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Western Cambodia is the epicentre of Plasmodium falciparum multidrug resistance and is facing high rates of dihydroartemisinin–piperaquine treatment failures. Genetic tools to detect the multidrug-resistant parasites are needed. Artemisinin resistance can be tracked using the K13 molecular marker, but no marker exists for piperaquine resistance. We aimed to identify genetic markers of piperaquine resistance and study their association with dihydroartemisinin–piperaquine treatment failures. We obtained blood samples from Cambodian patients infected with P falciparum and treated with dihydroartemisinin–piperaquine. Patients were followed up for 42 days during the years 2009–15. We established in-vitro and ex-vivo susceptibility profiles for a subset using piperaquine survival assays. We determined whole-genome sequences by Illumina paired-reads sequencing, copy number variations by qPCR, RNA concentrations by qRT-PCR, and protein concentrations by immunoblotting. Fisher’s exact and non-parametric Wilcoxon rank-sum tests were used to identify significant differences in single-nucleotide polymorphisms or copy number variants, respectively, for differential distribution between piperaquine-resistant and piperaquine-sensitive parasite lines. Whole-genome exon sequence analysis of 31 culture-adapted parasite lines associated amplification of the plasmepsin 2–plasmepsin 3 gene cluster with in-vitro piperaquine resistance. Ex-vivo piperaquine survival assay profiles of 134 isolates correlated with plasmepsin 2 gene copy number. In 725 patients treated with dihydroartemisinin–piperaquine, multicopy plasmepsin 2 in the sample collected before treatment was associated with an adjusted hazard ratio (aHR) for treatment failure of 20·4 (95% CI 9·1–45·5, p<0·0001). Multicopy plasmepsin 2 predicted dihydroartemisinin–piperaquine failures with 0·94 (95% CI 0·88–0·98) sensitivity and 0·77 (0·74–0·81) specificity. Analysis of samples collected across the country from 2002 to 2015 showed that the geographical and temporal increase of the proportion of multicopy plasmepsin 2 parasites was highly correlated with increasing dihydroartemisinin–piperaquine treatment failure rates (r=0·89 [95% CI 0·77–0·95], p<0·0001, Spearman’s coefficient of rank correlation). Dihydroartemisinin–piperaquine efficacy at day 42 fell below 90% when the proportion of multicopy plasmepsin 2 parasites exceeded 22%. Piperaquine resistance in Cambodia is strongly associated with amplification of plasmepsin 2–3, encoding haemoglobin-digesting proteases, regardless of the location. Multicopy plasmepsin 2 constitutes a surrogate molecular marker to track piperaquine resistance. A molecular toolkit combining plasmepsin 2 with K13 and mdr1 monitoring should provide timely information for antimalarial treatment and containment policies. Institut Pasteur in Cambodia, Institut Pasteur Paris, National Institutes of Health, WHO, Agence Nationale de la Recherche, Investissement d’Avenir programme, Laboratoire d’Excellence Integrative “Biology of Emerging Infectious Diseases”.

Triple artemisinin-based combination therapies (TACTs) have been proposed to delay the emergence of multidrug-resistant Plasmodium falciparum by combining two partner drugs with an artemisinin derivative. Among these, mefloquine–piperaquine (MQ–PPQ) is a leading candidate, based on the assumption that simultaneous resistance to both partner drugs would be difficult to develop. Here, we assess the efficacy and resistance potential of MQ–PPQ using Cambodian clinical isolates with distinct resistance profiles. We find that MQ resistance confers significant cross-tolerance to the MQ–PPQ combination, whereas PPQ-resistant and -sensitive strains remain susceptible. Under repeated MQ–PPQ pressure for four months, parasites rapidly acquire MQ–PPQ tolerance, driven by pfmdr1 amplification. Mechanistic investigations reveal that MQ inhibits PPQ accumulation in a dose-dependent manner, providing a functional explanation for the compromised efficacy of the combination. These findings demonstrate that MQ resistance alone can undermine MQ–PPQ TACT efficacy, calling into question the strategic rationale of this combination and underscoring the need for alternative regimens with a lower risk of resistance selection. Triple artemisinin-based combination therapies, including mefloquine–piperaquine (MQ–PPQ), may delay emergence of multidrug-resistant strains. Here the authors show that resistance to mefloquine alone reduces the efficacy of the MQ-PPQ combination therapy, and that the interaction between the two drugs further inhibits piperaquine’s activity.

Background The recent emergence of artemisinin resistance in Africa is drawing scrutiny toward the use of alternative anti-malarial therapy based on Artemisia annua and Artemisia afra phytotherapies. This study aimed to determine if either A. annua and A. afra extracts are active against artemisinin-resistant Plasmodium falciparum isolates and determine the selectivity of inhibitory phytotherapies. Methods Artemisia extracts were tested in vitro to mimic parasites exposure to extracts in population drinking Artemisia sp . teas. Artemisia extracts were tested in Ring Stage Survival Assays (RSA 0−3 h ) against Cambodian clinical isolates previously genetically and phenotypically characterized as artemisinin resistant or sensitive. Primary human hepatocytes and a human hepatoblastoma cell line (HepG2 cells) were used to assess the cytotoxicity of Artemisia extracts. Results The study revealed a substantially decreased in vitro activity of A. annua extracts when tested on artemisinin-resistant parasites mutated in the Pfkelch13 gene (RSA 50 0.137–2.56 g.L −1 ) compared to artemisinin-sensitive parasites (RSA 50 0.080 g.L −1 ). Conversely, the A. afra extracts have a similar activity on the isolates tested whether they are sensitive or resistant to artemisinin (RSA 50 0.537–0.758 g.L −1 ) However, the selectivity index for A. afra extracts was much lower than for A. annua extracts ( A. afra: 4.628, 4.305 and 6.076 vs A. annua: 387.625, 226.350 and 12.099, respectively for WT, C580Y and R539T). Conclusions Artemisia annua activity is driven by artemisinin, implicating the same resistance profiles and concerns associated with semisynthetic artemisinin derivatives. Artemisia afra showed artemisinin-independent antiplasmodial activity. However, the molecular basis of this activity is unknown and may not present a sufficient selectivity, thus further characterization of A. afra is essential.

Background The declining efficacy of dihydroartemisinin-piperaquine against Plasmodium falciparum in Cambodia, along with increasing numbers of recrudescent cases, suggests resistance to both artemisinin and piperaquine. Available in vitro piperaquine susceptibility assays do not correlate with treatment outcome. A novel assay using a pharmacologically relevant piperaquine dose/time exposure was designed and its relevance explored in retrospective and prospective studies. Methods The piperaquine survival assay (PSA) exposed parasites to 200 nM piperaquine for 48 hours and monitored survival 24 hours later. The retrospective study tested 32 culture-adapted, C580Y-K13 mutant parasites collected at enrolment from patients treated with a 3-day course of dihydroartemisinin-piperaquine and having presented or not with a recrudescence at day 42 (registered ACTRN12615000793516). The prospective study assessed ex vivo PSA survival rate alongside K13 polymorphism of isolates collected from patients enrolled in an open-label study with dihydroartemisinin-piperaquine for uncomplicated P. falciparum malaria in Cambodia (registered ACTRN12615000696594). Results All parasites from recrudescent cases had in vitro or ex vivo PSA survival rates ≥10 %, a relevant cut-off value for piperaquine-resistance. Ex vivo PSA survival rates were higher for recrudescent than non-recrudescent cases (39.2 % vs. 0.17 %, P <1 × 10 −7 ). Artemisinin-resistant K13 mutants with ex vivo PSA survival rates ≥10 % were associated with 32-fold higher risk of recrudescence (95 % CI, 4.5–224; P  = 0.0005). Conclusion PSA adequately captures the piperaquine resistance/recrudescence phenotype, a mainstay to identify molecular marker(s) and evaluate efficacy of alternative drugs. Combined ex vivo PSA and K13 genotyping provides a convenient monitor for both artemisinin and piperaquine resistance where dihydroartemisinin-piperaquine is used.