Optimizing the application of the AmpliSeq assays as molecular malaria surveillance tools in Peru and the Amazon basin using bioinformatics and laboratory approaches.

Malaria remains a significant health challenge globally, particularly in the Amazon Basin, which accounts for most malaria cases in Latin America (LATAM). Despite efforts in the region, malaria persists due to several challenges for control and elimination, including transmission in remote and border areas, human and environmental factors, difficulties in diagnosis and potential drug resistance. In Peru, the fourth-highest malaria burden country in LATAM, malaria occurs predominant in the Loreto region, with P. vivax (Pv) and P. falciparum (Pf) contributing to 85% and 15% of infections, respectively. A national malaria elimination program (NMEP) was launched in 2022 aiming to reduce malaria cases by 90% in 2030. Malaria molecular surveillance (MMS) has a big potential to contribute to decision-making by providing evidence for tailored strategies and interventions. MMS is not incorporated in Peru’s NMEP but the elimination plan took into account evidence from small scale molecular surveillance studies. Our team has developed novel MMS tools: Pv AmpliSeq v2 Peru and Pf AmpliSeq v1 Peru assays and validated them using hundreds of samples from Loreto. These are targeted next generation sequencing (NGS) assays based on deep amplicon sequencing that serve applications (“uses cases”) related to transmission dynamics (with country-specific and global SNP barcodes), drug resistance (with 11–17 resistance associated genes) and performance of rapid diagnostic tests (RDTs) based on histidine rich protein 2 and 3 (pfhrp2/3) genes in P. falciparum. With the AmpliSeq assays, we have described recent P. falciparum lineages and characterized the heterogeneous P. vivax transmission in Peru.

In this PhD project, I aim to optimize the application of these AmpliSeq assay in order to address the specific challenges of malaria elimination in Peru and the Amazon region. First, I will explore the population structure and parasite connectivity of P. vivax populations in LATAM (WP1), including new regions within Peru (Amazonas and Tumbes) and neighboring countries in LATAM (Brazil, Colombia, Ecuador, Venezuela) using our Pv AmpliSeq Peru assay. Moreover, I will improve the Pf AmpliSeq Peru assay by facing challenges related to it (WP2). These include the update of the bioinformatic algorithm for calling pfhrp2/3 gene deletions, the investigation of novel mutations in pfcrt gene (associated to chloroquine resistance) and the revision the Peru SNP barcode to have better resolution at the LATAM level. Finally, I will phenotypically characterize the drug susceptibility profiles of novel genetic variants in P. falciparum found in Peru with our AmpliSeq assay (WP3). This project will improve and standardize protocols and generate important data for MMS interpretation, essential before implementation of MMS tools in Peru and other countries in the region, ultimately contributing efforts towards malaria elimination.