Molecular markers of antimalarial drug resistance in pfk13, pfmdr1, pfdhfr, and pfdhps genes of Plasmodium falciparum in a rural municipality in Cubal, Benguela Province, Angola (2022-2023).

Abstract

Malaria remains a major cause of morbidity and mortality in Angola, where Plasmodium falciparum accounts for most infections. The widespread use of artemether-lumefantrine (AL) as first-line treatment and sulfadoxine-pyrimethamine (SP) for intermittent preventive treatment in pregnancy (IPTp) exerts selective pressure on local parasite populations. Molecular surveillance of drug resistance markers is essential to monitor susceptibility and anticipate changes that may influence treatment and prevention strategies. This study analyzed pfk13, pfmdr1, pfdhfr, and pfdhps polymorphisms in P. falciparum isolates from Cubal, Benguela province, to assess parasite genetic evolution under AL and SP pressure. A cross-sectional study was conducted between 2022 and 2023 at Hospital Nossa Senhora da Paz (Cubal). A total of 139 real-time PCR-confirmed P. falciparum infections were included. Dried blood spot samples underwent sequencing of the genes involved in resistance. Mutation and haplotype frequencies associated with antimalarial resistance were determined. Among 120 pfk13 sequences, mutations were detected in 12 isolates (10%), including five nonsynonymous variants, but none corresponded to validated or candidate markers of artemisinin resistance. In pfmdr1 (100 sequences), Y184F was the most prevalent mutation (43%), N86Y was rare (1%), and D1246Y was not detected. Seven additional pfmdr1 variants were identified. Of the 101 isolates correctly sequenced for pfdhfr/pfdhps, 77.2% carried the triple pfdhfr N51I/C59R/S108N mutation. In pfdhps, A437G was nearly fixed (98%), K540E showed moderate prevalence (23.8%), and A581G was absent. The most common pfdhps haplotypes were ISGKAA (64.4%) and ISGEAA (22.8%). Combined pfdhfr/pfdhps profiles classified 58.4% of isolates as "partially resistant" (IRNG) and 17.8% as "fully resistant" (IRNGE), with no "super-resistant" haplotypes. Additional single mutations not associated with SP resistance were also detected. This study provides an updated molecular profile of antimalarial resistance in a rural area of Angola. No validated or candidate pfk13 markers of artemisinin resistance were identified. However, the predominance of pfmdr1 haplotypes linked to reduced lumefantrine susceptibility raises concerns regarding AL efficacy. Although SP remains suitable for IPTp, the presence of the "fully resistant" haplotype (IRNGE) of pfdhfr/pfdhps highlight potential risks to long-term effectiveness. These findings reinforce the need for integrated molecular surveillance and periodic therapeutic efficacy studies to guide malaria control policies in rural Angola.