Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing

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Wild-living chimpanzees and gorillas harbor a multitude of Plasmodium species, including six of the subgenus Laverania , one of which served as the progenitor of Plasmodium falciparum . Despite the magnitude of this reservoir, it is unknown whether apes represent a source of human infections. Here, we used Plasmodium species-specific PCR, single-genome amplification, and 454 sequencing to screen humans from remote areas of southern Cameroon for ape Laverania infections. Among 1,402 blood samples, we found 1,000 to be Plasmodium mitochondrial DNA (mtDNA) positive, all of which contained human parasites as determined by sequencing and/or restriction enzyme digestion. To exclude low-abundance infections, we subjected 514 of these samples to 454 sequencing, targeting a region of the mtDNA genome that distinguishes ape from human Laverania species. Using algorithms specifically developed to differentiate rare Plasmodium variants from 454-sequencing error, we identified single and mixed-species infections with P. falciparum , Plasmodium malariae , and/or Plasmodium ovale . However, none of the human samples contained ape Laverania parasites, including the gorilla precursor of P. falciparum . To characterize further the diversity of P. falciparum in Cameroon, we used single-genome amplification to amplify 3.4-kb mtDNA fragments from 229 infected humans. Phylogenetic analysis identified 62 new variants, all of which clustered with extant P. falciparum , providing further evidence that P. falciparum emerged following a single gorilla-to-human transmission. Thus, unlike Plasmodium knowlesi -infected macaques in southeast Asia, African apes harboring Laverania parasites do not seem to serve as a recurrent source of human malaria, a finding of import to ongoing control and eradication measures.

Section: Discussionmentioning

confidence: 52%

Plasmodium falciparum -like parasites infecting wild apes in southern Cameroon do not represent a recurrent source of human malaria

Sundararaman

Liu²,

Keele

et al. 2013

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“…No significant SNPs showed r 2 > 0.5 with any adjacent SNPs with MAF >0.05. Whole-genome sequencing of 227 P. falciparum field isolates indicated that r 2 fell below 0.10 within 1 kb in all parasite populations studied (18). The markers in our GWAS were, on average, 7 kb apart, so it may be that other loci associated with parasite clearance were not detected because they were not in LD with a genotyped SNP.…”

Section: Discussionmentioning

confidence: 98%

Genetic loci associated with delayed clearance of Plasmodium falciparum following artemisinin treatment in Southeast Asia

Takala-Harrison

Clark

Jacob

et al. 2012

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251

239

The recent emergence of artemisinin-resistant Plasmodium falciparum malaria in western Cambodia could threaten prospects for malaria elimination. Identification of the genetic basis of resistance would provide tools for molecular surveillance, aiding efforts to contain resistance. Clinical trials of artesunate efficacy were conducted in Bangladesh, in northwestern Thailand near the Myanmar border, and at two sites in western Cambodia. Parasites collected from trial participants were genotyped at 8,079 single nucleotide polymorphisms (SNPs) using a P. falciparum-specific SNP array. Parasite genotypes were examined for signatures of recent positive selection and association with parasite clearance phenotypes to identify regions of the genome associated with artemisinin resistance. Four SNPs on chromosomes 10 (one), 13 (two), and 14 (one) were significantly associated with delayed parasite clearance. The two SNPs on chromosome 13 are in a region of the genome that appears to be under strong recent positive selection in Cambodia. The SNPs on chromosomes 10 and 13 lie in or near genes involved in postreplication repair, a DNA damage-tolerance pathway. Replication and validation studies are needed to refine the location of loci responsible for artemisinin resistance and to understand the mechanism behind it; however, two SNPs on chromosomes 10 and 13 may be useful markers of delayed parasite clearance in surveillance for artemisinin resistance in Southeast Asia.drug resistance | genome-wide association | molecular markers

“…To test whether infections were monoclonal or polyclonal, we used the F WS statistic (86,87). To enable direct F WS comparisons between P. falciparum and P. vivax isolates, which had different numbers of loci, we bootstrapped each calculation 1,000 times, randomly selecting 5,000 variable sites for each isolate and each replicate.…”

Section: Discussionmentioning

confidence: 99%

Selective sweep suggests transcriptional regulation may underlie Plasmodium vivax resilience to malaria control measures in Cambodia

Parobek

Lin

Saunders

et al. 2016

Cambodia, in which both Plasmodium vivax and Plasmodium falciparum are endemic, has been the focus of numerous malaria-control interventions, resulting in a marked decline in overall malaria incidence. Despite this decline, the number of P. vivax cases has actually increased. To understand better the factors underlying this resilience, we compared the genetic responses of the two species to recent selective pressures. We sequenced and studied the genomes of 70 P. vivax and 80 P. falciparum isolates collected between 2009 and 2013. We found that although P. falciparum has undergone population fracturing, the coendemic P. vivax population has grown undisrupted, resulting in a larger effective population size, no discernable population structure, and frequent multiclonal infections. Signatures of selection suggest recent, species-specific evolutionary differences. Particularly, in contrast to P. falciparum, P. vivax transcription factors, chromatin modifiers, and histone deacetylases have undergone strong directional selection, including a particularly strong selective sweep at an AP2 transcription factor. Together, our findings point to different population-level adaptive mechanisms used by P. vivax and P. falciparum parasites. Although population substructuring in P. falciparum has resulted in clonal outgrowths of resistant parasites, P. vivax may use a nuanced transcriptional regulatory approach to population maintenance, enabling it to preserve a larger, more diverse population better suited to facing selective threats. We conclude that transcriptional control may underlie P. vivax's resilience to malaria control measures. Novel strategies to target such processes are likely required to eradicate P. vivax and achieve malaria elimination.D uring the last decade, western Cambodia has been the focus of numerous and multimodal interventions to control the spread of artemisinin-resistant Plasmodium falciparum (1, 2). Such interventions, including increased vector control, increased surveillance, and improved access to quality artemisinin-combination therapy (ACT), would be expected to curtail coendemic Plasmodium vivax as well. However, even as P. falciparum infections in Cambodia decreased by 81% between 2009 and 2013, P. vivax cases have increased, making it the predominant species in the Mekong region (3-6). This scenario, repeated in Brazil and other areas of coendemicity, has led to growing awareness that P. vivax, although infecting the same populations and transmitted by the same mosquito vectors, will likely be the more challenging species to eradicate (6-9). In this study, we use population genomics to gain insight into the evolutionary factors underlying P. vivax's resilience to malaria control measures.Population genetic studies have previously hinted at the resilience of P. vivax populations in comparison with P. falciparum. Studies of microsatellites and highly variable antigens of sympatric P. vivax and P. falciparum populations in Southeast Asia and the Southwest Pacific have consistently shown P. viv...