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

Malaria Molecular Epidemiology Unit, Institut Pasteur du Cambodge, Phnom Penh, Cambodia.
The Lancet Infectious Diseases (Impact Factor: 22.43). 09/2013; 13(12). DOI: 10.1016/S1473-3099(13)70252-4
Source: PubMed


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

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Available from: Didier Menard, May 22, 2015
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    • "Understanding the mechanism of action of ARTs in early rings is important given the recent emergence of resistance to ARTs in South East Asia (Tun et al., 2015). This manifests as slowed parasite clearance in patients, which correlates with decreased in vitro drug sensitivity of the youngest rings (Witkowski et al., 2013). In this work we present a detailed analysis of the roles of free iron and haem in the activity of ARTs at different stages of intraerythrocytic development. "
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    • "Seven SNPs were assessed using a PCR-LDR-FMA (PCR-Ligase Detection Reaction-Fluorescence Microspheres Assay). Protocols, PCR/nested PCR primer sequences, and LDR probe sequences (assays No. 8, 9, 12, 16, 19, 20 and 24) are available in [22]. Nearby villages were pooled together in 20 clusters based on a visual inspection of the distribution of villages in the province. "
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