Malaria molecular surveillance in the Peruvian Amazon with novel highly multiplexed Plasmodium falciparum Ampliseq assay

Abstract

AbstractBackgroundMalaria molecular surveillance has great potential to support local national malaria control programs (NMCPs) to inform policy for malaria control and elimination. Molecular markers associated with drug resistance are good predictors of treatment responses. In addition, molecular detection of deletions in hrp2 and hrp3 genes are indicative of potential failure of HRP2-based rapid diagnostic tests. However, there is an urgent need for feasible, cost-effective and fast molecular surveillance tools that NMCPs can implement.MethodsHere we present a new 3-day workflow for targeted resequencing of markers in 13 resistance-associated genes, hrp2&3, a country-specific 28 SNP-barcode for population genetic analysis, and ama1. The assay was applied to control isolates and retrospective samples collected between 2003-2018 in the Loreto region (n = 254) in Peru. Pf AmpliSeq libraries were prepared using a multiplex PCR simultaneously amplifying a high number of targets from dried blood spots and sequenced at high coverage (median 1336, range 20-43795).ResultsThere was no evidence suggesting the emergence of artemisinin resistance in Peru. However, alleles in ubp1 and coronin contributed to recent genetic differentiation of the parasite population. After 2008, predominant parasite lineages in Peru are resistant to sulfadoxine-pyrimethamine (sextuple dhfr/dhps mutant) and chloroquine (SVMNT in crt and NDFCDY in mdr1) and can escape HRP2 based RDTs.ConclusionsThese findings indicate a parasite population under drug pressure, and demonstrates the added value of molecular surveillance systems and offers a highly multiplexed surveillance tool. The targets in the assay can be easily adjusted to suit the needs of other settings.FundingThis work was funded by the Belgium Development Cooperation (DGD) under the Framework Agreement Program between DGD and ITM (FA4 Peru, 2017-2021) and the sample collections in 2018 were supported by VLIR-UOS (project PE2018TEA470A102; University of Antwerp). Funding for the sample collections lead by the U.S. Naval Medical Research Unit 6 (NAMRU-6) in 2011 and 2012 was provided by the Armed Forces Health Surveillance Division (AFHSD) and its Global Emerging Infections Surveillance and Response (GEIS) Section (P0144_20_N6_01, 2020-2021).