Plasmodium vivaxgenomic surveillance in the Peruvian Amazon with Pv AmpliSeq assay

Abstract

BackgroundPlasmodium vivaxis the most predominant malaria species in Latin America, constituting 71.5% of malaria cases in 2021. With several countries aiming for malaria elimination, it is crucial to prioritize effectiveness of national control programs by optimizing the utilization of available resources and strategically implementing necessary changes. To support this, there is a need for innovative approaches such as genomic surveillance tools that can investigate changes in transmission intensity, imported cases and sources of reintroduction, and can detect molecular markers associated with drug resistance.Methodology/Principal FindingsHere, we apply a modified highly-multiplexed deep sequencing assay: Pv AmpliSeq v2 Peru. The tool targets a newly developed 41-SNP Peru barcode for parasite population analysis within Peru, the 33-SNP vivaxGEN-geo panel for country-level classification, and 11 putative drug resistance genes. It was applied to 230 samples from the Peruvian Amazon (2007 – 2020), generating baseline surveillance data. We observed a heterogenousP. vivaxpopulation with high diversity and gene flow in peri-urban areas of Maynas province (Loreto region) with a temporal drift. In comparison, in an indigenous isolated area, the parasite population was genetically differentiated (FST=0.07-0.09) with moderate diversity and high relatedness between isolates in the community. In a remote border community, a clonalP. vivaxcluster was identified, with distinct haplotypes in drug resistant genes andama1, more similar to Brazilian isolates, likely representing an introduction ofP. vivaxfrom Brazil at that time. To test its applicability for Latin America, we evaluated the SNP Peru barcode inP. vivaxgenomes from the region and demonstrated the capacity to capture local population clustering at within-country level.Conclusions/SignificanceTogether this data shows thatP. vivaxtransmission is heterogeneous in different settings within the Peruvian Amazon. Genetic analysis is a key component for regional malaria control, offering valuable insights that should be incorporated into routine surveillance.Author SummaryLatin America is aiming towards malaria elimination. Genomic surveillance is crucial for a country’s malaria strategy, aiding in understanding and stopping the spread of the disease. While widely used for another malaria species (Plasmodium falciparum), limited tools exist for trackingP. vivax, a significant player in malaria-endemic areas outside of Africa, and the primary cause of malaria in Latin America.In this study, we used a new tool, Pv AmpliSeq v2 Peru assay, to examine the genetic makeup of malaria parasites in the Peruvian Amazon. This tool helps us see how the parasites from different areas are connected and tracks markers that could indicate resistance to drugs. We found that the parasites from remote areas in the Amazon were genetically different from parasites in areas surrounding the main city of Iquitos, and parasites in a remote border community were genetically more similar to Brazilian parasites.We also show that the Pv AmpliSeq v2 Peru assay can be used to study parasites from other countries in Latin America, highlighting the broader application in the region. Considering that parasites are not constrained by borders and can easily spread between neighboring countries, a regional approach can be crucial for malaria elimination.

Genomic surveillance of malaria parasites in an indigenous community in the Peruvian Amazon

“…Using Ampliseq, our team reported Pf lineages with low genetic diversity, double pfhrp2/3 deletion, and resistance haplotypes, except for ART, predominant after 2014 11 . In addition, we showed that Pv transmission is heterogeneous in different settings in the Peruvian Amazon, with high diversity in areas close to Loreto and lower diversity in border areas 13 . However, our molecular surveillance studies mainly focus on urban areas or accessible rural communities.…”

“…Pv and Pf AmpliSeq Peru library preparation was performed as previously described 11,13,26 using the AmpliSeq Library PLUS kit (Illumina). Brie y, each sample (7.5 µl of DNA) was ampli ed by PCR using two different sets of primer panels.…”

“…Recently, we validated two targeted next-generation sequencing assays (Pf and Pv AmpliSeq Peru) for malaria molecular surveillance 11,13 . The AmpliSeq assays include country-speci c SNP barcodes, molecular resistance markers, and pfhrp2/3 genes.…”

“…Pf populations showed low to moderate genetic diversity across time and space 5,6 , with some outbreaks [7][8][9] and predominant lineages since the mid-2010s 10,11 . On the other hand, Pv populations are more diverse than Pf, with a variable prevalence of polyclonal infections (10-80%) and gene ow even among distant areas 5,[12][13][14] .…”

“…Mutations in pvmdr1 and pvcrt have also been found, with no correlation to resistant phenotypes 5,13 .…”