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.
Antimalarial resistance is rapidly spreading across parts of southeast Asia where dihydroartemisinin–piperaquine is used as first-line treatment for Plasmodium falciparum malaria. The first published ...reports about resistance to antimalarial drugs came from western Cambodia in 2013. Here, we analyse genetic changes in the P falciparum population of western Cambodia in the 6 years before those reports.
We analysed genome sequence data on 1492 P falciparum samples from 11 locations across southeast Asia, including 464 samples collected in western Cambodia between 2007 and 2013. Different epidemiological origins of resistance were identified by haplotypic analysis of the kelch13 artemisinin resistance locus and the plasmepsin 2–3 piperaquine resistance locus.
We identified more than 30 independent origins of artemisinin resistance, of which the KEL1 lineage accounted for 140 (91%) of 154 parasites resistant to dihydroartemisinin–piperaquine. In 2008, KEL1 combined with PLA1, the major lineage associated with piperaquine resistance. By 2013, the KEL1/PLA1 co-lineage had reached a frequency of 63% (24/38) in western Cambodia and had spread to northern Cambodia.
The KEL1/PLA1 co-lineage emerged in the same year that dihydroartemisinin–piperaquine became the first-line antimalarial drug in western Cambodia and spread rapidly thereafter, displacing other artemisinin-resistant parasite lineages. These findings have important implications for management of the global health risk associated with the current outbreak of multidrug-resistant malaria in southeast Asia.
Wellcome Trust, Bill & Melinda Gates Foundation, Medical Research Council, UK Department for International Development, and the Intramural Research Program of the National Institute of Allergy and Infectious Diseases.
Summary Background Artemisinin resistance in Plasmodium falciparum threatens to reduce the efficacy of artemisinin combination therapies (ACTs), thus compromising global efforts to eliminate malaria. ...Recent treatment failures with dihydroartemisinin-piperaquine, the current first-line ACT in Cambodia, suggest that piperaquine resistance may be emerging in this country. We explored the relation between artemisinin resistance and dihydroartemisinin–piperaquine failures, and sought to confirm the presence of piperaquine-resistant P falciparum infections in Cambodia. Methods In this prospective cohort study, we enrolled patients aged 2–65 years with uncomplicated P falciparum malaria in three Cambodian provinces: Pursat, Preah Vihear, and Ratanakiri. Participants were given standard 3-day courses of dihydroartemisinin–piperaquine. Peripheral blood parasite densities were measured until parasites cleared and then weekly to 63 days. The primary outcome was recrudescent P falciparum parasitaemia within 63 days. We measured piperaquine plasma concentrations at baseline, 7 days, and day of recrudescence. We assessed phenotypic and genotypic markers of drug resistance in parasite isolates. The study is registered with ClinicalTrials.gov , number NCT01736319. Findings Between Sept 4, 2012, and Dec 31, 2013, we enrolled 241 participants. In Pursat, where artemisinin resistance is entrenched, 37 (46%) of 81 patients had parasite recrudescence. In Preah Vihear, where artemisinin resistance is emerging, ten (16%) of 63 patients had recrudescence and in Ratanakiri, where artemisinin resistance is rare, one (2%) of 60 patients did. Patients with recrudescent P falciparum infections were more likely to have detectable piperaquine plasma concentrations at baseline compared with non-recrudescent patients, but did not differ significantly in age, initial parasite density, or piperaquine plasma concentrations at 7 days. Recrudescent parasites had a higher prevalence of kelch13 mutations, higher piperaquine 50% inhibitory concentration (IC50 ) values, and lower mefloquine IC50 values; none had multiple pfmdr1 copies, a genetic marker of mefloquine resistance. Interpretation Dihydroartemisinin–piperaquine failures are caused by both artemisinin and piperaquine resistance, and commonly occur in places where dihydroartemisinin–piperaquine has been used in the private sector. In Cambodia, artesunate plus mefloquine may be a viable option to treat dihydroartemisinin–piperaquine failures, and a more effective first-line ACT in areas where dihydroartemisinin–piperaquine failures are common. The use of single low-dose primaquine to eliminate circulating gametocytes is needed in areas where artemisinin and ACT resistance is prevalent. Funding National Institute of Allergy and Infectious Diseases.
Summary Background As the prevalence of artemisinin-resistant Plasmodium falciparum malaria increases in the Greater Mekong subregion, emerging resistance to partner drugs in artemisinin combination ...therapies seriously threatens global efforts to treat and eliminate this disease. Molecular markers that predict failure of artemisinin combination therapy are urgently needed to monitor the spread of partner drug resistance, and to recommend alternative treatments in southeast Asia and beyond. Methods We did a genome-wide association study of 297 P falciparum isolates from Cambodia to investigate the relationship of 11 630 exonic single-nucleotide polymorphisms (SNPs) and 43 copy number variations (CNVs) with in-vitro piperaquine 50% inhibitory concentrations (IC50 s), and tested whether these genetic variants are markers of treatment failure with dihydroartemisinin–piperaquine. We then did a survival analysis of 133 patients to determine whether candidate molecular markers predicted parasite recrudescence following dihydroartemisinin–piperaquine treatment. Findings Piperaquine IC50 s increased significantly from 2011 to 2013 in three Cambodian provinces (2011 vs 2013 median IC50 s: 20·0 nmol/L IQR 13·7–29·0 vs 39·2 nmol/L 32·8–48·1 for Ratanakiri, 19·3 nmol/L 15·1–26·2 vs 66·2 nmol/L 49·9–83·0 for Preah Vihear, and 19·6 nmol/L 11·9–33·9 vs 81·1 nmol/L 61·3–113·1 for Pursat; all p≤10−3 ; Kruskal-Wallis test). Genome-wide analysis of SNPs identified a chromosome 13 region that associates with raised piperaquine IC50 s. A non-synonymous SNP (encoding a Glu415Gly substitution) in this region, within a gene encoding an exonuclease, associates with parasite recrudescence following dihydroartemisinin–piperaquine treatment. Genome-wide analysis of CNVs revealed that a single copy of the mdr1 gene on chromosome 5 and a novel amplification of the plasmepsin 2 and plasmepsin 3 genes on chromosome 14 also associate with raised piperaquine IC50 s. After adjusting for covariates, both exo-E415G and plasmepsin 2–3 markers significantly associate (p=3·0 × 10−8 and p=1·7 × 10−7 , respectively) with decreased treatment efficacy (survival rates 0·38 95% CI 0·25–0·51 and 0·41 0·28–0·53, respectively). Interpretation The exo-E415G SNP and plasmepsin 2–3 amplification are markers of piperaquine resistance and dihydroartemisinin–piperaquine failures in Cambodia, and can help monitor the spread of these phenotypes into other countries of the Greater Mekong subregion, and elucidate the mechanism of piperaquine resistance. Since plasmepsins are involved in the parasite’s haemoglobin-to-haemozoin conversion pathway, targeted by related antimalarials, plasmepsin 2–3 amplification probably mediates piperaquine resistance. Funding Intramural Research Program of the US National Institute of Allergy and Infectious Diseases, National Institutes of Health, Wellcome Trust, Bill & Melinda Gates Foundation, Medical Research Council, and UK Department for International Development.
Plasmodium falciparum resistance to artemisinin derivatives in southeast Asia threatens malaria control and elimination activities worldwide. To monitor the spread of artemisinin resistance, a ...molecular marker is urgently needed. Here, using whole-genome sequencing of an artemisinin-resistant parasite line from Africa and clinical parasite isolates from Cambodia, we associate mutations in the PF3D7_1343700 kelch propeller domain ('K13-propeller') with artemisinin resistance in vitro and in vivo. Mutant K13-propeller alleles cluster in Cambodian provinces where resistance is prevalent, and the increasing frequency of a dominant mutant K13-propeller allele correlates with the recent spread of resistance in western Cambodia. Strong correlations between the presence of a mutant allele, in vitro parasite survival rates and in vivo parasite clearance rates indicate that K13-propeller mutations are important determinants of artemisinin resistance. K13-propeller polymorphism constitutes a useful molecular marker for large-scale surveillance efforts to contain artemisinin resistance in the Greater Mekong Subregion and prevent its global spread.
Artemisinin resistance in Plasmodium falciparum threatens global efforts to control and eliminate malaria. Polymorphisms in the kelch domain–carrying protein K13 are associated with artemisinin ...resistance, but the underlying molecular mechanisms are unknown. We analyzed the in vivo transcriptomes of 1043 P. falciparum isolates from patients with acute malaria and found that artemisinin resistance is associated with increased expression of unfolded protein response (UPR) pathways involving the major PROSC and TRiC chaperone complexes. Artemisinin-resistant parasites also exhibit decelerated progression through the first part of the asexual intraerythrocytic development cycle. These findings suggest that artemisinin-resistant parasites remain in a state of decelerated development at the young ring stage, whereas their up-regulated UPR pathways mitigate protein damage caused by artemisinin. The expression profiles of UPR-related genes also associate with the geographical origin of parasite isolates, further suggesting their role in emerging artemisinin resistance in the Greater Mekong Subregion.
Multidrug resistant Plasmodium falciparum in Southeast Asia endangers regional malaria elimination and threatens to spread to other malaria endemic areas. Understanding mechanisms of piperaquine ...(PPQ) resistance is crucial for tracking its emergence and spread, and to develop effective strategies for overcoming it. Here we analyze a mechanism of PPQ resistance in Cambodian parasites. Isolates exhibit a bimodal dose-response curve when exposed to PPQ, with the area under the curve quantifying their survival in vitro. Increased copy number for plasmepsin II and plasmepsin III appears to explain enhanced survival when exposed to PPQ in most, but not all cases. A panel of isogenic subclones reinforces the importance of plasmepsin II-III copy number to enhanced PPQ survival. We conjecture that factors producing increased parasite survival under PPQ exposure in vitro may drive clinical PPQ failures in the field.
Resistance to anti-malarial drugs hampers control efforts and increases the risk of morbidity and mortality from malaria. The efficacy of standard therapies for uncomplicated Plasmodium falciparum ...and Plasmodium vivax malaria was assessed in Chumkiri, Kampot Province, Cambodia.
One hundred fifty-one subjects with uncomplicated falciparum malaria received directly observed therapy with 12 mg/kg artesunate (over three days) and 25 mg/kg mefloquine, up to a maximum dose of 600 mg artesunate/1,000 mg mefloquine. One hundred nine subjects with uncomplicated vivax malaria received a total of 25 mg/kg chloroquine, up to a maximum dose of 1,500 mg, over three days. Subjects were followed for 42 days or until recurrent parasitaemia was observed. For P. falciparum infected subjects, PCR genotyping of msp1, msp2, and glurp was used to distinguish treatment failures from new infections. Treatment failure rates at days 28 and 42 were analyzed using both per protocol and Kaplan-Meier survival analysis. Real Time PCR was used to measure the copy number of the pfmdr1 gene and standard 48-hour isotopic hypoxanthine incorporation assays were used to measure IC50 for anti-malarial drugs.
Among P. falciparum infected subjects, 47.0% were still parasitemic on day 2 and 11.3% on day 3. The PCR corrected treatment failure rates determined by survival analysis at 28 and 42 days were 13.1% and 18.8%, respectively. Treatment failure was associated with increased pfmdr1 copy number, higher initial parasitaemia, higher mefloquine IC50, and longer time to parasite clearance. One P. falciparum isolate, from a treatment failure, had markedly elevated IC50 for both mefloquine (130 nM) and artesunate (6.7 nM). Among P. vivax infected subjects, 42.1% suffered recurrent P. vivax parasitaemia. None acquired new P. falciparum infection.
The results suggest that artesunate-mefloquine combination therapy is beginning to fail in southern Cambodia and that resistance is not confined to the provinces at the Thai-Cambodian border. It is unclear whether the treatment failures are due solely to mefloquine resistance or to artesunate resistance as well. The findings of delayed clearance times and elevated artesunate IC50 suggest that artesunate resistance may be emerging on a background of mefloquine resistance.
Summary Background Artemisinin-resistant Plasmodium falciparum has been reported in Pailin, western Cambodia, detected as a slow parasite clearance rate in vivo. Emergence of this phenotype in ...western Thailand and possibly elsewhere threatens to compromise the effectiveness of all artemisinin-based combination therapies. Parasite genetics is associated with parasite clearance rate but does not account for all variation. We investigated contributions of both parasite genetics and host factors to the artemisinin-resistance phenotype in Pursat, western Cambodia. Methods Between June 19 and Nov 28, 2009, and June 26 and Dec 6, 2010, we enrolled patients aged 10 years or older with uncomplicated falciparum malaria, a density of asexual parasites of at least 10 000 per μL of whole blood, no symptoms or signs of severe malaria, no other cause of febrile illness, and no chronic illness. We gave participants 4 mg/kg artesunate at 0, 24, and 48 h, 15 mg/kg mefloquine at 72 h, and 10 mg/kg mefloquine at 96 h. We assessed parasite density on thick blood films every 6 h until undetectable. The parasite clearance half-life was calculated from the parasite clearance curve. We genotyped parasites with 18 microsatellite markers and patients for haemoglobin E, α-thalassaemia, and a mutation of G6PD , which encodes glucose-6-phosphate dehydrogenase. To account for the possible effects of acquired immunity on half-life, we used three surrogates for increased likelihood of exposure to P falciparum : age, sex, and place of residence. This study is registered with ClinicalTrials.gov , number NCT00341003. Findings We assessed 3504 individuals from all six districts of Pursat province seeking treatment for malaria symptoms. We enrolled 168 patients with falciparum malaria who met inclusion criteria. The geometric mean half-life was 5·85 h (95% CI 5·54–6·18) in Pursat, similar to that reported in Pailin (p=0·109). We identified two genetically different parasite clone groups: parasite group 1 (PG1) and parasite group 2 (PG2). Non-significant increases in parasite clearance half-life were seen in patients with haemoglobin E (0·55 h; p=0·078), those of male sex (0·96 h; p=0·064), and in 2010 (0·68 h; p=0·068); PG1 was associated with a significant increase (0·79 h; p=0·033). The mean parasite heritability of half-life was 0·40 (SD 0·17). Interpretation Heritable artemisinin resistance is established in a second Cambodian province. To accurately identify parasites that are intrinsically susceptible or resistant to artemisinins, future studies should explore the effect of erythrocyte polymorphisms and specific immune responses on half-life variation. Funding Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health.
Artemisinin resistance is associated with delayed parasite clearance half-life in vivo and correlates with ring-stage survival under dihydroartemisinin in vitro. Both phenotypes are associated with ...mutations in the PF3D7_1343700 pfkelch13 gene. Recent spread of artemisinin resistance and emerging piperaquine resistance in Southeast Asia show that artemisinin combination therapy, such as dihydroartemisinin-piperaquine, are losing clinical effectiveness, prompting investigation of drug resistance mechanisms and development of strategies to surmount emerging anti-malarial resistance.
Sixty-eight parasites isolates with in vivo clearance data were obtained from two Tracking Resistance to Artemisinin Collaboration study sites in Cambodia, culture-adapted, and genotyped for pfkelch13 and other mutations including pfmdr1 copy number; and the RSA
survival rates and response to antimalarial drugs in vitro were measured for 36 of these isolates.
Among these 36 parasites one isolate demonstrated increased ring-stage survival for a PfKelch13 mutation (D584V, RSA
= 8%), previously associated with slow clearance but not yet tested in vitro. Several parasites exhibited increased ring-stage survival, yet lack pfkelch13 mutations, and one isolate showed evidence for piperaquine resistance.
This study of 68 culture-adapted Plasmodium falciparum clinical isolates from Cambodia with known clearance values, associated the D584V PfKelch13 mutation with increased ring-stage survival and identified parasites that lack pfkelch13 mutations yet exhibit increased ring-stage survival. These data suggest mutations other than those found in pfkelch13 may be involved in conferring artemisinin resistance in P. falciparum. Piperaquine resistance was also detected among the same Cambodian samples, consistent with reports of emerging piperaquine resistance in the field. These culture-adapted parasites permit further investigation of mechanisms of both artemisinin and piperaquine resistance and development of strategies to prevent or overcome anti-malarial resistance.
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