Emergence of chloroquine-sensitive and rising resistance to first-line artemisinin partner drugs in Malawi.

The emergence and spread of resistance to malaria drugs pose a major threat to malaria control efforts. This study assessed the prevalence of molecular markers associated with resistance to key antimalarials in clinical isolates from Mzuzu and Lilongwe in Malawi. These two regions have high human mobility and are strategically located near the border with Zambia and Tanzania, respectively. A total of 1582 blood samples were collected from individuals who visited hospitals for diagnosis between December 2020 and June 2021. infections were confirmed using nested and quantitative PCR, and drug resistance marker genes (, , , , and ) were sequenced by Sanger sequencing. No resistance-associated mutations were detected in and genes, supporting continued susceptibility to artemisinin derivatives and chloroquine (CQ). However, the -NFD haplotype, linked to reduced lumefantrine (LUM) susceptibility, was present in 159/371 (42.9%) isolates. Notably, the quadruple - mutant haplotype (AIRNVI-SGEAA), associated with high-level sulfadoxine-pyrimethamine (SP) resistance, was found in 287/328 (87.5%) of isolates. These findings highlight the ongoing risk of declining efficacy of LUM partner drugs in artemisinin-based combination therapies (ACT) and reduced SP effectiveness for intermittent preventive treatment in pregnancy (IPTp). The absence of mutations, together with the presence of wild-type pfmdr1 alleles lacking CQ-related mutation, suggests the re-emergence of CQ-sensitive parasites. Continuous molecular surveillance, alongside clinical efficacy studies, is essential to inform treatment policies and prevent the spread of drug-resistant malaria in Malawi.