Molecular surveillance of the Pfmdr1 N86Y allele among Congolese pregnant women with asymptomatic malaria

Background Malaria in pregnancy is associated with considerable morbidity and mortality. Regular surveillance of artemisinin-based combination therapy tolerance, or molecular makers of resistance, is vital for effective malaria treatment, control and eradication programmes. Plasmodium falciparum multiple drug resistance-1 gene (Pfmdr1) N86Y mutation is associated with reduced susceptibility to lumefantrine. This study assessed the prevalence of Pfmdr1 N86Y in Brazzaville, Republic of Congo. Methods A total 1001 of P. falciparum-infected blood samples obtained from asymptomatic malaria pregnant women having a normal child delivery at the Madibou Integrated Health Centre were analysed. Pfmdr1 N86Y genotyping was conducted using PCR-restriction fragment length polymorphism. Results The wild type Pfmdr1 N86 allele was predominant (> 68%) in this study, whereas a few isolates carrying the either the mutant allele (Pfmdr1 86Y) alone or both alleles (mixed genotype). The dominance of the wildtype allele (pfmdr1 N86) indicates the plausible decline P. falciparum susceptibility to lumefantrine. Conclusion This study gives an update on the prevalence of Pfmdr1 N86Y alleles in Brazzaville, Republic of Congo. It also raises concern on the imminent emergence of resistance against artemether–lumefantrine in this setting. This study underscores the importance to regular artemether–lumefantrine efficacy monitoring to inform the malaria control programme of the Republic of Congo.

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Background
Plasmodium falciparum malaria among pregnant women is a major public health concern in sub-Saharan Africa. Pregnant women have substantial risks and malaria in pregnant women are related to preterm delivery, intrauterine growth restriction, low birth weight and maternal anaemia. The World Health Organization (WHO) recommends the use of intermittent preventive treatment (IPTp) with sulfadoxine-pyrimethamine (SP) for pregnant women and also for infants (IPTi) [1]. Currently, artemisinin-based combination therapy (ACT) is the first-line treatment for P. falciparum uncomplicated malaria. Resistance against chloroquine (CQ) and its successor, sulfadoxine-pyrimethamine, had devasting consequences in sub-Saharan Africa in 1990s and 2000, particularly among children below 5 years [2].
After the introduction of ACT, malaria mortality and morbidity has globally declined until 2015. Even though ACT is still efficacious, there is sensitive concern on the Open Access Malaria Journal *Correspondence: ffntoumi@hotmail.com; fntoumi@fcrm-congo.com 2 Congolese Foundation for Medical Research, Brazzaville, Republic of Congo Full list of author information is available at the end of the article potential spread of artemisinin-resistant P. falciparum parasites from Southeast Asia to sub-Saharan Africa, reminiscent of the spread of CQ and SP resistance [3][4][5][6][7]. The WHO recommends routine surveillance of anti-malarial drug efficacy once every 2 years. However, the efforts to monitor the emergence and spread of anti-malarial drug resistance in resource-limited settings are hampered due to high clinical trial costs. Molecular surveillance of distinct point mutation(s) in P. falciparum genes linked to anti-malarial treatment failure offers a cost-effective tool to monitor spatial and temporal emergence and spread of resistant parasites. High prevalence of gene mutations associated with CQ (P. falciparum chloroquine transporter, Pfcrt) and SP (P. falciparum dihydrofolate reductase gene; Pfdhfr, and P. falciparum dihydropteroate synthase; Pfdhps) resistance informed, in part, the decision to replace these anti-malarial drugs with ACT, including in the Republic of Congo [8][9][10][11].
The P. falciparum multidrug resistance 1 protein (PfMDR1), also known as P-glycoprotein homologue, is a transmembrane protein of the P. falciparum digestive vacuole (DV) [12]. It is involved in the transport of substrates into digestive vacuole of the parasite, including anti-malarial drugs [13]. Distinct changes in the sequence and/or amplification of the copy number of the Pfmdr1 gene alters P. falciparum susceptibility to several anti-malarial drugs [14].
In particular, the Pfmdr1 N86Y single nucleotide polymorphism (SNP) has been implicated in P. falciparum resistance to chloroquine and amodiaquine [15]. Pfmdr1 N86Y is mostly abundant in African settings. High prevalence of Pfmdr1 86 N and 86Y alleles is currently being driven by ACT-linked P. falciparum selection. Previous studies have shown that parasites carrying Pfmdr1 N86 are less susceptible to lumefantrine [16,17], artemether-lumefantrine (AL) selects for Pfmdr1 86 N, whereas artesunate-amodiaquine (ASAQ) and piperaquine is selective for Pfmdr1 86Y [16,[18][19][20]. Since this phenomenon indicates potential decline of malaria parasite sensitivity or increased tolerance to ACT partner drugs, Pfmdr1 N86Y genotyping has been proposed as a useful marker to guide rotation of ACT medicines in a given geographical area [20,21].
The present study aimed to genotype and to determine the prevalence of Pfmdr1 N86Y in Brazzaville, Republic of Congo among pregnant women using maternal peripheral, placental, and cord blood. The study aims to provide factual data as a useful measure for the refinement and adaption of the current malaria treatment policy with the long-term goal of reducing malaria in the Republic of Congo.

Sample collection
This study analysed a total of 101 matched blood samples (maternal peripheral, placenta, and cord blood) collected from pregnant women with asymptomatic malaria who had a normal child delivery at the Madibou Integrated Health Center, Brazzaville, between March 2014 and April 2015 [21]. The study was conducted in Brazzaville, the capital of the Republic of Congo with 1.8 million inhabitants [22]. Malaria transmission in this area is perennial with P. falciparum being the predominant Plasmodium species [22,23]. AL and ASAQ are the first-line and second-line anti-malarial drugs for uncomplicated P. falciparum malaria in the Republic of Congo, respectively [24].

Pfmdr1 genotyping
Total genomic DNA was isolated using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Amplification of P. falciparum merozoite surface protein 2 gene (Pfmsp2) was used to determine P. falciparum multiplicity of infection (MOI), as described previously [25]. Nested-PCR followed by a restriction fragment length polymorphism (PCR-RFLP) were used to genotype Pfmd1 N86Y, as described earlier [26]. In brief, Pfmdr1 primary and nested PCRs were amplified by adding 2 µl DNA template into a PCR master mix (50 µl) containing 1X PCR buffer, 2.8 mM MgCl 2 , 200 µM dNTPs, 5 pM of each primer, 1UTaq DNA polymerases (Qiagen, Hilden, Germany). The primer pairs for the primary PCR were A1 (5′-CGG AGT GAC CAA ATC TGG GA-3′) and A3 (5′-GGG AAT CTG GTG GTA ACA GC-3′) and for the secondary PCR were A2 (5′-TTG AAG AAC AGA AAT TAC ATG ATG A-3′) and A4 (5′-AAA GAT GGT AAC CTC AGT ATC AAA GAA GAG -3′). The thermal cycler conditions were as follows: initial denaturation at 94 °C for 2 min, followed by 40 cycles at 94 °C for 1 min, 45 °C for 1 min, 72 °C for 1 min and a final extension at 72 °C for 5 min. The secondary reaction was amplified using the product of the primary reaction as a template. DNA extracted P. falciparum laboratory strains (3D7 and Dd2) and PCR grade water were used as positive and negative controls, respectively.
The Pfmdr1 N86Y mutation was identified by digesting Pfmdr1 A2/A4 secondary PCR products (10 µl) using ApoI (New England Biolabs Inc., Ipswich, Massachusetts, USA) restriction enzyme for 15 min at 50 °C following manufacturer's instructions. The resulting DNA fragments were separated and resolved by gel electrophoresis on a 2% agarose gel stained with SYBR green at 100 V for 45 min. ApoI digests Pfmdr1PCR product when Pfmdr1N86 (wild type allele) is present. The PCR amplification was performed three consecutive times for a given sample in order to get a successful amplification. Also, the nested PCR products were subjected to RFLP using ApoI twice (with independent PCR products) to reconfirm the Pfmdr1 N86Y alleles. Additionally, few random samples were chosen and were subjected to sanger sequencing.

Data analyses
Chi square and Fisher exact tests were applied to compare the proportions of Pfmdr1 N86Y alleles in this study. The statistical significance was set at p-value< 0.05.

Ethical considerations
This study was approved by the Institutional Ethics Committee of Fondation Congolaise pour la Recherche Médicale, FCRM, Brazzaville, Republic of Congo. Written informed consent was obtained from all participants before samples collection. The objectives of the study including the study procedures, sample to be taken, study benefits, potential risks and discomforts were explained. Newly opened needle and syringe were used for each subject.

Results
The baseline characteristics of participants recruited in this study is summarized in Table 1. The mean age of participants was 23.7 ± 5.75 years. Overall, 24% of the pregnant women did not take intermittent preventive treatment during pregnancy and most of the participants (70%) had > 1 parity. Of the 101 matched samples analysed in this study, Pfmdr1 was successfully amplified in 59 (58%), 38 (38%) and 21 (21%) maternal peripheral blood, placental blood and cord blood samples, respectively. Figure 1 shows an electrophoresis gel of PCR products before and after digestion with specific enzyme restriction. Pfmsp2 genotyping showed mean multiplicity of infection (MOI) was 1.06 ± 0.24. High prevalence of Pfmdr1 wild type allele (N86) was observed among the different sample types. Pfmdr1 N86 was present in 70% (41/59), 58% (22/38) and 86% (18/21) of the peripheral blood, placenta blood and cord blood samples, respectively. The remaining samples had the mutant allele (Pfm-dr186Y). Three peripheral blood samples, two placental blood samples and one cord blood sample had both the wild type and mutant Pfmdr1 N86Y alleles. The Pfmdr1 N86 allele prevalence was similar (p-value> 0.05) among different sample types, parity and the number of intermittent preventive treatment during pregnancy ( Table 2).

Discussion
Anti-malarial drug resistance is a major obstacle in malaria reduction/eradication globally. Molecular surveillance is important to identify resistant phenotypes and to constantly monitor for any anti-malarial drug resistance. This study was set out to determine the prevalence of Pfmdr1 N86Y mutation among pregnant women having a normal child delivery at the Madibou Integrated Health Centre, Brazzaville Republic of Congo. The Pfmsp2 gene used to determine MOI showed mean multiplicity of infection as 1.06 ± 0.24. In areas with constant transmission of malaria, MOI may increase as immunity develops. MOI in pregnant women is a factor for the acquisition and maintenance of immunity against malaria. In this study, only using msp2 genotyping, only one set of parasite clones were predominantly present among the pregnant women investigated. However, there are possibilities that these individuals may harbour more than one parasite, and this could be explained only by additional msp1 genotyping for K1, MAD20, and RO33 alleles.
The frequency of Pfmdr1 86Y (mutated allele) in this study was lower than previously estimated in this setting in 2010 (73%) and in 2015 (27%) [27,28]. These findings are also comparable to Pfmdr1 86Y allele (23%) global frequency and most parts of Africa (17 to 24%), except Central Africa, where high resistant allele frequency (44%) has been observed [29]. In Southeast Asia, however, the frequency of Pfmdr1 86Y is much lower than observed in this study whereas it has almost reached fixation in Papua New Guinea [29].
Pfmdr1 N86Y mutation is known to modulate P. falciparum susceptibility to various anti-malarial drugs by regulating the influx of the drugs into the parasite's digestive vacuole. Previous studies have shown that parasite carrying this mutation are less susceptible to 4-aminoquinolines, namely chloroquine, amodiaquine and piperaquine, in vitro [30] and increase the risk of chloroquine or amodiaquine therapeutic failure [15]. On the other hand, the Pfmdr1 86Y mutation enhance malaria parasite susceptibility to lumefantrine, mefloquine and the active derivative of artemisinin, dihydroartemisinin [30]. The converse impact of Pfmdr1 N86Y on P. falciparum response to longer-acting partner drugs of ACT implies that wide spread use of AL and ASAQ, particularly in Africa, exert opposite selection pressure on P. falciparum populations and allele frequency [31,32]. Changes in malaria treatment policies greatly influence the frequency of mutations that modulate P. falciparum susceptibility to anti-malarial drugs, including Pfmdr1 N86Y [31]. The introduction of ACT in the early 2000s and cessation of chloroquine use in the 1990s led to drastic changes in Pfmdr1 N86Y allele frequency in various malaria-endemic settings [27,33,34] [31]. In areas where ASAQ is the primary treatment for malaria, the decline of Pfmdr1 86Y allele frequency is slow owing to the reduced susceptibility of parasites carrying this mutation to amodiaquine. Previous studies demonstrate that parasites carrying Pfmdr1 N86 tolerate higher lumefantrine levels and have short-time to reinfection or recrudescence in patients with high lumefantrine concentration following AL treatment [16,17]. Even though there is no evidence directly linking Pfmdr1 N86 to AL treatment failure and AL is still highly efficacious, parasite tolerance to lumefantrine is a clear warning sign for plausible emergence of resistance against AL. In this context, Pfmdr1 86 N can be used to track lumefantrine selective pressure in a given area [17]. The findings show that Pfmdr1N 86 allele is approaching fixation in the Republic of Congo and could provide the genetic background needed for the emergence of resistance against lumefantrine threatening AL usefulness in this setting. However, this possibility could be averted by concurrent use of AL and ASAQ as first-line treatment for uncomplicated P. falciparum malaria. Such a strategy is supported by evidence showing the opposite effect of both Pfmdr1 N86Y alleles on P. falciparum susceptibility to AL and ASAQ [18].

Conclusion
This study offers an update on the frequency of Pfmdr1 N86Y alleles in Brazzaville, Republic of Congo and provides evidence supporting the concomitant deployment or rotation of AL and ASAQ as the primary treatment for uncomplicated P. falciparum malaria. This will be helpful to halt any further selection of Pfmdr1 alleles that dampen parasite susceptibility and safeguard AL efficacy.