Novel phenotypic assays for the detection of artemisinin-resistant Plasmodium falciparum malaria in Cambodia: in-vitro and ex-vivo drug-response studies

Abstract

Background: Artemisinin resistance in Plasmodium falciparum lengthens parasite clearance half-life during artemisinin monotherapy or artemisinin-based combination therapy. Absence of in-vitro and ex-vivo correlates of artemisinin resistance hinders study of this phenotype. We aimed to assess whether an in-vitro ring-stage survival assay (RSA) can identify culture-adapted P falciparum isolates from patients with slow-clearing or fast-clearing infections, to investigate the stage-dependent susceptibility of parasites to dihydroartemisinin in the in-vitro RSA, and to assess whether an ex-vivo RSA can identify artemisinin-resistant P falciparum infections.

Methods: We culture-adapted parasites from patients with long and short parasite clearance half-lives from a study done in Pursat, Cambodia, in 2010 (registered with ClinicalTrials.gov, number NCT00341003) and used novel in-vitro survival assays to explore the stage-dependent susceptibility of slow-clearing and fast-clearing parasites to dihydroartemisinin. In 2012, we implemented the RSA in prospective parasite clearance studies in Pursat, Preah Vihear, and Ratanakiri, Cambodia (NCT01736319), to measure the ex-vivo responses of parasites from patients with malaria. Continuous variables were compared with the Mann-Whitney U test. Correlations were analysed with the Spearman correlation test.

Findings: In-vitro survival rates of culture-adapted parasites from 13 slow-clearing and 13 fast-clearing infections differed significantly when assays were done on 0-3 h ring-stage parasites (10·88% vs 0·23%; p=0·007). Ex-vivo survival rates significantly correlated with in-vivo parasite clearance half-lives (n=30, r=0·74, 95% CI 0·50-0·87; p<0·0001).

Interpretation: The in-vitro RSA of 0-3 h ring-stage parasites provides a platform for the molecular characterisation of artemisinin resistance. The ex-vivo RSA can be easily implemented where surveillance for artemisinin resistance is needed.

Funding: Institut Pasteur du Cambodge and the Intramural Research Program, NIAID, NIH.

Figure 1. Dihydroartemisinin survival assays

Synchronisation and timing of DHA exposure (A) for four in-vitro survival assays—RSA, previously described by Witkowski and colleagues,20 RSA^{0–3 h}, RSA^{9–12 h}, and TSA^{18–21 h}—done on culture-adapted Plasmodium falciparum isolates. During their 48 h cycle of intraerythrocytic development, parasites circulate as ring-stages (0–18 h) and then sequester by specifically adhering to the endothelium of microvessels, where they mature into trophozoites (18–36 h) and schizonts (36–48 h). Because of sequestration, clinical studies assess the clearance rate of circulating ring-stage parasites only. In individual patients, the actual age-distribution of parasites circulating in peripheral blood is unknown and can vary from patient to patient. The timing of dihydroartemisinin exposure for the ex-vivo survival assay (B) done on circulating, ring-stage parasites (0–18 h) obtained directly from the blood of patients with uncomplicated malaria. This assay thus measures the dihydroartemisinin susceptibility of the parasite isolate at the same developmental stage and at the same time as the in-vivo parasite clearance study. DHA=dihydroartemisnin. RSA=ring-stage survival assay. TSA=trophozoite-stage survival assay.

Figure 2. In-vitro survival after exposure to dihydroartemisinin

Results are expressed as the proportion of viable Plasmodium falciparum parasites after a 6 h exposure of 0–3 h rings (RSA^{0–3 h}), 9–12 h rings (RSA3^{9–12 h}), and 18–21 h trophozoites (TSA^{18–21 h}) to 700 nM dihydroartemisinin compared with dimethyl sulfoxide. These assays were done on culture-adapted parasite isolates obtained from 13 patients with fast-clearing infections (filled circles) and 13 patients with slow-clearing infections (open circles) in Pursat in 2010. The horizontal lines represent the medians and whiskers the IQRs. The solid lines show stage-dependent survival pattern of parasites from slow-clearing infections and the dotted lines the stage-dependent survival pattern of parasites from fast-clearing infections. RSA=ring-stage survival assay. TSA=trophozoite-stage survival assay.

Figure 3. Correlation of in-vivo parasite clearance half-lives and ex-vivo dihydroartemisinin survival rates

Ex-vivo ring-stage survival assays (RSAs) were done on parasite isolates obtained directly from patients with malaria in Pursat, Preah Vihear, and Ratanakiri in 2012. Results from the ex-vivo RSAs are expressed as the proportion of viable parasites after a 6 h exposure to 700 nM dihydroartemisinin compared with dimethyl-sulfoxide-exposed controls. Results from the parasite clearance studies are expressed as the parasite clearance half-life in hours. The proportion of viable parasites in ex-vivo RSAs correlated significantly with the parasite clearance half-life (r=0·74, 95% CI 0·50–0·87; p<0·0001) in Pursat (red), Preah Vihear (blue), and Ratanakiri (green).