A Cross-Sectional Survey of Drug-Resistance Polymorphisms in Plasmodium falciparum K13, Plasmepsin 2 and Pfmdr-1 in Sentinel Sites in Myanmar

Abstract

Background: Plasmodium falciparum has developed resistance against artemisinin and partner drugs that have been widely used globally as artemisinin-based combination therapies (ACT). Such resistance, poses the greatest challenge to the prospect of malaria elimination in the Greater Mekong Subregion (GMS). Genetic polymorphisms in P. falciparum that confer resistance to the drugs that are part of two most commonly used ACT in the GMS, artemisinin-lumefantrine (AL) and dihydroartemisisnin-piperaquine (DP). Single point mutations in Kelch propeller domain of falciparum chromosome 13 (K13) for artemisinin resistance, and copy number variations of plasmepsin 2/3 are associated with piperaquine resistance, and Pfmdr-1 for mefloquine and lumefantrine resistance. Although the efficacy is high for ACTs in Myanmar, molecular markers of resistance to either of the drugs in ACT can still be present and may indicate that the drugs will be at risk in the near future. Therefore, this study aimed to detect genetic polymorphisms in K13, Pfmdr-1, and plasmepsin 2 (Pfpm-2) that mediate ACT treatment outcomes in Myanmar. Methods: The current study uses a cross-sectional study design and retrospective analysis of laboratory samples collected in previous therapeutic efficacy studies (TES) conducted during 2014 to 2018 in nine sentinel malaria endemic remote townships in Myanmar. The ACTs used in these TES were artemether-lumefantrine, dihydroartemisinin-piperaquine, and pyronaridine-artesunate. 176 samples were randomly selected out of 651 samples from nine TES sites because of the time constraint. K13 genotyping was done by Sanger sequencing, and the copy numbers of Pfpm-2 and Pfmdr-1 were quantified by real-time polymerase chain reaction. Results: Among 176 randomly selected pre-treatment parasites, we observed non- synonymous mutation in the K13 gene in 25% (42/169; 95% CI: 18.3, 31.4). Overall, 23% (39/169; 95% CI: 16.7, 29.4) of infections harbored a K13 mutation that has been validated as associated with artemisinin resistance. Among these, 58.9% (23/39) encoded the F446I substitution. The prevalence of parasites harboring the C580Y mutation that is the most closely associated with artemisinin resistance was 6.5% (11/169; 95% CI: 2.8, 10.2), and was present in 4 out of 9 study sites. Only 1 sample 0.6% (1/172; 95% CI: 0, 1.7) harbored more than one copy of Pfpm-2; this parasite also contained the K13 C580Y mutation conferring artemisinin resistance. No parasites harbored more than one copy of Pfmdr-1. Conclusion: Consistent with the high efficacy of ACTs in Myanmar, there were little evidence of resistance to artemisinin or partner drugs by analysis of molecular markers. However, there was remarkable amount of K13 molecular markers (C580Y, F446I, R561H) seen in this study. These observed K13 markers have already been confirmed and validated by WHO. This finding may be a warning sign of developing artemisinin resistance which may, in turn, have an effect on the malaria elimination process in Myanmar. Emergence of drug resistant malaria in GMS threatens the malaria elimination effort in the region as well as globally. Continued monitoring of artemisinin and its partner drugs resistance is needed to prevent the spread of drug resistant malaria.