Induction of Artesunate Resistance in Plasmodium falciparum 3D7 Strain Using Intermittent Exposure Method and Comparing P.fk13 Sequence between Susceptible and Resistant Strains

Sahar  Barati; Afsaneh Motevalli  Haghi; Mehdi  Nateghpour; Zahra  Zamani; Sadegh  Khodaveisi; Soudabeh  Etemadi

doi:10.18502/ijpa.v18i4.14244

Sahar Barati Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
Afsaneh Motevalli Haghi Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
Mehdi Nateghpour Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
Zahra Zamani Biochemistry Department, Pasteur Institute of Iran, Pasteur Avenue, Tehran, Iran
Sadegh Khodaveisi Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
Soudabeh Etemadi Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran

DOI: https://doi.org/10.18502/ijpa.v18i4.14244

Keywords: Artesunate resistance; Plasmodium falciparum; Intermittent exposure; Malaria

Abstract

Background: Resistance to artemisinin has threatened major achievements in malaria control, more investigations is needed about resistant strains and related genes. We aimed to induce resistance to artesunate in the Plasmodium falciparum 3D7 strain using intermittent exposure method and comparing P.fk13 gene sequence between susceptible and resistance strains.

Methods: P. falciparum 3D7 strain was cultured according to Trager & Jensen method with some modifications. Serial concentrations between 10^-2mol/l, to 10^-7mol/l were prepared, then P. falciparum 3D7 was exposed to each of the dilution to determine IC50 and lethal dose. Sensitivity reduction process was started from the concentration of 10^-7mol/l and ended at 10^-2mol/l. Exposed parasites were collected after at least 27 days after cultivation in each drug concentration. DNA extraction, PCR and sequencing process were performed to investigate any possible mutations in the P.fk13 gene sequence.

Results: Effectiveness of 10^-2mol/l concentration of artemisinin was found as a lethal dose. IC50 value was equal to 5˟10^-4 mol/l. The resistant strain was provided in the lab, sequenced and registered in the gene bank as P.f Art ^-2, (accession number MH796123. 1). Alignment of this registered sample showed no mutation in P.f kelch13 gene in comparison with standard strain submitted in the GenBank.

Conclusion: Resistance to artesunate in malaria parasite may occur but with no mutation in the P.f kelch13 gene. Therefore, whole genome sequencing should be applied to determine mutations in resistant strains.