Genetic Analysis and Species Specific Amplification of the Artemisinin Resistance-Associated Kelch Propeller Domain in P. falciparum and P. vivax

Abstract

Plasmodium falciparum resistance to artemisinin has emerged in the Greater Mekong Subregion and now poses a threat to malaria control and prevention. Recent work has identified mutations in the kelch propeller domain of the P. falciparum K13 gene to be associated artemisinin resistance as defined by delayed parasite clearance and ex vivo ring stage survival assays. Species specific primers for the two most prevalent human malaria species, P. falciparum and P. vivax, were designed and tested on multiple parasite isolates including human, rodent, and non- humans primate Plasmodium species. The new protocol described here using the species specific primers only amplified their respective species, P. falciparum and P. vivax, and did not cross react with any of the other human malaria Plasmodium species. We provide an improved species specific PCR and sequencing protocol that could be effectively used in areas where both P. falciparum and P. vivax are circulating. To design this improved protocol, the kelch gene was analyzed and compared among different species of Plasmodium. The kelch propeller domain was found to be highly conserved across the mammalian Plasmodium species.

Emergence and Spread of kelch13 Mutations Associated with Artemisinin Resistance in Plasmodium falciparum Parasites in 12 Thai Provinces from 2007 to 2016

“…Malaria diagnosis was confirmed using the previously described photoinduced electron transfer fluorogenic (PET)-PCR method (34,35). The K13 propeller domain was amplified using a P. falciparumspecific nested-PCR method as previously described (36). Briefly, two microliters of genomic DNA was amplified using 0.5 M each primer, 0.2 mM deoxynucleoside triphosphate (dNTP), 3 and 2 mM MgCl 2 for the primary and secondary reactions, respectively, and 1 U High Fidelity Phusion Taq (New England BioLabs).…”

Molecular Epidemiology of Plasmodium falciparum kelch13 Mutations in Senegal Determined by Using Targeted Amplicon Deep Sequencing

“…The Pfk13 gene (PF3D7_1343700) was amplified using a high-fidelity polymerase (New England BioLabs, USA [catalog no. M0530L]) using species-specific primers (primary PCR primers) as previously described (32). The PCR amplicon size was 2,120 bp prior to shearing and adaptor and index addition using the Nextera XT v2 kit (Illumina, USA 1 and 2).…”

High levels of imported asymptomatic malaria but limited local transmission in KwaZulu-Natal, a South African malaria-endemic province nearing malaria elimination

“…Samples from any positive master pool were tested individually, followed by AluI enzyme restriction digestion for species determination. All falciparum-positive samples were assessed for the markers associated with artemisinin resistance [18] and lumefantrine tolerance [19,20] as well as genotyped using 26 neutral microsatellite markers [21]. All collected blood samples, irrespective of malaria status, were also assessed for gametocyte carriage using the reverse transcriptase PCR method of Mlambo et al [22].…”