Genetic Diversity and Lack of Artemisinin Selection Signature on the Plasmodium falciparum ATP6 in the Greater Mekong Subregion

The recent detection of clinical Artemisinin (ART) resistance manifested as delayed parasite clearance in the Cambodia-Thailand border area raises a serious concern. The mechanism of ART resistance is not clear; but the P. falciparum sarco/endoplasmic reticulum Ca²⁺-ATPase (PfSERCA or PfATP6) has been speculated to be the target of ARTs and thus a potential marker for ART resistance. Here we amplified and sequenced pfatp6 gene (∼3.6 Kb) in 213 samples collected after 2005 from the Greater Mekong Subregion, where ART drugs have been used extensively in the past. A total of 24 single nucleotide polymorphisms (SNPs), including 8 newly found in this study and 13 nonsynonymous, were identified. However, these mutations were either uncommon or also present in other geographical regions with limited ART use. None of the mutations were suggestive of directional selection by ARTs. We further analyzed pfatp6 from a worldwide collection of 862 P. falciparum isolates in 19 populations from Asia, Africa, South America and Oceania, which include samples from regions prior to and after deployments ART drugs. A total of 71 SNPs were identified, resulting in 106 nucleotide haplotypes. Similarly, many of the mutations were continent-specific and present at frequencies below 5%. The most predominant and perhaps the ancestral haplotype occurred in 441 samples and was present in 16 populations from Asia, Africa, and Oceania. The 3D7 haplotype found in 54 samples was the second most common haplotype and present in nine populations from all four continents. Assessment of the selection strength on pfatp6 in the 19 parasite populations found that pfatp6 in most of these populations was under purifying selection with an average d_N/d_S ratio of 0.333. Molecular evolution analyses did not detect significant departures from neutrality in pfatp6 for most populations, challenging the suitability of this gene as a marker for monitoring ART resistance.     

Stable allele frequency distribution of the polymorphic region of SURFIN(4.2) in Plasmodium falciparum isolates from Thailand

Plasmodium falciparum SURFIN_4.2 (PFD1160w) is a polymorphic protein expressed on the surface of parasite-infected erythrocytes. Such molecules are expected to be under strong host immune pressure, thus we analyzed the nucleotide diversity of the N-terminal extracellular region of SURFIN_4.2 using P. falciparum isolates obtained from a malaria hypoendemic area of Thailand. The extracellular region of SURFIN_4.2 was divided into four regions based on the amino acid sequence conservation among SURFIN members and the level of polymorphism among SURFIN_4.2 sequences; N-terminal segment (Nter), a cysteine-rich domain (CRD), a variable region 1 (Var1), and a variable region 2 (Var2). Comparison between synonymous and non-synonymous substitutions, Tajima's D test, and Fu and Li's D* and F* tests detected signatures of positive selection on Var2 and to a lesser extent Var1, suggesting that these regions were likely under host immune pressure. Strong linkage disequilibrium was detected for nucleotide pairs separated by a distance of more than 1.5 kb, and 7 alleles among 19 alleles detected in 1988–1989 still circulated 14 years later, suggesting low recombination of the analyzed surf_4.2 sequence region in Thailand. The allele frequency distribution of polymorphic areas in Var2 did not differ between two groups collected in different time points, suggesting the allele frequency distribution of this region was stable for 14 years. The observed allele frequency distribution of SURFIN_4.2 Var2 may be fixed in Thai P. falciparum population as similar to the observation for P. falciparum merozoite surface protein 1, for which a stable allele frequency distribution was reported.