Background: Antimalarial drug resistance is one of the important challenges for governments in the fight against malaria. Molecular surveillance of antimalarial drug resistance supports early detection of how the recommended treatments work. This allows immediate action to reduce any threat and prevent it from spreading. Therefore, the aim of this study was to evaluate the frequency of dihydrofolate reductase (dhfr) mutants in Plasmodium falciparum resistance to pyrimethamine in Iranian malaria patients.

Materials and Methods: In 2020, 27 patients (21 males and 5 females) with imported P. falciparum cases were studied. The nested-PCR technique first confirmed the species in all samples and then amplification was done by the semi-nested-PCR method in order to detect single nucleotide polymorphisms (SNPs) in dhfr gene related to pyrimethamine resistance.

Results: All samples in the 18S rRNA gene had species-specific bands for P. falciparum strains. In the sequence analysis of pfdhfr gene amplification after comparison with the standard strain (wild type), 21 patients had a double mutation (C59R+S108N) and six patients had a triple mutation (N51I+C59R+S108N) of pyrimethamine resistance.

Conclusion: The results of this study showed that the susceptibility of P. falciparum to pyrimethamine in the treatment of malaria is significantly reducing. These findings can raise concerns about pyrimethamine resistance in P. falciparum. Due to the high emergence of double and triple mutants related to pyrimethamine resistance, the malaria surveillance and treatment systems in Iran, the use of pyrimethamine should be considered.