Background
The second highest rate of global malaria prevalence is documented in the Democratic Republic of Congo (DRC) [
1]. Affecting a number of risk groups, malaria is also a major concern in pregnancy. More than half of the pregnant women at risk of malaria infection live in sub-Saharan Africa [
2]. The infection leads to maternal anaemia and low birth weight (LBW), which results in infant mortality [
2]. For this reason, the World Health Organization (WHO) recommends the administration of intermittent preventive treatment in pregnancy with sulfadoxine-pyrimethamine (IPTp-SP) [
3]. The WHO guidelines that were updated in 2013 ensure a minimum of three doses of SP in pregnancy [
3].
In the DRC, approximately 97% of the population lives in areas with stable malaria transmission that lasts for 8–12 months of the year [
4]. In the country, IPTp was adopted in 2003 and SP has been used to prevent malaria in pregnant women and the newborns. Nevertheless, IPTp coverage has been reported to be low. The percentage of women receiving at least two doses of SP only increased from 5 to 14% over 2007–2013 period [
4]. IPTp guidelines in the DRC were also revised in 2013 according to the WHO recommendations. The current target is administration of two and three doses of IPTp to a minimum of 60 and 30% of pregnant women, respectively [
4].
Increasing resistance is of concern because this can limit the antimalarial activity of SP [
5]. Previously in the DRC, SP treatment failure by day 28 was reported to be between 2 and 60%. Thus, SP was replaced by artemisinin-based combination therapy (ACT) as the first-line treatment of malaria [
2]. Point mutations in
Plasmodium falciparum dihydrofolate reductase
(Pfdhfr) and dihydropteroate synthase
(Pfdhps) genes, encoding the related DHFR and DHPS enzymes in the folate-pathway, cause resistance against pyrimethamine and sulfadoxine, respectively [
6]. Notably, SP resistance is initiated by single polymorphisms, and augmented by accumulation of mutations in
Pfdhfr and
Pfdhps genes [
7]. Five mutations have been widely reported in Africa, which are N51I, C59R, S108N in
Pfdhr; with A437G and K540E in
Pfdhps gene [
8]. In addition to K540E, A581G polymorphism in
Pfdhps has been typically detected in East Africa [
5]. Mutant
Pfdhfr‐
Pfdhps haplotypes lead to high level resistance which is associated with decreased efficiency of IPTp-SP [
5]. Moreover, the emergence of a sextuple mutant genotype, characterized with triple
Pfdhfr (N51I + C59R + S108N) and triple
Pfdhps (A437G + K540E + A581G) mutations, has been associated with reduced birth weight [
9].
So far, a number of reports have shown the gene polymorphisms associated with SP resistance in the DRC [
10‐
13]. However the molecular basis of resistance among women receiving IPTp-SP in the country remains unclear. Thus, the present study aimed to identify the point mutations in
Pfdhfr and
Pfdhps genes in
P. falciparum isolates recovered from newly delivered women who received two doses of SP prophylaxis during pregnancy in the DRC.
Discussion
In the regions where malaria is endemic, 25% of pregnant women are estimated to be infected with
Plasmodium parasites [
14]. Therefore, maintenance of IPTp-SP in those regions is crucial for protecting both pregnant women and their infants against the adverse outcomes of malaria [
3]. Considering the high prevalence of malaria infection in the DRC [
1], pregnant women also constitute a risk group for the disease.
Despite the effect of IPTp-SP on birth weight was evaluated previously in the DRC [
2], there is a lack of research addressing the molecular basis of SP resistance. Thus, the present study was carried out to identify the
Pfdhfr and
Pfdhps mutations in
P. falciparum strains isolated from 48 individuals that received two doses of SP during pregnancy.
In the study, the highest percentage (100.0%) of polymorphism in
Pfdhfr gene was noted to be S108N, which was followed by N51I (85.4%) and C59R (60.4%) (Table
1). On the contrary, I164L mutation was not detected, and this finding is compatible with those reported elsewhere [
12,
13]. The prevalence of S108N was consistent with that of a previous report (99.1%) from the DRC, however N51I and C59R rates found here differed from that study (97.9 and 80.7%, respectively) [
13]. On the contrary, the rate of C59R allele was consistent with that of another study (66%) from the DRC [
12]. Furthermore, findings of the present study support the hypothesis that pyrimethamine resistance is initiated by the codon 108 mutation, and improved by the additional polymorphisms at the other
Pfdhfr codons [
7].
In the present study, 47.9% of the isolates were found to harbour the triple
Pfdhfr mutation (C
IRNI haplotype) (Table
2). This is lower than previously reported rates of 57.8–78.2% [
11], however similar to the findings (46.2%) of another study [
10], which were both conducted in the DRC. The prevalence of C
IC
NI (37.5%) and CNC
NI (2.1%) was comparable with those reported elsewhere; however the rate of CN
RNI (12.5%) in this study was found to be relatively higher [
10]. In contrast with other studies where the rates of wild-type
Pfdhfr were reported to be 1.0–7.6% [
10‐
12], the CNCSI haplotype was not detected in the present study.
The
Pfdhps A437G mutation was found in all (100.0%)
P. falciparum isolates sequenced (Table
1). The prevalence of this polymorphism was reported to be 93.1% [
13] and 72% [
12] by former studies in the DRC. In contrast to the A437G allele, K540E in the present study was less prevalent (2.1%) than previous findings (9.5%) [
13]. An increased rate of K540E polymorphism (67%) was detected by another study in Rutshuru, the DRC [
12], however this was attributed to the close proximity of the study area to Eastern Africa where SP resistance is high [
15]. Therefore, the result presented here supports the previous studies that documented the rate of K540E to be less than 50% in the DRC [
15]. On the contrary, the rate of A581G mutation in the present study (2.1%) is different from the previous data, where it was reported to be higher than 10% in the DRC [
15]. Also, no polymorphism was detected at the codon 613, which is consistent with earlier reports [
12]. Taken together, the results presented here are in parallel with previous data which suggest that sulfadoxine resistance may be initiated by the mutations at codons 436 or 437, and augmented by the additional polymorphisms at the other
Pfdhps codons [
7].
In this study, S
GKAA carrying the single A437G mutant allele was the most prevalent (97.9%)
Pfdhps haplotype (Table
2). This result is higher than the recently documented percentage (76%) in the DRC [
5]. On the contrary, the rate of S
GEGA haplotype (437G/540E/581G) was low (2.1%) in the present study. A similar percentage (3.3%) was also noted by another study in the DRC [
16]. The present result is consistent with the studies where double- and triple-mutant
Pfdhps haplotypes were reported to be less common in Central Africa [
5]. Recently, a higher percentage (8%) of S
GEGA has been detected in Kinshasa (located in the west of the DRC), but this was lower compared to the eastern countries, Tanzania (15%) and Uganda (13%) [
5]. Of note, a study showed that S
GEGA haplotypes from the DRC and the eastern countries (Malawi and Tanzania) separated into distinct lineages. These haplotypes lead to high levels of sulfadoxine resistance, thus compromising the efficacy of SP both in pregnant women and the infants [
17]. Moreover, even the haplotypes in the DRC were found to be genetically and geographically clustered. Single-mutant haplotypes were shown to be more prevalent in the west, while the double- and triple-mutant haplotypes predominated in the east of the DRC [
16]. Considering that the study setting, Bandundu city, is situated in the west of the DRC, this can explain the reason of low rate of S
GEGA in contrast to the high number of
Pfdhps single-mutant haplotypes found in the present survey. On the other hand, unlike the former studies which reported the prevalence of wild-type
Pfdhps as high as 75.6% in the DRC [
11], no sensitive strain (SAKAA) was found in the current study.
The combination of
Pfdhfr–
Pfdhps mutant alleles generated five different haplotypes in this study. C
IRNI-S
GKAA was found to be the most prevalent haplotype (47.9%). This quadruple mutation was shown to occur at rates greater than 50% in African countries including the DRC. Also, this quadruple haplotype was associated with treatment failure [
15].
The quintuple mutation (51I/59R/108N + 437G/540E) was previously associated with SP treatment failure and detected at rates of 27.1 and 43% in the studies from the DRC [
10,
12]. Unlike those studies, here
Pfdhfr 51I/108N allele was detected in combination with
Pfdhps 437G/540E/581G in one (2.1%) isolate, which generated another type of quintuple mutant haplotype (C
IC
NI-S
GEGA). This haplotype was also documented by a previous study from Tanzania [
9]. Owing to the association between K540E and A581G mutations and worsened SP resistance in Eastern Africa [
5], the mutant strains harbouring these alleles in the DRC should be carefully considered.
The triple mutant haplotypes (C
IC
NI-S
GKAA and CN
RNI-S
GKAA) were identified in 35.4 and 12.5% of the isolates, respectively. A study from India found the AN
RNI-S
GKAA allele combination (A: alanine at codon 16 of
Pfdhfr) in 0.92% of their isolates, and related this haplotype with low-level SP resistance [
7]. Lastly, the mutant allele combination (CNC
NI-S
GKAA) was detected at low levels (2.1%) in the present study. This result is lower than that reported for double mutant haplotype (16.7%) in the DRC [
10]. Similar to the single mutant allele in
Pfdhfr or
Pfdhps, the double mutant haplotype seems to occur at the initial stage of SP resistance [
7].
In the present study, no
Pfdhfr-
Pfdhps sextuple mutant was detected, which is a promising result. Previously, sextuple haplotypes were associated with reduced birth weights [
9]. Furthermore, newborns of women with the sextuple mutant haplotypes were found to have lower birth weights compared to those born to mothers with less mutated parasite infections [
9]. Due to the lack of birth weight data in the study, no assessment was done between the mutation levels and the birth outcomes. Another limitation of this study is the relatively low number of participants. Yet, the mutations in both
Pfdhfr and
Pfdhps genes were confirmed by DNA sequencing in all 48 (100.0%)
P. falciparum isolates. The study results also indicate that the S
GEGA haplotype is present in the DRC. Considering that the S
GEGA haplotypes are associated with decreased IPTp-SP outcomes [
17], these mutations should be carefully monitored in the DRC.