Novel phenotypic assays for the detection of artemisinin-resistant Plasmodium falciparum malaria in Cambodia: in-vitro and ex-vivo drug-response studies
ABSTRACT 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 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.
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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ABSTRACT: Background Artemisinin-based combination therapy (ACT) is the recommended first-line treatment of falciparum malaria in all endemic countries. Artemisinin resistance in Plasmodium falciparum has been confirmed in the Greater Mekong subregion (GMS). Dihydroartemisinin-piperaquine (DAPQ) is the most commonly used ACT in China. To understand the DAPQ sensitivity of P. falciparum, DAPQ resistance was monitored in vivo along the China-Myanmar border from 2007 to 2013.Methods Eligible patients with mono-infections of P. falciparum were recruited to this study after obtaining full informed consent. DAPQ tablets for different categories of kg body weight ranges were given once a day for three days. Patients were followed up for 42 days. Polymerase chain reaction (PCR) was conducted to distinguish between re-infection and recrudescence, to confirm the Plasmodium species. The data were entered and analysed by the Kaplan-Meier method. Treatment outcome was assessed according to the WHO recommended standards.Results243 patients were completed valid follow-up. The fever clearance time (FCT) and asexual parasite clearance times (APCT) were, respectively, 36.5¿±¿10.9 and 43.5¿±¿11.8 hours, and there was an increasing trend of both FCT (F¿=¿268.41, P¿<¿0.0001) and APCT (F¿=¿88.6, P¿<¿0.0001) from 2007 to 2013. Eight (3.3%, 95% confidence interval, 1.4¿6.4%) patients present parasitaemia on day three after medication; however they were spontaneous cure on day four. 241 (99.2%; 95% CI, 97.1¿99.9%) of the patients were adequate clinical and parasitological response (ACPR) and the proportions of ACPR had not changed significantly from 2007 to 2013 (X2¿=¿2.81, P¿=¿0.7288).Conclusion In terms of efficacy, DAPQ is still an effective treatment for falciparum malaria. DAPQ sensitivity in P. falciparum had not significantly changed along the China-Myanmar border of Yunnan Province, China. However more attentions should be given to becoming slower fever and parasite clearance.Malaria Journal 02/2015; 14(1):47. DOI:10.1186/s12936-015-0584-8 · 3.49 Impact Factor
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ABSTRACT: The emergence of drug-resistant parasites is a serious threat faced by malaria control programmes. Understanding the genetic basis of resistance is critical to the success of treatment and intervention strategies. A novel locus associated with antimalarial resistance, pcap2-mu (encoding the mu chain of the AP2 adaptor complex), was recently identified in studies on the rodent malaria parasite Plasmodium chabaudi. Furthermore, analysis in Kenyan malaria patients of polymorphisms in the Plasmodium falciparum homologue pfap2-mu found evidence that differences in the amino acid encoded by codon 160 are associated with enhanced parasite survival in vivo following combination treatments which included artemisinin derivatives. Here we characterize the role of pfap2-mu in mediating the in vitro antimalarial drug response of P. falciparum by generating transgenic parasites constitutively expressing either the wild-type Ser160 or the 160Asn mutant form of pfap2-mu. Transgenic parasites carrying the pfap2-mu 160Asn allele were significantly less sensitive to dihydroartemisinin using a standard 48-hour in vitro test, providing direct evidence of an altered parasite response to artemisinin. Our data also provide evidence that pfap2-mu variants can modulate parasite sensitivity to quinine. No evidence was found that pfap2-mu variants contribute to the slow clearance phenotype exhibited by P. falciparum in Cambodian patients treated with artesunate monotherapy. These findings provide compelling evidence that pfap2-mu can modulate P. falciparum responses to multiple drugs. We propose that this gene should be evaluated further as a potential molecular marker of antimalarial resistance. Copyright © 2015, American Society for Microbiology. All Rights Reserved.Antimicrobial Agents and Chemotherapy 02/2015; DOI:10.1128/AAC.04067-14 · 4.45 Impact Factor