Reduced Polymorphism in the Kelch Propeller Domain in Plasmodium vivax Isolates from Cambodia

Abstract

Polymorphism in the ortholog gene of the Plasmodium falciparum K13 gene was investigated in Plasmodium vivax isolates collected in Cambodia. All of them were Sal-1 wild-type alleles except two (2/284, 0.7%), and P. vivax K12 polymorphism was reduced compared to that of the P. falciparum K13 gene. Both mutant allele isolates had the same nonsynonymous mutation at codon 552 (V552I) and were from Ratanak Kiri province. These preliminary data should encourage additional studies for associating artemisinin or chloroquine resistance and K12 polymorphism. In areas in which malaria is endemic and Plasmodium falciparum and Plasmodium vivax are present, such as in Southeast Asia and Pacific Oceania, both species share the same vectors and human hosts, either successively or concomitantly (mixed infections) (1, 2). These two species, in this context, often undergo similar mutation-driven evolution and natural selection. This includes nucleotide substitution, gene duplication/deletion, chromosomal change, and genome duplication (3). In terms of drug resistance, regardless of the fundamental biological differences between the two Plasmodium species, it is well known that antimalarial drug pressure induces a strong selection of resistant parasites for both of these parasite populations. For instance, sequencing of the dhfr gene in P. vivax isolates collected in areas where sulfadoxine-pyrimethamine was used to treat falciparum malaria and the alignment of these alleles with the P. falciparum dhfr gene have clearly demonstrated that mutations in codons 57, 58, 61, 117, and 173 were involved in pyrimethamine resistance and corresponded to the codons 51, 59, 108, and 164 found in P. falciparum pyrimethamine-resistant strains (4). More recently, we observed high frequencies of P. falciparum and P. vivax isolates with increased mdr-1 copy numbers in areas where mefloquine has been extensively used as the first-line treatment in falciparum-uncomplicated malaria, while in areas where mefloquine has never been used, P. falciparum and P. vivax isolates with increased mdr-1 copy numbers are rare (5). These studies clearly show that antimalarial drugs used to treat falciparum malaria have a significant impact on sympatric Plasmodium species, such as P. vivax.Since 2001, artemisinin combination therapies (ACTs) have been recommended as first-line treatment in the national treatment guidelines of most countries in which malaria is endemic. In 2008, the emergence of artemisinin-resistant P. falciparum parasites was observed in Southeast Asia (6-15). Recent molecular and biological studies showed that artemisinin resistance was associated with P. falciparum early ring stages and mutations in the PF3D7_1343700 kelch propeller domain (K13-propeller) (8,14,15). To date, although the role of the P. falciparum K13 protein remains unknown, two pieces of evidence suggest that it is involved in the cellular response to oxidative stress (8): (i) its homology to the KEAP1 human protein, which is involved in cell adaptation to oxidative stress (16), a...

Longitudinal Pooled Deep Sequencing of the Plasmodium vivax K12 Kelch Gene in Cambodia Reveals a Lack of Selection by Artemisinin

“…Previous studies have found a similarly low number of mutations in the K12 gene among Cambodian P. vivax (2/284 clinical isolates). 15,17 This standing variation in K12 is similar to the current understanding of K13 mutations in African falciparum populations. 9,15 We have developed and validated a rapid pooled NGS approach for surveillance of K12 mutations.…”

“…The V552I mutation shared between the OM2011 and KS2011 pools has been previously reported in Ratanakiri Province among samples collected in 2013. 17 The highest MAF detected in this study was 8.80% from the OM region in 2010. Most mutations FIGURE 1.…”

“…16 As such, previous surveillance of the P. vivax K12 gene may have overlooked minority strain polymorphisms and missed emerging resistance at low frequency in the population. 17 In addition, using an NGS with pooled clinical isolates has proven to be accurate, sensitive, and cost-effective as compared with Sanger sequencing. 15,16 Whole blood from 304 patients presenting with uncompli- 63°C with a ΔT of 1°C per two cycles, and an extension of 72°C for 3 minutes.…”

“…Although we found only a small number of ephemeral nonsynonymous mutations, our results show that pooled NGS surveillance for allele frequency assessment may provide a more complete picture of gene diversity than traditional Sanger sequencing methods. 17 In addition, this study provides a baseline to measure future changes to the P. vivax K12. Cambodia's recent shift to dihydroartemisinin-piperaquine as first-line therapy for vivax malaria in 2012 will result in strong selective pressure on the parasite and may eventually result in selection of mutations in this gene.…”

Genetic diversity of the Pvk12 gene in Plasmodium vivax from the China-Myanmar border area

“…Although ACT is still highly efficacious for treating vivax malaria [14, 15], vigilance is required on potential emergence of resistance in this parasite [16]. A recent study aiming to determine whether similar mutations mediating artemisinin resistance are also present in the Pfk13 orthologue in P. vivax (here named Pvk12 gene as this gene is located on chromosome 12) found that parasites with a Pvk12 mutation (V552I) was circulating in Cambodia at a very low frequency [17]. To further examine the genetic diversity of this gene in the GMS, recent collections of clinical isolates of P. vivax from both sides of the China-Myanmar border were examined.…”