K13 Propeller Mutations in Plasmodium falciparum Populations in Regions of Malaria Endemicity in Vietnam from 2009 to 2016

Abstract

The spread of artemisinin-resistant Plasmodium falciparum compromises the therapeutic efficacy of artemisinin combination therapies (ACTs) and is considered the greatest threat to current global initiatives to control and eliminate malaria. This is particularly relevant in Vietnam, where dihydroartemisinin-piperaquine (DP) is the recommended ACT for P. falciparum infection. The propeller domain gene of K13, a molecular marker of artemisinin resistance, was successfully sequenced in 1,060 P. falciparum isolates collected at 3 malaria hot spots in Vietnam between 2009 and 2016. Eight K13 propeller mutations (Thr474Ile, Tyr493His, Arg539Thr, Ile543Thr, Pro553Leu, Val568Gly, Pro574Leu, and Cys580Tyr), including several that have been validated to be artemisinin resistance markers, were found. The prevalences of K13 mutations were 29% (222/767), 6% (11/188), and 43% (45/105) in the Binh Phuoc, Ninh Thuan, and Gia Lai Provinces of Vietnam, respectively. Cys580Tyr became the dominant genotype in recent years, with 79.1% (34/43) of isolates in Binh Phuoc Province and 63% (17/27) of isolates in Gia Lai Province carrying this mutation. K13 mutations were associated with reduced ring-stage susceptibility to dihydroartemisinin (DHA) in vitro and prolonged parasite clearance in vivo An analysis of haplotypes flanking K13 suggested the presence of multiple strains with the Cys580Tyr mutation rather than a single strain expanding across the three sites.

Keywords: K13 mutation; Plasmodium falciparum; RSA; Vietnam; artemisinin resistance; frequency; parasite clearance.

FIG 1

Prevalence of K13 mutations in P. falciparum parasites in Binh Phuoc Province, 2009 to 2016.

FIG 2

Prevalence of K13 mutations in P. falciparum parasites in Ninh Thuan (A) and Gia Lai (B) Provinces. Results for only two seasons in Krong Pa, Gia Lai, are shown. Samples from the first season were from a different commune (Iato, Gia Lai).

FIG 3

Association between K13 mutations and parasite clearance half-life in Vietnam, 2009 to 2016. The median (interquartile range) half-life for each mutation (shown as gray lines) is as follows: 1.96 h (1.63 to 2.73 h) for the wild type, 6.8 h (3.69 to 7.42 h) for Tyr493His, 6 h (4.81 to 7.04 h) for Arg539Thr, 6.7 h (5.9 to 8.02 h) for Ile543Thr, 6.4 h (4.39 to 7.08 h) for Pro553Leu, 6.8 h (6.22 to 7.59 h) for Val568Gly, and 6.9 h (6.33 to 8.14 h) for Cys580Tyr.

FIG 4

Ring-stage survival after exposure to dihydroartemisinin. Forty parasites were tested. Red dots, parasites with the Cys580Tyr mutation; green dots, parasites without the Cys580Tyr mutation. The median and interquartile range of the proportion of viable parasite are shown as gray lines.

FIG 5

Allelic heterozygosity at microsatellite loci flanking the K13 gene in Cys580Tyr mutant parasites from Binh Phuoc (green), Ninh Thuan (red), and Gia Lai (blue) Provinces.

FIG 6

Locations of the three regions in Vietnam where malaria is endemic. The regions of endemicity (Binh Phuoc, Ninh Thuan, and Gia Lai Provinces) are shown in red. The locations of sample collection are marked.