Plasmepsin II-III copy number accounts for bimodal piperaquine resistance among Cambodian Plasmodium falciparum.
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ABSTRACT:
SUBMITTER: Bopp S
PROVIDER: S-EPMC5931971 | biostudies-literature | 2018 May
REPOSITORIES: biostudies-literature
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Plasmepsin II-III copy number accounts for bimodal piperaquine resistance among Cambodian Plasmodium falciparum.
Bopp Selina S Magistrado Pamela P Wong Wesley W Schaffner Stephen F SF Mukherjee Angana A Lim Pharath P Dhorda Mehul M Amaratunga Chanaki C Woodrow Charles J CJ Ashley Elizabeth A EA White Nicholas J NJ Dondorp Arjen M AM Fairhurst Rick M RM Ariey Frederic F Menard Didier D Wirth Dyann F DF Volkman Sarah K SK
Nature communications 20180502 1
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 the ...[more]
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Project description:BACKGROUND:Long regarded as an epicenter of drug-resistant malaria, Southeast Asia continues to provide new challenges to the control of Plasmodium falciparum malaria. Recently, resistance to the artemisinin combination therapy partner drug piperaquine has been observed in multiple locations across Southeast Asia. Genetic studies have identified single nucleotide polymorphisms as well as copy number variations in the plasmepsin 2 and plasmepsin 3 genes, which encode haemoglobin-degrading proteases that associate with clinical and in vitro piperaquine resistance. RESULTS:To accurately and quickly determine the presence of copy number variations in the plasmepsin 2/3 genes in field isolates, this study developed a quantitative PCR assay using TaqMan probes. Copy number estimates were validated using a separate SYBR green-based quantitative PCR assay as well as a novel PCR-based breakpoint assay to detect the hybrid gene product. Field samples from 2012 to 2015 across three sites in Cambodia were tested using DNA extracted from dried blood spots and whole blood to monitor the extent of plasmepsin 2/3 gene amplifications, as well as amplifications in the multidrug resistance transporter 1 gene (pfmdr1), a marker of mefloquine resistance. This study found high concordance across all methods of copy number detection. For samples derived from dried blood spots, a success rate greater than 80% was found in each assay, with more recent samples performing better. Evidence of extensive plasmepsin 2/3 copy number amplifications was observed in Pursat (94%, 2015) (Western Cambodia) and Preah Vihear (87%, 2014) (Northern Cambodia), and lower levels in Ratanakiri (16%, 2014) (Eastern Cambodia). A shift was observed from two copies of plasmepsin 2 in Pursat in 2013 to three copies in 2014-2015 (25% to 64%). Pfmdr1 amplifications were absent in all samples from Preah Vihear and Ratanakiri in 2014 and absent in Pursat in 2015. CONCLUSIONS:The multiplex TaqMan assay is a robust tool for monitoring both plasmepsin 2/3 and pfmdr1 copy number variations in field isolates, and the SYBR-green and breakpoint assays are useful for monitoring plasmepsin 2/3 amplifications. This study shows increasing levels of plasmepsin 2 copy numbers across Cambodia from 2012 to 2015 and a complete reversion of multicopy pfmdr1 parasites to single copy parasites in all study locations.
| S-EPMC7218657 | biostudies-literature
Project description:Artemisinin derivatives and their partner drugs in artemisinin combination therapies (ACTs) have played a pivotal role in global malaria mortality reduction during the last two decades. The loss of artemisinin efficacy due to evolving drug-resistant parasites could become a serious global health threat. Dihydroartemisinin-piperaquine is a well tolerated and generally highly effective ACT. The implementation of a partner drug in ACTs is critical in the control of emerging artemisinin resistance. Even though artemisinin is highly effective in parasite clearance, it is labile in the human body. A partner drug is necessary for killing the remaining parasites when the pulses of artemisinin have ceased. A population of Plasmodium falciparum parasites in Cambodia and adjacent countries has become resistant to piperaquine. Increased copy number of the genes encoding the haemoglobinases Plasmepsin II and Plasmepsin III has been linked with piperaquine resistance by genome-wide association studies and in clinical trials, leading to the use of increased plasmepsin II/plasmepsin III copy number as a molecular marker for piperaquine resistance. Here we demonstrate that overexpression of plasmepsin II and plasmepsin III in the 3D7 genetic background failed to change the susceptibility of P. falciparum to artemisinin, chloroquine and piperaquine by both a standard dose-response analysis and a piperaquine survival assay. Whilst plasmepsin copy number polymorphism is currently implemented as a molecular surveillance resistance marker, further studies to discover the molecular basis of piperaquine resistance and potential epistatic interactions are needed.
| S-EPMC6304341 | biostudies-literature
Project description:BackgroundHigh rates of dihydroartemisinin-piperaquine (DHA-PPQ) treatment failures have been documented for uncomplicated Plasmodium falciparum in Cambodia. The genetic markers plasmepsin 2 (pfpm2), exonuclease (pfexo) and chloroquine resistance transporter (pfcrt) genes are associated with PPQ resistance and are used for monitoring the prevalence of drug resistance and guiding malaria drug treatment policy.MethodsTo examine the relative contribution of each marker to PPQ resistance, in vitro culture and the PPQ survival assay were performed on seventeen P. falciparum isolates from northern Cambodia, and the presence of E415G-Exo and pfcrt mutations (T93S, H97Y, F145I, I218F, M343L, C350R, and G353V) as well as pfpm2 copy number polymorphisms were determined. Parasites were then cloned by limiting dilution and the cloned parasites were tested for drug susceptibility. Isobolographic analysis of several drug combinations for standard clones and newly cloned P. falciparum Cambodian isolates was also determined.ResultsThe characterization of culture-adapted isolates revealed that the presence of novel pfcrt mutations (T93S, H97Y, F145I, and I218F) with E415G-Exo mutation can confer PPQ-resistance, in the absence of pfpm2 amplification. In vitro testing of PPQ resistant parasites demonstrated a bimodal dose-response, the existence of a swollen digestive vacuole phenotype, and an increased susceptibility to quinine, chloroquine, mefloquine and lumefantrine. To further characterize drug sensitivity, parental parasites were cloned in which a clonal line, 14-B5, was identified as sensitive to artemisinin and piperaquine, but resistant to chloroquine. Assessment of the clone against a panel of drug combinations revealed antagonistic activity for six different drug combinations. However, mefloquine-proguanil and atovaquone-proguanil combinations revealed synergistic antimalarial activity.ConclusionsSurveillance for PPQ resistance in regions relying on DHA-PPQ as the first-line treatment is dependent on the monitoring of molecular markers of drug resistance. P. falciparum harbouring novel pfcrt mutations with E415G-exo mutations displayed PPQ resistant phenotype. The presence of pfpm2 amplification was not required to render parasites PPQ resistant suggesting that the increase in pfpm2 copy number alone is not the sole modulator of PPQ resistance. Genetic background of circulating field isolates appear to play a role in drug susceptibility and biological responses induced by drug combinations. The use of latest field isolates may be necessary for assessment of relevant drug combinations against P. falciparum strains and when down-selecting novel drug candidates.
| S-EPMC7382038 | biostudies-literature
Project description:The combination of piperaquine and dihydroartemisinin has recently become the official first-line therapy in several Southeast Asian countries. The pharmacokinetic mismatching of these drugs, whose plasma half-lives are ~20 days and ~1 h, respectively, implies that recrudescent or new infections emerging shortly after treatment cessation will encounter piperaquine as a monotherapy agent. This creates substantial selection pressure for the emergence of resistance. To elucidate potential resistance determinants, we subjected cloned Plasmodium falciparum Dd2 parasites to continuous piperaquine pressure in vitro (47 nM; ~2-fold higher than the Dd2 50% inhibitory concentration [IC(50)]). The phenotype of outgrowth parasites was assayed in two clones, revealing an IC(50) against piperaquine of 2.1 ?M and 1.7 ?M, over 100-fold greater than that of the parent. To identify the genetic determinant of resistance, we employed comparative whole-genome hybridization analysis. Compared to the Dd2 parent, this analysis found (in both resistant clones) a novel single-nucleotide polymorphism in P. falciparum crt (pfcrt), deamplification of an 82-kb region of chromosome 5 (that includes pfmdr1), and amplification of an adjacent 63-kb region of chromosome 5. Continued propagation without piperaquine selection pressure resulted in "revertant" piperaquine-sensitive parasites. These retained the pfcrt polymorphism and further deamplified the chromosome 5 segment that encompasses pfmdr1; however, these two independently generated revertants both lost the neighboring 63-kb amplification. These results suggest that a copy number variation event on chromosome 5 (825600 to 888300) is associated with piperaquine resistance. Transgene expression studies are underway with individual genes in this segment to evaluate their contribution to piperaquine resistance.
| S-EPMC3147642 | biostudies-literature
Project description:Piperaquine (PPQ) is widely used in combination with dihydroartemisinin (DHA) as a first-line treatment against malaria parasites. Multiple genetic drivers of PPQ resistance have been reported, including mutations in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and increased copies of plasmepsin II/III (pm2/3). We generated a cross between a Cambodia-derived multi-drug resistant KEL1/PLA1 lineage isolate (KH004) and a drug susceptible parasite isolated in Malawi (Mal31). Mal31 harbors a wild-type (3D7-like) pfcrt allele and a single copy of pm2/3, while KH004 has a chloroquine-resistant (Dd2-like) pfcrt allele with an additional G367C substitution and four copies of pm2/3. We recovered 104 unique recombinant progeny and examined a targeted set of progeny representing all possible combinations of variants at pfcrt and pm2/3 for detailed analysis of competitive fitness and a range of PPQ susceptibility phenotypes, including PPQ survival assay (PSA), area under the dose-response curve (AUC), and a limited point IC50 (LP-IC50). We find that inheritance of the KH004 pfcrt allele is required for PPQ resistance, whereas copy number variation in pm2/3 further enhances resistance but does not confer resistance in the absence of PPQ-R-associated mutations in pfcrt. Deeper investigation of genotype-phenotype relationships demonstrates that progeny clones from experimental crosses can be used to understand the relative contributions of pfcrt, pm2/3, and parasite genetic background, to a range of PPQ-related traits and confirm the critical role of the PfCRT G367C substitution in PPQ resistance.
| S-EPMC10515748 | biostudies-literature
Project description:The malaria parasite replicates within erythrocytes. The pathogenesis of clinical malaria is in large part due to the capacity of the parasite to remodel its host cell. To do this, intraerythrocytic stages of Plasmodium falciparum export more than 300 proteins that dramatically alter the morphology of the infected erythrocyte as well as its mechanical and adhesive properties. P. falciparum plasmepsin V (PfPMV) is an aspartic protease that processes proteins for export into the host erythrocyte and is thought to play a key role in parasite virulence and survival. However, although standard techniques for gene disruption as well as conditional protein knockdown have been previously attempted with the pfpmv gene, complete gene removal or knockdown was not achieved so direct genetic proof that PMV is an essential protein has not been established. Here we have used a conditional gene excision approach combining CRISPR-Cas9 gene editing and DiCre-mediated recombination to functionally inactivate the pfpmv gene. The resulting mutant parasites displayed a severe growth defect. Detailed phenotypic analysis showed that development of the mutant parasites was arrested early in the ring-to-trophozoite transition in the erythrocytic cycle following gene excision. Our findings are the first to elucidate the effects of PMV gene disruption, showing that it is essential for parasite viability in asexual blood stages. The mutant parasites can now be used as a platform to further dissect the Plasmodium protein export pathway.
| S-EPMC6281190 | biostudies-literature
Project description:The combination of piperaquine and dihydroartemisinin has recently become the official first-line therapy in several Southeast Asian countries. The pharmacokinetic mismatching of these drugs, whose plasma half lives are ∼20 days and ∼1 hr respectively, implies that recrudescent or new infections emerging shortly after treatment cessation will encounter piperaquine as a monotherapy agent. This creates substantial selection pressure for the emergence of resistance. To elucidate potential resistance determinants, we subjected cloned Plasmodium falciparum Dd2 parasites to continuous piperaquine pressure in vitro (47 nM, ∼two-fold higher than the Dd2 IC(50) value). The phenotype of outgrowth parasites was assayed in two clones, revealing an IC(50) value against piperaquine of 2.1 μM and 1.7 μM, over 100-fold greater than the parent. To identify the genetic determinant of resistance, we employed comparative whole-genome hybridization analysis. Compared to the Dd2 parent, this analysis found (in both resistant clones) a novel single nucleotide polymorphism in pfcrt, deamplification of an 82 kb region of chromosome 5 (that includes pfmdr1), and amplification of an adjacent 63 kb region of chromosome 5. Continued propagation without piperaquine selection pressure resulted in derivation of "revertant" piperaquine-sensitive parasites. These retained the pfcrt polymorphism and further deamplified the chromosome 5 segment that encompasses pfmdr1; however, these two independently generated revertants both lost the neighboring 63 kb amplification. These results suggest that a copy number variation event on chromosome 5 (825600-888300) is associated with piperaquine resistance. Transgenic studies are underway with individual genes in this segment to evaluate their contribution to piperaquine resistance.
2011-05-23 | GSE27907 | GEO
Project description:BACKGROUND:Gene copy number variation (CNV) is responsible for several important phenotypes of the malaria parasite Plasmodium falciparum, including drug resistance, loss of infected erythrocyte cytoadherence and alteration of receptor usage for erythrocyte invasion. Despite the known effects of CNV, little is known about its extent throughout the genome. RESULTS:We performed a whole-genome survey of CNV genes in P. falciparum using comparative genome hybridisation of a diverse set of 16 laboratory culture-adapted isolates to a custom designed high density Affymetrix GeneChip array. Overall, 186 genes showed hybridisation signals consistent with deletion or amplification in one or more isolate. There is a strong association of CNV with gene length, genomic location, and low orthology to genes in other Plasmodium species. Sub-telomeric regions of all chromosomes are strongly associated with CNV genes independent from members of previously described multigene families. However, approximately 40% of CNV genes were located in more central regions of the chromosomes. Among the previously undescribed CNV genes, several that are of potential phenotypic relevance are identified. CONCLUSION:CNV represents a major form of genetic variation within the P. falciparum genome; the distribution of gene features indicates the involvement of highly non-random mutational and selective processes. Additional studies should be directed at examining CNV in natural parasite populations to extend conclusions to clinical settings.
| S-EPMC2732925 | biostudies-literature
Project description:BackgroundThe emergence and spread of artemisinin resistance in Plasmodium falciparum poses a threat to malaria eradication, including China's plan to eliminate malaria by 2020. Piperaquine (PPQ) resistance has emerged in Cambodia, compromising an important partner drug that is widely used in China in the form of dihydroartemisinin (DHA)-PPQ. Several mutations in a P. falciparum gene encoding a kelch protein on chromosome 13 (k13) are associated with artemisinin resistance and have arisen spread in the Great Mekong subregion, including the China-Myanmar border. Multiple copies of the plasmepsin II/III (pm2/3) genes, located on chromosome 14, have been shown to be associated with PPQ resistance.MethodsThe therapeutic efficacy of DHA-PPQ for the treatment of uncomplicated P. falciparum was evaluated along the China-Myanmar border from 2010 to 2014. The dry blood spots samples collected in the efficacy study prior DHA-PPQ treatment and from the local hospital by passive detection were used to amplify k13 and pm2. Polymorphisms within k13 were genotyped by capillary sequencing and pm2 copy number was quantified by relative-quantitative real-time polymerase chain reaction. Treatment outcome was evaluated with the World Health Organization protocol. A linear regression model was used to estimate the association between the day 3 positive rate and k13 mutation and the relationship of the pm2 copy number variants and k13 mutations.ResultsDHA-PPQ was effective for uncomplicated P. falciparum infection in Yunnan Province with cure rates > 95%. Twelve non synonymous mutations in the k13 domain were observed among the 268 samples with the prevalence of 44.0% and the predominant mutation was F446I with a prevalence of 32.8%. Only one sample was observed with multi-copies of pm2, including parasites with and without k13 mutations. The therapeutic efficacy of DHA-PPQ was > 95% along the China-Myanmar border, consistent with the lack of amplification of pm2.ConclusionDHA-PPQ for uncomplicated P. falciparum infection still showed efficacy in an area with artemisinin-resistant malaria along the China-Myanmar border. There was no evidence to show PPQ resistance by clinical study and molecular markers survey. Continued monitoring of the parasite population using molecular markers will be important to track emergence and spread of resistance in this region.
| S-EPMC7488220 | biostudies-literature
Project description:BackgroundToday, the development of new and well-tolerated anti-malarial drugs is strongly justified by the emergence of Plasmodium falciparum resistance. In 2014-2015, a phase 2b clinical study was conducted to evaluate the efficacy of a single oral dose of Artefenomel (OZ439)-piperaquine (PPQ) in Asian and African patients presenting with uncomplicated falciparum malaria.MethodsBlood samples collected before treatment offered the opportunity to investigate the proportion of multidrug resistant parasite genotypes, including P. falciparum kelch13 mutations and copy number variation of both P. falciparum plasmepsin 2 (Pfpm2) and P. falciparum multidrug resistance 1 (Pfmdr1) genes.ResultsValidated kelch13 resistance mutations including C580Y, I543T, P553L and V568G were only detected in parasites from Vietnamese patients. In Africa, isolates with multiple copies of the Pfmdr1 gene were shown to be more frequent than previously reported (21.1%, range from 12.4% in Burkina Faso to 27.4% in Uganda). More strikingly, high proportions of isolates with multiple copies of the Pfpm2 gene, associated with piperaquine (PPQ) resistance, were frequently observed in the African sites, especially in Burkina Faso and Uganda (> 30%).ConclusionsThese findings were considered to sharply contrast with the recent description of increased sensitivity to PPQ of Ugandan parasite isolates. This emphasizes the necessity to investigate in vitro susceptibility profiles to PPQ of African isolates with multiple copies of the Pfpm2 gene and estimate the risk of development of PPQ resistance in Africa. Trial registration Clinicaltrials.gov reference: NCT02083380. Study title: Phase II efficacy study of artefenomel and piperaquine in adults and children with P. falciparum malaria. https://clinicaltrials.gov/ct2/results?cond=&term=NCT02083380&cntry=&state=&city=&dist= . FSFV: 23-Jul-2014; LSLV: 09-Oct-2015.
| S-EPMC6457011 | biostudies-literature