Drug resistance gene polymorphisms of Plasmodium falciparum Kinshasa clinical isolates

Background Reducing the burden of Plasmodium falciparum infection in endemic African countries relies on the sustained effectiveness of artemisinin (ART)-based combination therapies. ART derivatives remain the key frontline treatment, while sulfadoxine-pyrimethamine (SP) is used for prevention. The recent discovery in Africa of P. falciparum kelch 13 ( k13 ) substitutions linked to ART resistance (ART-R), distinct from the k13 ART-R alleles reported in Southeast Asia, suggests local emergence of ART-R. The emergence and spread of ART-R, together with widespread high-level pyrimethamine resistance in Africa, represent major public health concerns. Molecular surveillance provides a comprehensive approach to detect, analyze, and track resistance, thereby guiding evidence-based malaria control policies. Methods From 2022 to 2023, 303 patients diagnosed with P. falciparum malaria via rapid diagnostic tests were enrolled in Kinshasa, the Democratic Republic of the Congo (DRC). Blood samples collected as dried blood spots from 171 microscopy-confirmed patients underwent PCR amplification and sequencing of the k13 encoding the propeller domain. In addition, k13 sequence outside the propeller domain and 12 key drug resistance loci, including coronin, KIC4, KIC5, KIC7, PX1 , AP2-Mu , eEF2 , mdr1 , mdr2 , crt , dhfr , and dhps were examined using 31 whole genome sequencing data sets obtained from culture-adapted Kinshasa isolates. Results One out of 171 isolates carried the A578S substitution in the K13 propeller domain, a variant previously shown not to confer ART-R in transgenic parasites. Whole-genome sequencing revealed both previously reported and novel polymorphisms across multiple candidate genes involved in drug resistance. Notably, isolates with an increased number of Asn residues between H136 and L143 of the 3D7 K13 sequence were detected; this variant has been reported in other African countries and linked to reduced susceptibility to DHA in vitro . PX1 M1701I and D1705N mutations, recently described as being linked to decreased ex vivo susceptibility to DHA were also detected. These markers suggest potential k13 -independents effects on ART susceptibility in Kinshasa. Conclusions Our findings provide crucial insight into the current genetic landscape of P. falciparum isolates circulating in Kinshasa and emphasize the importance of molecular surveillance to monitor potential drug resistance markers and inform timely updates to national treatment guidelines.