in pregnant women at ANC-1 (13.2%) compared to that in patients with uncomplicated malaria two years before (pre-treatment prevalence of 8.3% in 2010–2012) [
50]. An early study conducted by the time of adoption of ACT as a first-line treatment for uncomplicated falciparum malaria in the country (2005), reported a higher prevalence of
pfmdr1 86Y mutant allele (35–40%) in children aged 6–59 months with uncomplicated malaria [
51]. Surprisingly, no
pfmdr1 184F mutant allele was detected among the study population while a prevalence of about 50% was reported among
P. falciparum uncomplicated malaria patients in 2010–2012 [
50]. In the same study, only three
pfmdr1 1246Y mutant alleles were detected in 660 isolates, corresponding to a prevalence of 0.4%.
It has been shown that wild-type sequences of
pfmdr1 N86Y, Y184F and D1246Y codons are selected by prior use of AL treatment in malaria patients [
47,
48,
51‐
53]. By contrast, selection of the
pfmdr1 184F mutant allele has been observed in prior therapy with AL in malaria patients in Uganda [
47]. To explore the potential selection of wild-type/mutant sequences of
pfmdr1 polymorphisms following AL treatments during pregnancy, the prevalence of
pfmdr1 86Y mutant alleles at ANC-1 was compared to that at delivery. No significant difference of the
pfmdr1 86Y mutant allele was found neither in the general study population (
P = 0.77) nor in sub-groups represented by women infected both at ANC-1 and at delivery (P = 0.25), women who received the standard IPTp-SP (
P = 0.63) and women who benefitted from additional screening and treatment of
P. falciparum infections (asymptomatic infections) using AL (
P = 0.38).
To further assess the potential selection of
pfmdr1 N86Y mutant/wild-type alleles by specific factors, factors associated with
pfmdr1 86Y mutant allele carriage at ANC-1 and at delivery were investigated. Among the variables evaluated, none was significantly associated with
pfmdr1 86Y mutant allele carriage at ANC-1. However,
Plasmodium falciparum infection at delivery with high parasitaemia was significantly associated with nearly 5.5 times increase risk of
pfmdr1 86Y mutant allele carriage at delivery after adjusting by confounding factors (
P = 0.04). By contrast, uptake of at least three IPTp-SP doses and at least one AL treatment were found to be significant protective factors against
pfmdr1 86Y mutant allele carriage at delivery in multivariate analyses (75% reduction for both with
P = 0.04 and
P = 0.03, respectively). These results suggest a benefit in reducing the risk of aminoquinoline resistance marker carriage by high coverage of IPTp-SP doses, which has been shown to reduce parasite load in
P. falciparum infection during pregnancy [
54,
55]. Moreover, these findings suggest a positive selection of
pfmdr1 N86 wild-type allele at delivery following AL treatment during pregnancy in women receiving IPTp-SP, similar to that reported in non-pregnant women in Southeast Asia (Thailand) and East Africa (Kenya) [
30,
31] and in patients of all age groups in West Africa (Burkina Faso) [
50].
Studies have demonstrated that recent AL use has more of an impact on
pfmdr1 N86 wild-type allele prevalence than less recent AL use, as lumefantrine levels decline over time, resulting in less selection [
56,
57]. On the other hand, it has been shown that AL could select for
pfmdr1 N86 wild-type allele a few months post-treatment in children [
58]. This could be explained by genetic variations or other factors including the administration of AL with fatty foods, leading some individuals to exhibit longer artemether or lumefantrine half-lives than other, allowing longer periods of selection [
58,
59]. Although, no evidence of an impact of the timing of AL treatment to delivery on the selection of
pfmdr1 N86 wild-type allele was observed in this study, the existence of a specific selective window in pregnant women should not be ruled out given the limited number of
pfmdr1 86Y mutant allele carriers. In addition, the sub-group of women who experienced malaria infection both at ANC-1 and at delivery did not show a significant association with
pfmdr1 86Y mutant allele carriage at delivery and no significant difference was found for triple
dhfr 51/59/108 mutations carriage in this sub-group compared to those infected only at delivery (
P =
0.6), suggesting new infection parasite population after IPTp-SP and eventually AL treatments. In this regard, the lack of
Plasmodium falciparum genotyping to distinguish recrudescent parasites to new infection parasites could be seen as another limit of this study. Future investigations on factors associated with
pfmdr1 gene polymorphisms selection in pregnant women living in endemic countries should include large sample size and parasites population genotyping.
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