Molecular characterization of antimalarial resistance and hrp2/3 deletions in Plasmodium falciparum strains from Mtwara, Tanzania (2021–2024)

Abstract Background Malaria remains a significant public health challenge in sub-Saharan Africa and Southeast Asia, with Plasmodium falciparum responsible for the majority of infections and deaths, particularly among children aged < 5 years. In Tanzania, the widespread use of antimalarial drugs, such as sulfadoxine-pyrimethamine for preventive therapy and artemisinin-based combination therapies as first-line treatment, has prompted concerns about the emergence and spread of drug resistance. The prolonged use of these drugs may have influenced resistance patterns, varying over time. Moreover, some P. falciparum samples have been reported as rapid diagnostic test (RDT)-negative despite microscopic confirmation of P. falciparum infection, raising questions about whether such false-negative results are due to poor-quality RDT kits or genetic deletions in the parasite. Methods This study investigated drug resistance dynamics and potential false-negative RDT results in P. falciparum by analyzing molecular markers in pfdhps, pfdhfr , pfkelch13, and pfhrp2 / 3 , with the 18S rRNA gene used as a housekeeping control to verify DNA quality and confirm true parasite positivity. Results Molecular analysis revealed a decline in the triple pfdhfr mutation paired with double pfdhps mutations, alongside an increase in mixed dhfr–dhps haplotypes ( χ 2  = 28.94, p  < 0.001), indicating ongoing genetic diversification under drug pressure. Wild-type parasites were predominant (56–76%), and most pfkelch13 mutations were located in the non-propeller region, with no validated or candidate mutations associated with artemisinin-resistance detected. P. falciparum -infected samples that were RDT-negative showed a high frequency of both complete and partial deletions in pfhrp2 and pfhrp3 , potentially explaining RDT inconsistencies and false-negative results. Conclusions P. falciparum remains the dominant species in this high-transmission region, showing a persistently high rate of antifolate resistance. Significant associations between triple pfdhfr and double pfdhps resistance markers were observed between 2021 and 2024. The continued susceptibility to artemisinin emphasizes the need for ongoing molecular surveillance and monitoring of hrp2/hrp3 deletions to support malaria control strategies in Tanzania.