Author(s):
Brown, Noah ; da Silva, Clemente ; Webb, Caroline ; Matias, Daniela ; Dias, Brigite ; Cancio, Beatriz ; Silva, Miguel ; Viegas, Ruben ; Salvador, Crizolgo ; Chivale, Nordino ; Luis, Sonia ; Arnaldo, Paulo ; Zulawinska, Julia ; Moore, Christopher C ; Nogueira, Fatima ; Guler, Jennifer L
Date: 2024
Persistent ID: http://hdl.handle.net/10362/168813
Origin: Repositório Institucional da UNL
Subject(s): copy number variation; droplet digital PCR; drug resistance; malaria; multidrug resistance; plasmepsin; General Biochemistry,Genetics and Molecular Biology; Infectious Diseases; Parasitology; SDG 3 - Good Health and Well-being
Description
Funding text The authors would like to acknowledge Funda\u00E7\u00E3o para a Ci\u00EAncia e a Tecnologia for funds to GHTM (UID/04413/2020) and LA-REAL (LA/P/0117/2020) and Instituto CAM\u00D5ES (https://www.instituto-camoes.pt/) BOLSAS CAM\u00D5ES, FUNDA\u00C7\u00C3O MILLENNIUM BCP (https://www.fundacaomillenniumbcp.pt/en/), Par\u00F3quia de S\u00E3o Nicolau \u2013 Lisboa (to C.D.S.), R01AI150856 (to J.L.G.), the University of Virginia Global Infectious Disease Institute (to C.W. and N.B.), and Center for Global Health Equity (to C.W.). We thank Michelle Warthan (University of Virginia) for laboratory parasite culturing and Dr. Michael Klemba (Virginia Tech) for genomic DNA from the PM2GT clone F4 parasite line.
While the Plasmodium falciparum malaria parasite continues to cause severe disease globally, Mozambique is disproportionally represented in malaria case totals. Acquisition of copy number variations (CNVs) in the parasite genome contributes to antimalarial drug resistance through overexpression of drug targets. Of interest, piperaquine resistance is associated with plasmepsin 2 and 3 CNVs ( pfpmp2 and pfpmp3, respectively), while CNVs in the multidrug efflux pump, multidrug resistance-1 ( pfmdr1), increase resistance to amodiaquine and lumefantrine. These antimalarials are partner drugs in artemisinin combination therapies (ACTs) and therefore, CNV detection with accurate and efficient tools is necessary to track ACT resistance risk. Here, we evaluated ~300 clinically derived samples collected from three sites in Mozambique for resistance-associated CNVs. We developed a novel, medium-throughput, quadruplex droplet digital PCR (ddPCR) assay to simultaneously quantify the copy number of pfpmp3, pfpmp2, and pfmdr1 loci in these clinical samples. By using DNA from laboratory parasite lines, we show that this nanodroplet-based method is capable of detecting picogram levels of parasite DNA, which facilitates its application for low yield and human host-contaminated clinical surveillance samples. Following ddPCR and the application of quality control standards, we detected CNVs in 13 of 229 high-quality samples (prevalence of 5.7%). Overall, our study revealed a low number of resistance CNVs present in the parasite population across all three collection sites, including various combinations of pfmdr1, pfpmp2, and pfpmp3 CNVs. The potential for future ACT resistance across Mozambique emphasizes the need for continued molecular surveillance across the region.
