Lack of Artemisinin Resistance in Plasmodium falciparum in Uganda Based on Parasitological and Molecular Assays

Abstract

We evaluated markers of artemisinin resistance in Plasmodium falciparum isolated in Kampala in 2014. By standard in vitro assays, all isolates were highly sensitive to dihydroartemisinin (DHA). By the ring-stage survival assay, after a 6-h DHA pulse, parasitemia was undetectable in 40 of 43 cultures at 72 h. Two of 53 isolates had nonsynonymous K13-propeller gene polymorphisms but did not have the mutations associated with resistance in Asia. Thus, we did not see evidence for artemisinin resistance in Uganda.A rtemisinin-based combination therapy is now the standard for treating P. falciparum malaria. However, this regimen is threatened by resistance to artemisinins, which manifests as delayed clearance of parasitemia after therapy, in Southeast Asia (1, 2). Recently, resistance has been associated with increased parasitemia in culture, compared to that in sensitive parasites, 72 h after a 6-h pulse with dihydroartemisinin (DHA) and has been associated with polymorphisms in propeller-encoding domains of the Plasmodium falciparum kelch (K13; UniProt number PF3D7_1343700) gene (3-6). Although artemisinin resistance is evident to date only in Southeast Asia, the bulk of P. falciparum malaria occurs in sub-Saharan Africa. Clinical resistance has not been noted in Africa, as rapid parasite clearance has been the rule in many trials, including those in Uganda (7-9). In recent surveys in Uganda (10) and other locations in Africa (11-15), K13-propeller gene polymorphisms were identified, but these were not the mutations clearly associated with artemisinin resistance in Asian isolates. Results for a new correlate of artemisinin resistance, the ex vivo ring-stage survival assay (RSA) (3), have not been reported previously for P. falciparum isolates from Africa. We characterized artemisinin sensitivity by this assay and assessed K13 polymorphisms in isolates from Uganda. Parasites were collected from patients diagnosed with malaria from May to August 2014 at Mulago Hospital, Kampala. Samples were delinked from patient information, so clinical data were unavailable. After malaria diagnosis, excess blood collected in a heparinized tube as part of clinical care was promptly delivered to the laboratory. Giemsastained thin smears were made, and samples containing only P. falciparum isolates with parasitemia of Ն0.1% were placed in culture after washing of erythrocytes, as previously described (16); blood was also spotted onto filter paper. Samples with parasitemia of Ͼ1% were diluted with uninfected erythrocytes for a final parasitemia of 1%.The susceptibility of fresh isolates to DHA and chloroquine was assessed by a standard ex vivo histidine-rich protein 2-based enzyme-linked immunosorbent microplate assay, as previously described (17), except that drugs were freshly diluted from frozen stocks prior to each assay. Results from single assays were fitted to a variable-slope sigmoidal function using GraphPad Prism 6.0f to generate 50% inhibitory concentrations (IC 50 s). All data were visually assessed for parasite growth above...

Changing Antimalarial Drug Sensitivities in Uganda

“…As seen in prior studies from Uganda, occasional k13 mutations were seen (12, 29, 33), but these were not the mutations associated with artemisinin resistance in Southeast Asia, and prevalence was not associated with drug sensitivity or recent drug pressure. In addition, as seen recently in parasites from Kampala (29), the ex vivo DHA ring survival assay did not suggest artemisinin resistance in Ugandan parasites. Similarly, an increase in plasmepsin 2 copy number, a newly identified marker associated with piperaquine resistance, was also seen occasionally in Ugandan isolates, but it was not associated with piperaquine sensitivity in recent isolates or in a comparison of older isolates with relatively high or low piperaquine sensitivity.…”

“…The A578S mutation has been described in isolates from Uganda and other African countries, and it has not been associated with artemisinin resistance (29,32,33). Recent reports have identified amplification of a gene encoding plasmepsin 2 and a SNP in an exonuclease gene (PF3D7_1362500) that encodes an E415G mutation as markers of piperaquine resistance in Southeast Asia (24,25).…”

“…IC 50 s were derived by plotting percent growth against log drug concentration and fitting the data by variable slope, sigmoidal curve fit in Prism 6.0 (GraphPad Software, San Diego, CA). DHA drug susceptibility was measured with an ex vivo ring stage survival assay as previously described (29).…”

Antimalarial Drug Resistance: A Threat to Malaria Elimination

“…In Africa, highly diverse and low-frequent K13 mutant alleles have been observed, with no evidence of selection, and none of these were associated with clinical artemisinin resistance assessed by the presence of parasites on day 3 following artesunate monotherapy or a 3-d ACT course. It is thought that artemisinin resistance has not been established in Africa, supported by the additional absence of evidence of invasion by Asian K13 alleles validated as molecular marker of artemisinin resistance (C580Y, R539T, I543T, Y493H), confirming previous smaller-sized studies (Conrad et al 2014;Torrentino-Madamet et al 2014;Cooper et al 2015;Escobar et al 2015;Hawkes et al 2015;Isozumi et al 2015;Kamau et al 2015;Taylor et al 2015). Haplotyping studies on the most common African mutant 578A !…”

Genetic markers of artemisinin resistance in Plasmodium spp. parasites

“…• studies of the distribution of polymorphisms in the field, particularly in Africa [9][10][11][12][13];…”