Genetic Analysis of Artemisinin Resistance

Parasite DNA was analysed on day 0 for the presence of mutations in the Pfk13 propeller domain, which is associated with artemisinin resistance. The propeller domain was amplified in a nested-PCR assay, amplicons were sequenced according to Sanger’s method (Macrogen, Republic of Korea), and DNA sequences were analysed to identify specific single nucleotide polymorphisms (SNPs) related to artemisinin resistance [9 (link)]. The amino acid sequences were compared with the 3D7 wild-type amino acid sequences PF3D7_1343700. The presence of SNPs was confirmed by reading both the forward and the reverse strands. Parasites with mixed alleles were considered mutants. The number of copies of Pfpm2 genes was assessed by reverse transcriptase-PCR (sybr green dye). The full method has been described by Witkowski et al. [10 (link)].

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Warsame M., Hassan A.M., Hassan A.H., Jibril A.M., Khim N., Arale A.M., Gomey A.H., Nur Z.S., Osman S.M., Mohamed M.S., Abdulrahman A., Yusuf F.E., Amran J.G., Witkowski B, & Ringwald P. (2019). High therapeutic efficacy of artemether–lumefantrine and dihydroartemisinin–piperaquine for the treatment of uncomplicated falciparum malaria in Somalia. Malaria Journal, 18, 231.

Publication 2019

Sanger’s method

Alleles Amino acid sequences Artemisinin Assay Dna sequences Mutations Nested pcr Parasites Reverse transcriptase pcr Single nucleotide polymorphisms Sybr green

Corresponding Organization :

Other organizations : World Health Organization, Puntland State University, Institut Pasteur du Cambodge, University of Hargeisa, Institut Pasteur

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Variable analysis

independent variables

Presence of mutations in the Pfk13 propeller domain

dependent variables

Artemisinin resistance
Number of copies of Pfpm2 genes

control variables

3D7 wild-type amino acid sequences PF3D7_1343700

controls

Positive control: 3D7 wild-type amino acid sequences PF3D7_1343700
Negative control: Not specified

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