Population genomics and evidence of clonal replacement of Plasmodium falciparum in the Peruvian Amazon

Abstract

Previous studies have shown that P. falciparum parasites in South America have undergone population bottlenecks resulting in clonal lineages that are differentially distributed and that have been responsible for several outbreaks different endemic regions. In this study, we explored the genomic profile of 18 P. falciparum samples collected in the Peruvian Amazon Basin (Loreto) and 6 from the Peruvian North Coast (Tumbes). Our results showed the presence of three subpopulations that matched previously typed lineages in Peru: Bv1 (n = 17), Clonet D (n = 4) and Acre-Loreto type (n = 3). Gene coverage analysis showed that none of the Bv1 samples presented coverage for pfhrp2 and pfhrp3. Genotyping of drug resistance markers showed a high prevalence of Chloroquine resistance mutations S1034C/N1042D/D1246Y in pfmdr1 (62.5%) and K45T in pfcrt (87.5%). Mutations associated with sulfadoxine and pyrimethamine treatment failure were found on 88.8% of the Bv1 samples which were triple mutants for pfdhfr (50R/51I/108N) and pfdhps (437G/540E/581G). Analysis of the pfS47 gene that allows P. falciparum to evade mosquito immune responses showed that the Bv1 lineage presented one pfS47 haplotype exclusive to Loreto and another haplotype that was present in both Loreto and Tumbes. Furthermore, a possible expansion of Bv1 was detected since 2011 in Loreto. This replacement could be a result of the high prevalence of CQ resistance polymorphisms in Bv1, which could have provided a selective advantage to the indirect selection pressures driven by the use of CQ for P. vivax treatment.

Figure 1

Collection sites. Samples for this study came from P. falciparum specimens collected in the Peruvian North Coast (red dot) and Amazon Basin (blue bot) between 2006 and 2017. The map was created using ArcGIS online (ESRI Inc. Redlands, CA, USA. https://www.esri.com/) using open data obtained from GADM database of Global Administrative Areas, version 3.6. www.gadm.org.

Figure 2

Genomic wide scans of SNP density and Tajima’s D. The figure shows SNP density in quartiles (blue, orange, red and dark red) across the 14 P. falciparum chromosomes (internal dot plot) showing regions with high density on chromosomes 1, 2, 4, 5, 7, 8, 11, 13 and 14. Population scaled Tajima’s D shows regions under positive selection on chromosomes 1–6, 8, 10–14 (external blue line plot). Coordinates of genes under positive (red) or negative (blue) selection are shown as lines after the Tajimas’D line plot.

Figure 3

Read depth on pfhrp2 and pfhrp3 for all samples. (A) Three samples presented coverage for the pfhrp2 gene. (B) Four samples presented coverage for the pfhrp3 gene. None of the Bv1-type samples presented coverage on pfhrp2 nor pfhrp3. The y-axis denote read depth whereas the x-axis indicates the nucleotide position in base pairs in chromosomes 8 (A) and 13 (B), respectively. Pfhrp2 and pfhrp3 gene positions are showed as boxes on the x-axis. In order to facilitate visualization, read depth was plotted using a sliding window of 500 bp and samples with coverage along the gene have been plotted as green shade colored lines.

Figure 4

Population structure of P. falciparum samples. (A) Principal component analysis (PCA) showing three potential clusters for samples from Tumbes and Loreto. (B) Admixture analysis confirms the presence of these three Peruvian subpopulations, one of these (blue) that included all Bv1-type samples from the Peruvian Amazon Basin and North Coast regions and the other two corresponding to Clonet D and Acre-Loreto type, respectively. African reference samples were included as controls.

Figure 5

Maximum likelihood phylogenetic analysis. The phylogenetic tree shows the presence of three subpopulations from which only one (Bv1-type) was exclusively isolated since 2011 in the Peruvian Amazon Basin suggesting a population replacement in that region. Most of the branches of the tree are strongly supported by bootstrap values equal to 1000.

Figure 6

Structural diversity of pfS47 (A) Haplotype network for Peruvian isolates and control samples from Brazil, Africa and Asia. The network shows the distribution of the typed Peruvian lineages according to their pfS47 haplotype. (B) Four PfS47 circulating haplotypes in Peru based on three amino acid positions associated with evasion to mosquito immune response. D1, D2 and D3 represents protein domains and 68, 236 and 242 amino acid positions.