Culture-adapted Plasmodium falciparum isolates from UK travellers: in vitro drug sensitivity, clonality and drug resistance markers

Abstract

BackgroundThe screening of lead compounds against in vitro parasite cultures is an essential step in the development of novel anti-malarial drugs, but currently relies on laboratory parasite lines established in vitro during the last century. This study sought to establish in continuous culture a series of recent Plasmodium falciparum isolates to represent the current parasite populations in Africa, all of which are now exposed to artemisinin combination therapy.MethodsPre-treatment P. falciparum isolates were obtained in EDTA, and placed into continuous culture after sampling of DNA. One post-treatment blood sample was also collected for each donor to monitor parasite clonality during clearance in vivo. IC50 estimates were obtained for 11 anti-malarial compounds for each established parasite line, clonal multiplicity measured in vivo and in vitro, and polymorphic sites implicated in parasite sensitivity to drugs were investigated at the pfmdr1, pfcrt, pfdhfr, pfdhps and pfap2mu loci before and after treatment, and in the cultured lines.ResultsPlasmodium falciparum isolates from seven malaria patients with recent travel to three West African and two East African countries were successfully established in long-term culture. One of these, HL1211, was from a patient with recrudescent parasitaemia 14 days after a full course of artemether-lumefantrine. All established culture lines were shown to be polyclonal, reflecting the in vivo isolates from which they were derived, and at least two lines reliably produce gametocytes in vitro. Two lines displayed high chloroquine IC50 estimates, and carried the CVIET haplotype at codons 72–76, whereas the remaining five lines carried the CVMNK haplotype and were sensitive in vitro. All were sensitive to the endoperoxides dihydroartemisinin and OZ277, but IC50 estimates for lumefantrine varied, with the least sensitive parasites carrying pfmdr1 alleles encoding Asn at codon 86.ConclusionsThis study describes the establishment in continuous culture, in vitro drug sensitivity testing and molecular characterization of a series of multiclonal P. falciparum isolates taken directly from UK malaria patients following recent travel to various malaria-endemic countries in Africa. These “HL” isolates are available as an open resource for studies of drug response, antigenic diversity and other aspects of parasite biology.

Directional Selection at the pfmdr1, pfcrt, pfubp1, and pfap2mu Loci of Plasmodium falciparum in Kenyan Children Treated With ACT

“…Products were characterized by direct sequencing as described [22]. Ambiguous mixed haplotypes of pfmdr1 were excluded from the analysis as previously described [14].…”

Identification and Deconvolution of Cross-Resistance Signals from Antimalarial Compounds Using Multidrug-Resistant Plasmodium falciparum Strains

“…In particular, the addition of P. falciparum isolates recently adapted for laboratory culture to the panel was necessary to provide further segregation between resistance mediated by pfcrt and that mediated by pfdhfr, revealing a shortcoming in the ability of the original panel of strains to fully deconvolute cross-resistance signals. These two isolates are also evolutionarily closer to the current target parasite populations than the standard laboratory strains, most of which were culture adapted more than 20 years ago (30).…”

“…The multiclonal parasite lines HL1210 and HL1212 were isolated from patients presenting to the Hospital for Tropical Diseases in London, United Kingdom, in 2012 after returning from travel to Ghana and Nigeria, respectively, and are described in detail elsewhere (30). Both strains originated from areas of endemicity where ACT has been in use since the mid-2000s and thus likely have prior exposure to lumefantrine, dihydroartemisinin, and potentially other ACT partner drugs.…”

“…To evaluate the implication of pfcrt mutations in the resistance to this chemical series in a more direct manner, we took advantage of P. falciparum field isolates recently adapted to laboratory culture and fully characterized for key determinants of resistance (30). More particularly, we selected isolates HL1210 and HL1212, which share the same pfdhfr triple mutation haplotype and differ in their pfcrt sequences.…”

A Novel Multiplex qPCR Assay for Detection ofPlasmodium falciparumwithHistidine-rich Protein 2 and 3 (pfhrp2 and pfhrp3)Deletions in Polyclonal Infections

“…A culture-adapted isolate lacking both genes (3BD5, double deletion) was obtained from Thomas Wellems (NIAID, US). Parasite cultures of 3D7, Dd2, HB3 and 3BD5 were tightly synchronized as ring stage trophozoites in vivo to simulate infected peripheral blood similar to previously used methods (14,15). P. falciparum International standard (Pf INT), a reagent comprising lyophilised whole blood from a single hyperparasitaemic individual was obtained from NIBSC UK.…”