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

Abstract

Abstract. The emergence of artemisinin resistance among Plasmodium falciparum in the Greater Mekong subregion threatens malaria control interventions and is associated with multiple unique mutations in K13 (PF3D7_1343700). The aim of this study was to survey Cambodian Plasmodium vivax for mutations in the K13 ortholog (K12, PVX_083080) that might similarly confer artemisinin resistance. Extracted DNA from Cambodian isolates collected between 2009 and 2012 was pooled by province and year and submitted for next-generation sequencing. Single-nucleotide polymorphisms (SNPs) were identified using a pile-up approach that detected minority SNPs. Among the 14 pools, we found six unique SNPs, including three nonsynonymous SNPs, across six codons in K12. However, none of the SNPs were orthologous to artemisinin resistance-conferring mutations in PF3D7_1343700, and nonsynonymous changes did not persist through time within populations. These results suggest a lack of selection in the P. vivax population in Cambodia due to artemisinin drug pressure.

A deep sequencing approach to estimate Plasmodium falciparum complexity of infection (COI) and explore apical membrane antigen 1 diversity

“…In order to investigate pfama1 diversity at both the individual and population level, individual samples (representing a malaria infection in a single person), and pooled samples (representing population cluster samples) were targeted in this study. Pooling samples is a cost-effective approach to amplicon-based deep sequencing as it reduces the number of PCR reactions and library preparations, and this pooled approach has been utilized in several malaria population genetic studies [ 68 – 71 ]. This dual sample type (individual and population cluster) approach allows for the examination of COI using the individual samples and also powers spatial population genetic analyses combining the individual samples and the pooled population cluster samples.…”

Plasmodium vivax Biology: Insights Provided by Genomics, Transcriptomics and Proteomics

“…This is indicative of a large effective population size in P. vivax that shows signs of population specific natural selection, where P. vivax is constantly adapting to human host and mosquito vector regional differences, and more recently, to antimalarial drug pressures (Neafsey et al, 2012 ; Winter et al, 2015 ; Hupalo et al, 2016 ; Parobek et al, 2016 ). These results were also seen at the local level for a high number of P. vivax samples from the Asian-Pacific region (Brazeau et al, 2016 ; Parobek et al, 2016 ; Pearson et al, 2016 ) and, in a smaller scale, in South American samples (Winter et al, 2015 ). The non-uniform distribution of the single nucleotide polymorphisms (SNPs) in P. vivax , clearly enriched at subtelomeric regions, is being interpreted as a result of local high recombination rates.…”

“…Several studies have highlighted the great degree of diversity, which is shared between P. vivax samples of different geographic locations at the population level, strongly suggesting that recent evolutionary selective pressures are currently acting upon some genes at a few loci, mainly those related with drug resistance (Mu et al, 2005 ; Imwong et al, 2007a ; Gunawardena et al, 2010 ; Neafsey et al, 2012 ; Lin et al, 2013 ). Compared to P. falciparum natural isolates from similar geographical regions (Cheeseman et al, 2016 ), results exposed a greater genetic diversity in P. vivax natural isolates, both for SNP and microsatellites (Karunaweera et al, 2008 ; Neafsey et al, 2012 ; Orjuela-Sanchez et al, 2013 ; Taylor et al, 2013 ; Barry et al, 2015 ; Winter et al, 2015 ; Brazeau et al, 2016 ; Friedrich et al, 2016 ; Hupalo et al, 2016 ; Pearson et al, 2016 ). While microsatellite marker analysis has revealed the genetic diversity of P. vivax infections, they may provide over- or underestimates.…”

“…The tailored and more affordable P. vivax SNPs barcode project (Baniecki et al, 2015 ), transcriptome profiling microarrays (Boopathi et al, 2016 ) and other breakpoint-specific PCR approaches (Auburn and Barry, 2017 ) can be used as important tools to not only discriminate P. vivax infections and their origin, but also to deliver information of its population dynamics of transmission (Friedrich et al, 2016 ). Additionally, they are helping to characterize and discern between recrudescence, relapses (Lin et al, 2015 ), de novo infections (Friedrich et al, 2016 ) and monitoring drug resistance emergence (Auburn et al, 2016b ; Brazeau et al, 2016 ). As for P. falciparum (Cheeseman et al, 2016 ), this greater genetic/expression diversity may provide P. vivax with a wide range of alternative paths for successful host erythrocyte invasion (Figures 5A,B ) and immune system evasion (Figure 5C ), thus enhancing its capacity of adaptation and survival (Cornejo et al, 2015 ).…”

Polymorphisms in Plasmodium falciparum Kelch 13 and P. vivax Kelch 12 Genes in Parasites Collected from Three South Pacific Countries Prior to Extensive Exposure to Artemisinin Combination Therapies

“…A Kelch 13 homolog in Plasmodium vivax, Kelch 12, was identified, and a V552I polymorphism was observed in 0.7% of isolates from Cambodia (52). Three nonsynonomous polymorphisms were identified in Cambodian isolates in a separate study, and none were orthologs of artemisinin-resistant K13 mutations (53).…”