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 frontline and second-line antimalarial 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 2µl DNA template into a PCR master mix (50µl) containing 1X PCR buffer, 2.8mM MgCl2, 200µM dNTPs, 5pM of each primer, 1UTaq DNA polymerases (Qiagen, Hilden, Germany). The primer pairs 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 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 (10µl) using ApoI (New England Biolabs Inc., Ipswich, Massachusetts, USA) restriction enzyme for 15 minutes 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 100V for 45 mins. 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 analysesChi-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.