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

Abstract

Summary 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.

Adaptive evolution of malaria parasites in French Guiana: Reversal of chloroquine resistance by acquisition of a mutation in pfcrt

“…Our drug profiling data showed a significant loss of mdAQ resistance in field isolates harboring the PfCRT C350R mutation, consistent with earlier reports that highlighted the close correlation between CQ and mdAQ responses of PfCRT SVMNT parasites from South America (14). Our observation that mutant C350R isolates were nominally less susceptible to DHA, however, should be interpreted with caution, notably because in vitro artemisinin susceptibilities, as measured using IC 50 values, do not correlate with the clinical phenotype of delayed parasite clearance rates in southeast Asia (27,28).…”

Polymorphisms in the K13-Propeller Gene in Artemisinin-Susceptible Plasmodium falciparum Parasites from Bougoula-Hameau and Bandiagara, Mali

“…The SNPs described in this setting could be relevant as markers associated with artemisinin resistance. In vitro assessment of artemisinin resistance through the ring-stage survival assay 14 will be required to rule out resistance in these K13-propeller mutants from these high transmission areas of Mali, where resistant parasites can be cleared by the immune system. It is also possible that resistance requires the presence of additional mutations at secondary loci that are not yet present in Africa but that are present in southeast Asia (i.e., K13-propeller mutations are necessary but not sufficient for the manifestation of resistance).…”

Genetic markers of artemisinin resistance in Plasmodium spp. parasites

“…artesunate is activated for the rest of intra-erythrocytic development, and thus, K13 variant parasites are fully susceptible to prolonged exposure to artemisinin, as clearly seen in vitro [2,4,5] and in vivo [3,22,23].…”

“…Curiously, the in vivo slow-clearance phenotype of P. falciparum, spreading through the GMS, was not consistently reflected in estimates of the 50% effective concentration (EC 50 ) of dihydroartemisinin (DHA, the dominant parasiticidal artemisinin metabolite in vivo) against parasites cultured in vitro [2,4]. Only after the Pasteur Institute teams in Phnom Penh and Paris had devised an in vitro DHA susceptibility test specific for the earliest blood-stage parasites, the ring-stage survival assay (RSA) [5], Ariey et al [6] were then able to demonstrate that certain mutations in the pfk13 locus correlated with the ex vivo parasite phenotype in the RSA. This key locus was identified as a candidate for testing not by genome-wide association studies (GWASs), an approach that promised much [7] but delivered little [8] in terms of identifying specific markers of artemisinin susceptibility, but through 5 years of painstaking drug selection in the laboratory [6].…”