Emergence of chloroquine-sensitive <i>Plasmodium falciparum</i> and rising resistance to first-line artemisinin partner drugs in Malawi

The emergence and spread of <i>Plasmodium falciparum</i> 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 <i>P. falciparum</i> 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. <i>P. falciparum</i> infections were confirmed using nested and quantitative PCR, and drug resistance marker genes (<i>pfmdr1</i>, <i>pfcrt, pfk13</i>, <i>pfatp6</i>, <i>pfdhfr</i>, and <i>pfdhps</i>) were sequenced by Sanger sequencing. No resistance-associated mutations were detected in <i>pfk13</i> and <i>pfcrt</i> genes, supporting continued susceptibility to artemisinin derivatives and chloroquine (CQ). However, the <i>pfmdr1</i>-NFD haplotype, linked to reduced lumefantrine (LUM) susceptibility, was present in 159/371 (42.9%) isolates. Notably, the quadruple <i>pfdhfr</i>-<i>pfdhps</i> 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 <i>pfcrt</i> 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.