BACKGROUND Recent gains in reducing the global burden of malaria are threatened by the emergence of Plasmodium falciparum resistance to artemisinins. The discovery that mutations in portions of a P. falciparum gene encoding kelch (K13)–propeller domains are the major determinant of resistance has provided opportunities for monitoring such resistance on a global scale. METHODS We analyzed the K13-propeller sequence polymorphism in 14,037 samples collected in 59 countries in which malaria is endemic. Most of the samples (84.5%) were obtained from patients who were treated at sentinel sites used for nationwide surveillance of antimalarial resistance. We evaluated the emergence and dissemination of mutations by haplotyping neighboring loci. RESULTS We identified 108 nonsynonymous K13 mutations, which showed marked geographic disparity in their frequency and distribution. In Asia, 36.5% of the K13 mutations were distributed within two areas — one in Cambodia, Vietnam, and Laos and the other in western Thailand, Myanmar, and China — with no overlap. In Africa, we observed a broad array of rare nonsynonymous mutations that were not associated with delayed parasite clearance. The gene-edited Dd2 transgenic line with the A578S mutation, which expresses the most frequently observed African allele, was found to be susceptible to artemisinin in vitro on a ring-stage survival assay. CONCLUSIONS No evidence of artemisinin resistance was found outside Southeast Asia and China, where resistance-associated K13 mutations were confined. The common African A578S allele was not associated with clinical or in vitro resistance to artemisinin, and many African mutations appear to be neutral.

SummaryBackgroundWestern 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.MethodsWe 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.FindingsWhole-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%.InterpretationPiperaquine 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.FundingInstitut Pasteur in Cambodia, Institut Pasteur Paris, National Institutes of Health, WHO, Agence Nationale de la Recherche, Investissement d’Avenir programme, Laboratoire d’Excellence In...

BackgroundThe 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.MethodsThe 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).ResultsAll 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).ConclusionPSA 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12916-015-0539-5) contains supplementary material, which is available to authorized users.

BackgroundTo achieve the goal of malaria elimination in low transmission areas such as in Cambodia, new, inexpensive, high-throughput diagnostic tools for identifying very low parasite densities in asymptomatic carriers are required. This will enable a switch from passive to active malaria case detection in the field.MethodsDNA extraction and real-time PCR assays were implemented in an “in-house” designed mobile laboratory allowing implementation of a robust, sensitive and rapid malaria diagnostic strategy in the field. This tool was employed in a survey organized in the context of the MalaResT project (NCT01663831).ResultsThe real-time PCR screening and species identification assays were performed in the mobile laboratory between October and November 2012, in Rattanakiri Province, to screen approximately 5,000 individuals in less than four weeks and treat parasite carriers within 24–48 hours after sample collection. An average of 240 clinical samples (and 40 quality control samples) was tested every day, six/seven days per week. Some 97.7% of the results were available <24 hours after the collection. A total of 4.9% were positive for malaria. Plasmodium vivax was present in 61.1% of the positive samples, Plasmodium falciparum in 45.9%, Plasmodium malariae in 7.0% and Plasmodium ovale in 2.0%.ConclusionsThe operational success of this diagnostic set-up proved that molecular testing and subsequent treatment is logistically achievable in field settings. This will allow the detection of clusters of asymptomatic carriers and to provide useful epidemiological information. Fast results will be of great help for staff in the field to track and treat asymptomatic parasitaemic cases. The concept of the mobile laboratory could be extended to other countries for the molecular detection of malaria or other pathogens, or to culture vivax parasites, which does not support long-time delay between sample collection and culture.

Development of reliable, easy-to-use, rapid diagnostic tests (RDTs) to detect glucose-6-phosphate dehydrogenase (G6PD) deficiency at point of care is essential to deploying primaquine therapies as part of malaria elimination strategies. We assessed a kit under research and development called CareStart™ G6PD deficiency screening test (Access Bio, New Jersey, USA) by comparing its performance to quantitative G6PD enzyme activity using a standardized spectrophotometric method (‘gold standard’). Blood samples (n = 903) were collected from Cambodian adults living in Pailin province, western Cambodia. G6PD enzyme activities ranged from 0 to 20.5 U/g Hb (median 12.0 U/g Hg). Based on a normal haemoglobin concentration and wild-type G6PD gene, the normal values of G6PD enzymatic activity for this population was 3.6 to 20.5 U/g Hg (95th percentiles from 5.5 to 17.2 U/g Hg). Ninety-seven subjects (10.7%) had <3.6 U/g Hg and were classified as G6PD deficient. Prevalence of deficiency was 15.0% (64/425) among men and 6.9% (33/478) among women. Genotype was analyzed in 66 G6PD-deficient subjects and 63 of these exhibited findings consistent with Viangchang genotype. The sensitivity and specificity of the CareStart™ G6PD deficiency screening test was 0.68 and 1.0, respectively. Its detection threshold was <2.7 U/g Hg, well within the range of moderate and severe enzyme deficiencies. Thirteen subjects (1.4%, 12 males and 1 female) with G6PD enzyme activities <2 U/g Hg were falsely classified as “normal” by RDT. This experimental RDT test here evaluated outside of the laboratory for the first time shows real promise, but safe application of it will require lower rates of falsely “normal” results.