Molecular surveillance of the Pfmdr1 N86Y allele in P. falciparum isolates from Brazzaville, Republic of Congo

Background: Regular tolerance or resistance molecular makers is vital for effective malaria treatment, control and eradication programmes. P. 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 Center were analysed.Pfmdr1 N86Y genotyping was conducted using PCR-Restriction fragment length polymorphism (RFLP). 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. Our study underscores the importance to regular artemether−lumefantrine efficacy monitoring to inform malaria control programme of the Republic of Congo.

4 pyrimethamine (SP), had devasting consequences in sub-Saharan Africa in 1990s and 2000, particularly among children below 5 years [1]. Currently, artemisininbased combined therapy (ACT) is the first-line treatment for P. falciparum uncomplicated malaria alongside SP for intermittent preventive treatment-SP of pregnant women (ITPp) and infants (ITPi) [2].After the introduction of ACTs, malaria mortalities and morbidities global declined drastically until 2015. Even though ACTs are still highly efficacious, there is heightened concern on the potential spread of ACT resistance 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 antimalarial drug efficacy once every two years. However, high cost of clinical trials limit efforts to monitor the emergence and spread of antimalarial drug resistance in resource limited settings. Molecular surveillance of distinct point mutation(s) in P.
falciparum genes linked to antimalaria treatment failure offers a cost-effective tool to monitor spatiotemporal emergence and spread of resistant parasites. High prevalence of gene mutations associated with P. falciparum CQ (P. falciparum chloroquine transporter gene Pfcrt) and SP (P. falciparum dihydrofolate reductase gene; Pfdhfr and dihydropteroate synthase gene, Pfdhps) resistance informed, in part, the decision to replace these antimalarial drugs with ACTs including the Republic of Congo [8][9][10][11]. P. falciparum multidrug resistance1 (Pfmdr1), also known as P-glycoprotein homologue, is a transmembrane protein of the P. falciparum digestive vacuole (DV) [12]. It is involved transportation substrates into digestive vacuole of P. falciparum including antimalaria drugs [13]. Distinct changes in the sequence and/or amplification of the copy number ofPfmdr1gene alters P.
falciparumsusceptibility to several antimalarial 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 found in Africa. High prevalence of Pfmdr186N and 86Y alleles is currently being driven by ACT-linked P. falciparum opposite selection. Previous studies shown that parasites carrying Pfmdr1 N86 are less susceptible lumefantrine [16,17], artemisininlumefantrine, AL, select Pfmdr 186N whereas artesunate-amodiaquine, ASAQ, and piperaquine select 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 ACTs in a given geographical area [20,21]. The present study aimed to genotype and to determine the prevalence of Pfmdr1 N89Y in Brazzaville, Republic of Congo. Data for this study is useful 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 marched blood samples (maternal peripheral, placenta, and cord blood) collected from asymptomatic malaria pregnant women

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 and restriction fragment length polymorphism (PCR-RFLP) were used to genotype Pfmd1 N86Y as described previously [26]. In brief, Pfmdr1primary and nested PCRs were conducted by adding 2µl DNA template into a PCR master mix (50µl) containing 1X PCR buffer2.8mM MgCl 2 , 200µM dNTPs,5pM of each primer,1UTaq DNA polymerase. The primers for the primary PCR were A1 (5'-CGGAGTGACCAAATCTGGGA-3') and A3 (5'-GGGAATCTGGTGGTAACAGC-3') and for the secondary PCR were A2 (5'-TTGAAGAACAGAAATTACATGATGA-3') and A4 (5'-AAAGATGGTAACCTCAGTATCAAAGAAGAG-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 5min. The secondary reaction was conducted 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. 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 Table 1 shows that baseline characteristics of participants recruited in this study.
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 (IPTp) during pregnancy and most of the participants (70%) had >1 parity.
Of the 101 marched sample analysed in this study, Pfmdr1 was successfully amplified in 59 (58.4%), 38 (37.6%) and 21 (20.8%) maternal peripheral blood, placental blood and cord blood samples, respectively. Pfmsp2 genotyping showed  populations and allele frequency [31,32]. Changes in malaria treatment policies greatly influence the frequency of mutations that modulate P. falciparum susceptibility to antimalarial drugs including Pfmdr1 N86Y [31]. The introduction of ACTs 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]. For instance, the frequency ofPfmdr186Y has declined dramatically, in favour ofPfmdr1N86,in countries where AL is used as the first−line treatment for malaria. The increase in Pfmdr1 N86 allele frequency is faster when AL is used compared to AS−AQ usage [31].In areas where AS−AQ is the primary treatment for malaria, the decline of Pfmdr1 86Y allele frequency is slow owing to 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

Availability of data and material
All raw data provided in this work are available upon request to the corresponding author.

Ethical approval and consent to participate
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.   IPTp: Intermittent preventive treatment in pregnancy.