Gene encoding a deubiquitinating enzyme is mutated in artesunate- and chloroquine-resistant rodent malaria parasites.
ABSTRACT: Artemisinin- and artesunate-resistant Plasmodium chabaudi mutants, AS-ART and AS-ATN, were previously selected from chloroquine-resistant clones AS-30CQ and AS-15CQ respectively. Now, a genetic cross between AS-ART and the artemisinin-sensitive clone AJ has been analysed by Linkage Group Selection. A genetic linkage group on chromosome 2 was selected under artemisinin treatment. Within this locus, we identified two different mutations in a gene encoding a deubiquitinating enzyme. A distinct mutation occurred in each of the clones AS-30CQ and AS-ATN, relative to their respective progenitors in the AS lineage. The mutations occurred independently in different clones under drug selection with chloroquine (high concentration) or artesunate. Each mutation maps to a critical residue in a homologous human deubiquitinating protein structure. Although one mutation could theoretically account for the resistance of AS-ATN to artemisinin derivates, the other cannot account solely for the resistance of AS-ART, relative to the responses of its sensitive progenitor AS-30CQ. Two lines of Plasmodium falciparum with decreased susceptibility to artemisinin were also selected. Their drug-response phenotype was not genetically stable. No mutations in the UBP-1 gene encoding the P. falciparum orthologue of the deubiquitinating enzyme were observed. The possible significance of these mutations in parasite responses to chloroquine or artemisinin is discussed.
Project description:Resistance of Plasmodium falciparum to drugs such as chloroquine and sulfadoxine-pyrimethamine is a major problem in malaria control. Artemisinin (ART) derivatives, particularly in combination with other drugs, are thus increasingly used to treat malaria, reducing the probability that parasites resistant to the components will emerge. Although stable resistance to artemisinin has yet to be reported from laboratory or field studies, its emergence would be disastrous because of the lack of alternative treatments. Here, we report for the first time, to our knowledge, genetically stable and transmissible ART and artesunate (ATN)-resistant malaria parasites. Each of two lines of the rodent malaria parasite Plosmodium chabaudi chabaudi, grown in the presence of increasing concentrations of ART or ATN, showed 15-fold and 6-fold increased resistance to ART and ATN, respectively. Resistance remained stable after cloning, freeze-thawing, after passage in the absence of drug, and transmission through mosquitoes. The nucleotide sequences of the possible genetic modulators of ART resistance (mdr1, cg10, tctp, and atp6) of sensitive and resistant parasites were compared. No mutations in these genes were identified. In addition we investigated whether changes in the copy number of these genes could account for resistance but found that resistant parasites retained the same number of copies as their sensitive progenitors. We believe that this is the first report of a malaria parasite with genetically stable and transmissible resistance to artemisinin or its derivatives.
Project description:BACKGROUND:Plasmodium falciparum malaria is a public health problem worldwide. Malaria treatment policy has faced periodic changes due to emergence of drug resistant parasites. In Sudan chloroquine has been replaced by artesunate and sulfadoxine/pyrimethamine (AS/SP) in 2005 and to artemether-lumefantrine (AL) in 2017, due to the development of drug resistance. Different molecular markers have been used to monitor the status of drug resistant P. falciparum. This study aimed to determine the frequency of malaria drug resistance molecular markers in Southeast Sudan. METHODS:The samples of this study were day zero dried blood spot samples collected from efficacy studies in the Blue Nile State from November 2015 to January 2016. A total of 130 samples were amplified and sequenced using illumina Miseq platform. The molecular markers included were Pfcrt, Pfmdr1, Pfdhfr, Pfdhps, Pfk13, exonuclease and artemisinin resistant (ART-R) genetic background (Pfmdr2, ferroredoxine, Pfcrt and Pfarps10). RESULTS:Resistance markers for chloroquine were detected in 25.8% of the samples as mutant haplotype Pfcrt 72-76 CVIET and 21.7% Pfmdr1 86Y. Pfdhfr mutations were detected in codons 51, 59 and 108. The ICNI double-mutant haplotype was the most prevalent (69%). Pfdhps mutations were detected in codons 436, 437, 540, 581 and 613. The SGEGA triple-mutant haplotype was the most prevalent (43%). In Pfdhfr/Pfdhps combined mutation, quintuple mutation ICNI/SGEGA is the most frequent one (29%). Six of the seven treatment failure samples had quintuple mutation and the seventh was quadruple. This was significantly higher from the adequately responsive group (P?<?0.01). Pfk13 novel mutations were found in 7 (8.8%) samples, which were not linked to artemisinin resistance. Mutations in ART-R genetic background genes ranged from zero to 7%. Exonuclease mutation was not detected. CONCLUSION:In this study, moderate resistance to chloroquine and high resistance to SP was observed. Novel mutations of Pfk13 gene not linked to treatment failure were described. There was no resistance to piperaquine the partner drug of dihydroartemisinin/piperaquine (DHA-PPQ).
Project description:BACKGROUND:In 2005, Ghana replaced chloroquine with artemisinin-based combination therapy as the first-line treatment for uncomplicated malaria. The aim of this work was to determine for the first time, polymorphisms in the putative pfATPase6 and pftctp, pfmdr1, pfcrt genes in Ghanaian isolates, particularly at a time when there is no report on artemisinin resistance in malaria parasites from Ghana. The sensitivity of parasite isolates to anti-malaria drugs were also evaluated for a possible association with polymorphisms in these genes. METHODS:The prevalence of point mutations in the above Plasmodium falciparum genes were assessed from filter-paper blood blot samples by DNA sequencing. In vitro drug sensitivity test was carried out on some of the blood samples from volunteers visiting hospitals/clinics in southern Ghana using a modified version of the standard WHO Mark III micro-test. RESULTS:All successfully tested parasite isolates were sensitive to artesunate; while 19.4%, 29.0% and 51.6% were resistant to quinine, amodiaquine and chloroquine respectively. The geometric mean of IC50 value for artesunate was 0.73 nM (95% CI, 0.38-1.08), amodiaquine 30.69 nM (95% CI, 14.18-47.20) and chloroquine 58.73 nM (95% CI, 38.08-79.38). Twenty point mutations were observed in pfATPase6 gene, with no L263E and S769N. All mutations found were low in frequency, except D639G which was observed in about half of the isolates but was not associated with artesunate response (p = 0.42). The pftctp gene is highly conserved as no mutation was observed, while CVIET which is chloroquine-resistant genotype at codon 72-76 of the pfcrt gene was identified in about half of the isolates; this was consistent with chloroquine IC50 values (p = 0.001). Mutations were present in pfmdr1 gene but were not associated with artemisinin response (p = 1.00). CONCLUSION:The pfATPase6 gene is highly polymorphic with D639G appearing to be fixed in Ghanaian isolates. These may just be spontaneous mutations as all parasite isolates that were tested displayed satisfactory in vitro response to artesunate. However, there is no improvement in susceptibility of the parasites to chloroquine five years after its proscription.
Project description:The reduced in vivo sensitivity of Plasmodium falciparum has recently been confirmed in western Cambodia. Identifying molecular markers for artemisinin resistance is essential for monitoring the spread of the resistant phenotype and identifying the mechanisms of resistance. Four candidate genes, including the P. falciparum mdr1 (pfmdr1) gene, the P. falciparum ATPase6 (pfATPase6) gene, the 6-kb mitochondrial genome, and ubp-1, encoding a deubiquitinating enzyme, of artemisinin-resistant P. falciparum strains from western Cambodia were examined and compared to those of sensitive strains from northwestern Thailand, where the artemisinins are still very effective. The artemisinin-resistant phenotype did not correlate with pfmdr1 amplification or mutations (full-length sequencing), mutations in pfATPase6 (full-length sequencing) or the 6-kb mitochondrial genome (full-length sequencing), or ubp-1 mutations at positions 739 and 770. The P. falciparum CRT K76T mutation was present in all isolates from both study sites. The pfmdr1 copy numbers in western Cambodia were significantly lower in parasite samples obtained in 2007 than in those obtained in 2005, coinciding with a local change in drug policy replacing artesunate-mefloquine with dihydroartemisinin-piperaquine. Artemisinin resistance in western Cambodia is not linked to candidate genes, as was suggested by earlier studies.
Project description:Ever increasing multi-drug resistance by Plasmodium falciparum is creating new challenges in malaria chemotherapy. In the absence of licensed vaccines, treatment and prevention of malaria is heavily dependent on drugs. Potency, range of activity, safety, low cost and ease of administration are crucial issues in the design and formulation of antimalarials. We have tested three synthetic ozonides NAC89, LC50 and LCD67 in vitro and in vivo against multidrug resistant Plasmodium. In vitro, LC50 was at least 10 times more efficient inhibiting P. falciparum multidrug resistant Dd2 strain than chloroquine and mefloquine and as efficient as artemisinin (ART), artesunate and dihydroartemisinin. All three ozonides showed high efficacy in clearing parasitaemia in mice, caused by multi-drug resistant Plasmodium chabaudi strains, by subcutaneous administration, demonstrating high efficacy in vivo against ART and artesunate resistant parasites.
Project description:BACKGROUND:The problem of anti-malarial drug resistance is a long-term challenge faced by malaria control in Yunnan Province. Recently, the detection rates of chloroquine-resistant molecular markers (Plasmodium falciparum chloroquine resistant transporter, Pfcrt) and artemisinin-resistant molecular markers (P. falciparum kelch13 gene, ork13) were 85% and 35%, respectively. To understand the association of k13 gene mutation with artemisinin resistance in falciparum malaria cases, the difference in k13 gene differentiation between two populations and artemisinin resistance phenotype on falciparum malaria cases in Myanmar were analysed in this study. METHODS:This research involved all of falciparum malaria cases diagnosed continuously in Yunnan Province from 2013 to 2015 and some of falciparum malaria cases found in Lazar, Myanmar. Blood samples were taken from the former group for molecular epidemiological analysis of k13 gene mutations, and artemisinin resistance phenotypes of P. falciparum were observed in the latter group using the in vivo testing method recommended by the World Health Organization. Nested PCR was used to amplify the propeller domain of the k13 gene in P. falciparum, followed by sequencing. RESULTS:A total of 202 blood samples were collected from Yunnan Province and 382 blood samples were collected from falciparum malaria cases in Myanmar. 49 of 382 Myanmar cases were in vivo tested for artesunate resistance phenotype through full treatment course observation. At the same time, all the blood samples were screened for k13 gene mutation of P. falciparum. The genetic diversity of k13 was higher in the Plasmodium isolates from Yunnan Province than those from Myanmar cases. The genetic differentiation index of the two populations was 0.0410, where the intra- and inter-group variations were 95.9% and 4.1%, respectively. The odds ratio of artemisinin resistance phenotype and mutation at the locus 446 in k13 gene in Myanmar cases was 1.640, while the value was 1.840 based on the estimations of the mutations in the 12 loci. CONCLUSION:Although the Plasmodium isolates from Yunnan Province and those from Myanmar were collected from different sites, they still belong to the same geographical population. It is, therefore, reasonable to contrast the artemisinin resistance status of the Plasmodium population from Myanmar with the Plasmodium population from Yunnan Province. As a result, based on the molecular epidemiological investigation on k13 mutations of Plasmodium isolates in Yunnan Province and the determination of the artemisinin resistance on falciparum malaria cases in Myanmar, the positively genetic correlated was found between the k13 locus mutations with artemisinin resistance phenotype. This provides a basis for further monitoring the artemisinin resistance by detection some molecular markers in k13 gene of Plasmodium in Yunnan Province.
Project description:The emergence of artemisinin-resistant Plasmodium falciparum poses a major threat to current frontline artemisinin combination therapies. Artemisinin resistance is widely associated with mutations in the P. falciparum Kelch13 (PfKelch13) propeller region, leading to delayed parasite clearance and increased survival of early-ring-stage parasites. There is therefore a need to discover novel drugs that are effective against artemisinin-resistant P. falciparum In view of this, our study aimed to identify compounds from the Library of Pharmacologically Active Compounds1280 (LOPAC1280) that could increase the efficacy of artesunate and be used as a potential partner drug for treatment against artemisinin-resistant falciparum malaria. By using a modified ring-stage survival assay, we performed a high-throughput screening of the activities of the 1,280 compounds from the LOPAC library in combination with artesunate against the P. falciparum IPC 5202 field isolate harboring the R539T mutation in the PfKelch13 propeller region. The potencies of the hits against both the IPC 5202 and CamWT_C580Y field isolates were determined through dose-dependent isobologram analyses; CamWT_C580Y has the more prevalent C580Y mutation characteristic of strains with artemisinin resistance. We identified tyrphostin A9 to have synergistic and additive activity against both parasite strains when dosed in combination with artesunate. These findings provide promising novel artesunate combinations that can target the P. falciparum artemisinin-resistant ring stage and insights that may aid in obtaining a better understanding of the mechanism involved in artemisinin resistance.
Project description:Artemisinin-based combination therapies (ACTs) are highly effective for the treatment of Plasmodium falciparum malaria, yet their sustained efficacy is threatened by the potential spread of parasite resistance. Recent studies have provided evidence that artemisinins can inhibit the function of PfATP6, the P. falciparum ortholog of the ER calcium pump SERCA, when expressed in Xenopus laevis oocytes. Inhibition was significantly reduced in an L263E variant, which introduced the mammalian residue into a putative drug-binding pocket. To test the hypothesis that this single mutation could decrease P. falciparum susceptibility to artemisinins, we implemented an allelic-exchange strategy to replace the wild-type pfatp6 allele by a variant allele encoding L263E. Transfected P. falciparum clones were screened by PCR analysis for disruption of the endogenous locus and introduction of the mutant L263E allele under the transcriptional control of a calmodulin promoter. Expression of the mutant allele was demonstrated by reverse transcriptase (RT) PCR and verified by sequence analysis. Parasite clones expressing wild-type or L263E variant PfATP6 showed no significant difference in 50% inhibitory concentrations (IC(50)s) for artemisinin or its derivatives dihydroartemisinin and artesunate. Nonetheless, hierarchical clustering analysis revealed a trend toward reduced susceptibility that neared significance (artemisinin, P approximately = 0.1; dihydroartemisinin, P = 0.053 and P = 0.085; and artesunate, P = 0.082 and P = 0.162 for the D10 and 7G8 lines, respectively). Notable differences in the distribution of normalized IC(50)s provided evidence of decreased responsiveness to artemisinin and dihydroartemisinin (P = 0.02 for the D10 and 7G8 lines), but not to artesunate in parasites expressing mutant PfATP6.
Project description:BACKGROUND:Humanity has become largely dependent on artemisinin derivatives for both the treatment and control of malaria, with few alternatives available. A Plasmodium falciparum phenotype with delayed parasite clearance during artemisinin-based combination therapy has established in Southeast Asia, and is emerging elsewhere. Therefore, we must know how fast, and by how much, artemisinin-resistance can strengthen. METHODS:P. falciparum was subjected to discontinuous in vivo artemisinin drug pressure by capitalizing on a novel model that allows for long-lasting, high-parasite loads. Intravenous artesunate was administered, using either single flash-doses or a 2-day regimen, to P. falciparum-infected humanized NOD/SCID IL-2R?-/-immunocompromised mice, with progressive dose increments as parasites recovered. The parasite's response to artemisinins and other available anti-malarial compounds was characterized in vivo and in vitro. RESULTS:Artemisinin resistance evolved very rapidly up to extreme, near-lethal doses of artesunate (240 mg/kg), an increase of >?3000-fold in the effective in vivo dose, far above resistance levels reported from the field. Artemisinin resistance selection was reproducible, occurring in 80% and 41% of mice treated with flash-dose and 2-day regimens, respectively, and the resistance phenotype was stable. Measuring in vitro sensitivity proved inappropriate as an early marker of resistance, as IC50 remained stable despite in vivo resistance up to 30 mg/kg (ART-S: 10.7 nM (95% CI 10.2-11.2) vs. ART-R30: 11.5 nM (6.6-16.9), F?=?0.525, p?=?0.47). However, when in vivo resistance strengthened further, IC50 increased 10-fold (ART-R240 100.3 nM (92.9-118.4), F?=?304.8, p?<?0.0001), reaching a level much higher than ever seen in clinical samples. Artemisinin resistance in this African P. falciparum strain was not associated with mutations in kelch-13, casting doubt over the universality of this genetic marker for resistance screening. Remarkably, despite exclusive exposure to artesunate, full resistance to quinine, the only other drug sufficiently fast-acting to deal with severe malaria, evolved independently in two parasite lines exposed to different artesunate regimens in vivo, and was confirmed in vitro. CONCLUSION:P. falciparum has the potential to evolve extreme artemisinin resistance and more complex patterns of multidrug resistance than anticipated. If resistance in the field continues to advance along this trajectory, we will be left with a limited choice of suboptimal treatments for acute malaria, and no satisfactory option for severe malaria.
Project description:The emergence of artemisinin-resistant Plasmodium falciparum in Southeast Asia threatens malaria treatment efficacy. Mutations in a kelch protein encoded on P. falciparum chromosome 13 (K13) have been associated with resistance in vitro and in field samples from Cambodia.P. falciparum infections from artesunate efficacy trials in Bangladesh, Cambodia, Laos, Myanmar, and Vietnam were genotyped at 33 716 genome-wide single-nucleotide polymorphisms (SNPs). Linear mixed models were used to test associations between parasite genotypes and parasite clearance half-lives following artesunate treatment. K13 mutations were tested for association with artemisinin resistance, and extended haplotypes on chromosome 13 were examined to determine whether mutations arose focally and spread or whether they emerged independently.The presence of nonreference K13 alleles was associated with prolonged parasite clearance half-life (P = 1.97 × 10(-12)). Parasites with a mutation in any of the K13 kelch domains displayed longer parasite clearance half-lives than parasites with wild-type alleles. Haplotype analysis revealed both population-specific emergence of mutations and independent emergence of the same mutation in different geographic areas.K13 appears to be a major determinant of artemisinin resistance throughout Southeast Asia. While we found some evidence of spreading resistance, there was no evidence of resistance moving westward from Cambodia into Myanmar.