Clinical and In Vitro Resistance of Plasmodium falciparum to Artesunate-Amodiaquine in Cambodia

International audienceBackground Artesunate-amodiaquine is a potential therapy for uncomplicated malaria in Cambodia. Methods Between September 2016 and January 2017, artesunate-amodiaquine efficacy and safety were evaluated in a prospective, open-label, single-arm observational study at health centers in Mondulkiri, Pursat, and Siem Reap Provinces, Cambodia. Adults and children with microscopically confirmed Plasmodium falciparum malaria received oral artesunate-amodiaquine once daily for 3 days plus single-dose primaquine, with follow-up on days 7, 14, 21, and 28. The primary outcome was day-28 polymerase chain reaction (PCR)-adjusted adequate clinical and parasitological response (ACPR). An amodiaquine parasite survival assay (AQSA) was developed and applied to whole genome sequencing results to evaluate potential amodiaquine resistance molecular markers. Results In 63 patients, day-28 PCR-adjusted ACPR was 81.0% (95% confidence interval [CI], 68.9–88.7). Day 3 parasite positivity rate was 44.4% (28/63; 95% CI, 31.9–57.5). All 63 isolates had the K13(C580Y) marker for artemisinin resistance; 79.4% (50/63) had Pfpm2 amplification. The AQSA resistance phenotype (≥45% parasite survival) was expressed in 36.5% (23/63) of isolates and was significantly associated with treatment failure (P = .0020). Pfmdr1 mutant haplotypes were N86/184F/D1246, and Pfcrt was CVIET or CVIDT at positions 72–76. Additional Pfcrt mutations were not associated with amodiaquine resistance, but the G353V mutant allele was associated with ACPR compared to Pfmdr1 haplotypes harboring F1068L or S784L/R945P mutations (P = .030 and P = .0004, respectively). Conclusions For uncomplicated falciparum malaria in Cambodia, artesunate-amodiaquine had inadequate efficacy owing to amodiaquine-resistant P. falciparum. Amodiaquine resistance was not associated with previously identified molecular markers

In vitro activity of ferroquine against artemisinin-based combination therapy (ACT)-resistant Plasmodium falciparum isolates from Cambodia

International audienceBackgroundCambodia is the epicentre of resistance emergence for virtually all antimalarial drugs. Selection and spread of parasites resistant to artemisinin-based combination therapy (ACT) is a major threat for malaria elimination, hence the need to renew the pool of effective treatments.ObjectivesTo determine whether ACT resistance haplotypes could have an effect on ferroquine in vitro antimalarial activity.MethodsIn vitro susceptibility to ferroquine was measured for 80 isolates from Cambodia characterized for their molecular resistance profile to artemisinin, piperaquine and mefloquine.ResultsAmong the 80 isolates tested, the overall median (IQR) IC50 of ferroquine was 10.9 nM (8.7–18.3). The ferroquine median (IQR) IC50 was 8.9 nM (8.1–11.8) for Pfk13 WT parasites and was 12.9 nM (9.5–20.0) for Pfk13 C580Y parasites with no amplification of Pfpm2 and Pfmdr1 genes. The median (IQR) IC50 of ferroquine for Pfk13 C580Y parasites with amplification of the Pfpm2 gene was 17.2 nM (14.5–20.5) versus 9.1 nM (7.9–10.7) for Pfk13 C580Y parasites with amplification of the Pfmdr1 gene.ConclusionsFerroquine exerts promising efficacy against ACT-resistant isolates. Whereas Pfpm2 amplification was associated with the highest parasite tolerance to ferroquine, the susceptibility range observed was in accordance with those measured in ACT resistance-free areas. This enables consideration of ferroquine as a relevant therapeutic option against ACT-resistant malaria

Closing in on the Reservoir: Proactive Case Detection in High-Risk Groups as a Strategy to Detect Plasmodium falciparum Asymptomatic Carriers in Cambodia

International audienceBackground:In the frame of elimination strategies of Plasmodium falciparum (Pf), active case detection has been recommended as complementary approach to the existing passive case detection programs. We trialed a polymerase chain reaction (PCR)-based active detection strategy targeting asymptomatic individuals, named proactive case detection (PACD), with the aim of assessing its feasibility, the extra yield of Pf infections, and the at-risk population for Pf carriage status.Methods:A pilot of PACD was conducted in 3 villages in Chey Saen district (Preah Vihear province, Cambodia), from December 2015 to March 2016. Voluntary screening and treatment, following health promotion sensitization, was used as mobilization strategy.Results:A total of 2802 persons were tested, representing 54% of the population. PACD (n = 30) and the respective reactive case detection (RACD) (n = 3) identified 33 Pf carriers, approximately twice as many as the Pf infections (n = 17) diagnosed in passive case detection and respective RACD, by health centers and village malaria workers using PCR, in the same villages/period. Final positivity rate was 1.07% (30/2802). People spending nighttime in forests and plantations were found to be at increased risk for Pf infection (odds ratio [OR], 3.4 [95% CI, 1.6-7.2], P = .002 and OR, 2.3 [95% CI, 1.1-4.9], P = .03, respectively).Conclusions:We demonstrated the usefulness of the PACD component in identifying Pf asymptomatic carriers. Social mobilization and promotion led to good attendance of specific risk groups, identified to be, in the Cambodian context, individuals spending nighttime in forest and plantations

Prevalence and characterization of piperaquine, mefloquine and artemisinin derivatives triple-resistant Plasmodium falciparum in Cambodia

International audienceBackground In early 2016, in Preah Vihear, Northern Cambodia, artesunate/mefloquine was used to cope with dihydroartemisinin/piperaquine-resistant Plasmodium falciparum parasites. Following this policy, P. falciparum strains harbouring molecular markers associated with artemisinin, piperaquine and mefloquine resistance have emerged. However, the lack of a viable alternative led Cambodia to adopt artesunate/mefloquine countrywide, raising concerns about a surge of triple-resistant P. falciparum strains. Objectives To assess the prevalence of triple-resistant parasites after artesunate/mefloquine implementation countrywide in Cambodia and to characterize their phenotype. Methods For this multicentric study, 846 samples were collected from 2016 to 2019. Genotyping of molecular markers associated with artemisinin, piperaquine and mefloquine resistance was coupled with phenotypic analyses. Results Only four triple-resistant P. falciparum isolates (0.47%) were identified during the study period. These parasites combined the pfk13 polymorphism with pfmdr1 amplification, pfpm2 amplification and/or pfcrt mutations. They showed significantly higher tolerance to artemisinin, piperaquine and mefloquine and also to the mefloquine and piperaquine combination. Conclusions The use of artesunate/mefloquine countrywide in Cambodia has not led to a massive increase of triple-resistant P. falciparum parasites. However, these parasites circulate in the population, and exhibit clear resistance to piperaquine, mefloquine and their combination in vitro. This study demonstrates that P. falciparum can adapt to more complex drug associations, which should be considered in future therapeutic designs

A novel field-based molecular assay to detect validated artemisinin-resistant k13 mutants

Abstract Background Given the risk of artemisinin resistance spreading from the Greater Mekong sub-region, prospective monitoring in sub-Saharan Africa should be expedited. Molecular biology techniques used for monitoring rely on the detection of k13 validated mutants by using PCR and Sanger sequencing approach, usually not available in malaria endemic areas. Methods A semi-automated workflow based on the easyMAG® platform and the Argene Solution® (bioMérieux, Marcy l’Etoile, France) as a field-based surveillance tool operable at national level was developed in four steps. Clinical and analytical performances of this tool detecting five of the most frequent and validated k13 mutants (Y493H, I543T, R539T, F446I and C580Y) from dried blood spots (DBS) were compared to the gold standard approach (PCR and Sanger sequencing). Results By using the ARMS (amplification-refractory mutation system) strategy, the best multiplexing options were found in 3 separate real-time PCR duplexes (IC as internal control/I543T, C580Y/Y493H and F446I/R539T) with limits of detection ranging from 50 (C580Y) to 6.25 parasites/µL (Y493H). In field conditions, using 642 clinical DBS (from symptomatic patients and asymptomatic individuals) collected from Cambodia, Myanmar and Africa (Chad), the overall sensitivity and specificity of the K13 bMx prototype assay developed by bioMérieux were ≥ 90%. Areas under the ROC curves were estimated to be > 0.90 for all k13 mutants in samples from symptomatic patients. Conclusion The K13 ready-to-use bMx prototype assay, considered by the end-users as a user-friendly assay to perform (in shorter time than the K13 reference assay) and easy to interpret, was found to require less budget planning and had fewer logistical constraints. Its excellent performance qualifies the prototype as a reliable screening tool usable in malaria endemic countries recognized to be at risk of emergence or spread of validated k13 mutants. Additional multi-site studies are needed to evaluate the performances of the K13 bMx prototype assay in different epidemiological contexts such as Africa, India, or South America

Plasmodium falciparum dihydroartemisinin-piperaquine failures in Cambodia are associated with mutant K13 parasites presenting high survival rates in novel piperaquine in vitro assays: retrospective and prospective investigations

International audienceBACKGROUND: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

A surrogate marker of piperaquine-resistant Plasmodium falciparum malaria: a phenotype–genotype association study

International audienceBACKGROUND: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.METHODS: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.FINDINGS: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%.INTERPRETATION: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 policie

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Additional file 5. Overall clinical performances of the K13 reference assay obtained from 642 DBS collected from P. falciparum symptomatic patients in Cambodia, Myanmar and Africa and P. falciparum asymptomatic individuals in Cambodia. Cells coloured in green present concordant data obtained between the K13 bMx prototype assay and the K13 reference assay; Cells coloured in pale red present discordant data obtained between the K13 bMx prototype assay and the K13 reference assay

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Additional file 1. K13 bMx prototype assay 5-step workflow (24 DBS in less than 4 h)

MOESM7 of A novel field-based molecular assay to detect validated artemisinin-resistant k13 mutants

Additional file 7. Clinical performances of the K13 reference assay obtained from 65 DBS collected from P. falciparum asymptomatic individuals in Cambodia. Cells coloured in green present concordant data obtained between the K13 bMx prototype assay and the K13 reference assay; Cells coloured in pale red present discordant data obtained between the K13 bMx prototype assay and the K13 reference assay