Decreasing pfmdr1 copy number suggests that Plasmodium falciparum in Western Cambodia is regaining in vitro susceptibility to mefloquine.
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ABSTRACT:
SUBMITTER: Lim P
PROVIDER: S-EPMC4394809 | biostudies-literature | 2015 May
REPOSITORIES: biostudies-literature
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Decreasing pfmdr1 copy number suggests that Plasmodium falciparum in Western Cambodia is regaining in vitro susceptibility to mefloquine.
Lim Pharath P Dek Dalin D Try Vorleak V Sreng Sokunthea S Suon Seila S Fairhurst Rick M RM
Antimicrobial agents and chemotherapy 20150223 5
Dihydroartemisinin-piperaquine is the current frontline artemisinin combination therapy (ACT) for Plasmodium falciparum malaria in Cambodia but is now failing in several western provinces. To investigate artesunate plus mefloquine (AS+MQ) as a replacement ACT, we measured the prevalence of multiple pfmdr1 copies--a molecular marker for MQ resistance--in 844 P. falciparum clinical isolates collected in 2008 to 2013. The pfmdr1 copy number is decreasing in Western Cambodia, suggesting that P. falc ...[more]
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Project description:Mefloquine is widely used in combination with artemisinin derivatives for the treatment of falciparum malaria. Mefloquine resistance in Plasmodium falciparum has been related to increased copy numbers of multidrug-resistant gene 1 (pfmdr1). We studied the ex vivo dynamics of pfmdr1 gene amplification in culture-adapted P. falciparum in relation to mefloquine resistance and parasite fitness. A Thai P. falciparum isolate (isolate TM036) was assessed by the use of multiple genetic markers as a single genotype. Resistance was selected by exposure to stepwise increasing concentrations of mefloquine up to 30 ng/ml in continuous culture. The pfmdr1 gene copy numbers increased as susceptibility to mefloquine declined (P = 0.03). No codon mutations at positions 86, 184, 1034, 1042, and 1246 in the pfmdr1 gene were detected. Two subclones of selected parasites (average copy numbers, 2.3 and 3.1, respectively) showed a fitness disadvantage when they were grown together with the original parasites containing a single pfmdr1 gene copy in the absence of mefloquine; the multiplication rates were 6.3% and 8.7% lower, respectively (P < 0.01). Modeling of the dynamics of the pfmdr1 copy numbers over time in relation to the relative fitness of the parasites suggested that net pfmdr1 gene amplification from one to two copies occurs once in every 10(8) parasites and that amplification from two to three copies occurs once in every 10(3) parasites. pfmdr1 gene amplification in P. falciparum is a frequent event and confers mefloquine resistance. Parasites with multiple copies of the pfmdr1 gene have decreased survival fitness in the absence of drug pressure.
| S-EPMC2663078 | biostudies-literature
Project description:BackgroundIn 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.ObjectivesTo assess the prevalence of triple-resistant parasites after artesunate/mefloquine implementation countrywide in Cambodia and to characterize their phenotype.MethodsFor 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.ResultsOnly 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.ConclusionsThe 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.
| S-EPMC9890270 | biostudies-literature
Project description:The declining efficacy of artemisinin derivatives against Plasmodium falciparum in western Cambodia is a major concern. The knowledge gap in the understanding of the mechanisms involved hampers designing monitoring tools. Here, we culture-adapted 20 isolates from Pailin and Ratanakiri (areas of artemisinin resistance and susceptibility in western and eastern Cambodia, respectively) and studied their in vitro response to dihydroartemisinin. No significant difference between the two sets of isolates was observed in the classical isotopic test. However, a 6-h pulse exposure to 700 nM dihydroartemisinin (ring-stage survival assay -RSA]) revealed a clear-cut geographic dichotomy. The survival rate of exposed ring-stage parasites (ring stages) was 17-fold higher in isolates from Pailin (median, 13.5%) than in those from Ratanakiri (median, 0.8%), while exposed mature stages were equally and highly susceptible (0.6% and 0.7%, respectively). Ring stages survived drug exposure by cell cycle arrest and resumed growth upon drug withdrawal. The reduced susceptibility to artemisinin in Pailin appears to be associated with an altered in vitro phenotype of ring stages from Pailin in the RSA.
| S-EPMC3553720 | biostudies-literature
Project description:BackgroundLumefantrine and mefloquine are used worldwide in artemisinin-based combination therapy (ACT) of malaria. Better understanding of drug susceptibility and resistance is needed and can be obtained from studies of genetic crosses.MethodsDrug response phenotypes of a cross between Plasmodium falciparum lines 803 (Cambodia) and GB4 (Ghana) were obtained as half-maximal effective concentrations (EC50s) and days to recovery (DTR) after 24 h exposure to 500 nM lumefantrine. EC50s of mefloquine, halofantrine, chloroquine, and dihydroartemisinin were also determined. Quantitative trait loci (QTL) analysis and statistical tests with candidate genes were used to identify polymorphisms associated with response phenotypes.ResultsLumefantrine EC50s averaged 5.8-fold higher for the 803 than GB4 parent, and DTR results were 3-5 and 16-18 days, respectively. In 803 × GB4 progeny, outcomes of these two lumefantrine assays showed strong inverse correlation; these phenotypes also correlated strongly with mefloquine and halofantrine EC50s. By QTL analysis, lumefantrine and mefloquine phenotypes mapped to a chromosome 5 region containing codon polymorphisms N86Y and Y184F in the P. falciparum multidrug resistance 1 protein (PfMDR1). Statistical tests of candidate genes identified correlations between inheritance of PfK13 Kelch protein polymorphism C580Y (and possibly K189T) and lumefantrine and mefloquine susceptibilities. Correlations were detected between lumefantrine and chloroquine EC50s and polymorphisms N326S and I356T in the CVIET-type P. falciparum chloroquine resistance transporter (PfCRT) common to 803 and GB4.ConclusionsCorrelations in this study suggest common mechanisms of action in lumefantrine, mefloquine, and halofantrine responses. PfK13 as well as PfMDR1 and PfCRT polymorphisms may affect access and/or action of these arylaminoalcohol drugs at locations of hemoglobin digestion and heme metabolism. In endemic regions, pressure from use of lumefantrine or mefloquine in ACTs may drive selection of PfK13 polymorphisms along with versions of PfMDR1 and PfCRT associated with lower susceptibility to these drugs.
| S-EPMC7677662 | biostudies-literature
Project description:Previous microsatellite analyses of sympatric populations of Plasmodium vivax and Plasmodium falciparum in Brazil revealed higher diversity in the former species. However, it remains unclear whether regional species-specific differences in prevalence and transmission levels might account for these findings. Here, we examine sympatric populations of P. vivax (n=87) and P. falciparum (n=164) parasites from Pursat province, Western Cambodia, where both species are similarly prevalent. Using 10 genome-wide microsatellites for P. falciparum and 13 for P. vivax, we found that the P. vivax population was more diverse than the sympatric P. falciparum population (average virtual heterozygosity [HE], 0.87 vs. 0.66, P=0.003), with more multiple-clone infections (89.6% vs. 47.6%) and larger mean number of alleles per marker (16.2 vs. 11.1, P=0.07). Both populations showed significant multi-locus linkage disequilibrium suggestive of a predominantly clonal mode of parasite reproduction. The higher microsatellite diversity found in P. vivax isolates, compared to sympatric P. falciparum isolates, does not necessarily result from local differences in transmission level and may reflect differences in population history between species or increased mutation rates in P. vivax.
| S-EPMC3691688 | biostudies-literature
Project description:BackgroundArtemisinin-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.MethodsBetween 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.FindingsWe 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).InterpretationHeritable 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.FundingDivision of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health.
| S-EPMC3786328 | biostudies-literature
Project description:The emergence of artesunate-mefloquine (AS+MQ)-resistant Plasmodium falciparum in the Thailand-Cambodia region is a major concern for malaria control. Studies indicate that copy number increase and key alleles in the pfmdr1 gene are associated with AS+MQ resistance. In the present study, we investigated evidence for a selective sweep around pfmdr1 because of the spread of adaptive mutation and/or multiple copies of this gene in the P. falciparum population in Cambodia.We characterized 13 microsatellite loci flanking (+/-99 kb) pfmdr1 in 93 single-clone P. falciparum infections, of which 31 had multiple copies and 62 had a single copy of the pfmdr1 gene.Genetic analysis revealed no difference in the mean (+/- standard deviation) expected heterozygosity (H(e)) at loci around single (0.75+/-0.03) and multiple (0.76+/-0.04) copies of pfmdr1. Evidence of genetic hitchhiking with the selective sweep of certain haplotypes was seen around mutant (184F) pfmdr1 allele, irrespective of the copy number. There was an overall reduction of 28% in mean H(e) (+/-SD) around mutant allele (0.56+/-0.05), compared with wild-type allele (0.84+/-0.02). Significant linkage disequilibrium was also observed between the loci flanking mutant pfmdr1 allele.The 184F mutant allele is under selection, whereas amplification of pfmdr1 gene in this population occurs on multiple genetic backgrounds.
| S-EPMC3096139 | biostudies-literature
Project description:BackgroundArtemisinin-based combination therapy (ACT) is recommended as a means of prolonging the effectiveness of first-line malaria treatment regimens. Different brands of mefloquine (MQ) have been reported to be non-bioequivalent; this could result in sub-therapeutic levels of mefloquine with decreased efficacy. In 2002, mefloquine-artesunate (MQ-AS) combination therapy was adopted as the first-line treatment for uncomplicated Plasmodium falciparum malaria in the Amazon region of Peru. Although MQ resistance has yet to be reported from the Peruvian Amazon, it has been reported from other countries in the Amazon Region. Therefore, continuous monitoring is warranted to ensure that the first-line therapy remains efficacious. This study examines the in vivo efficacy and pharmacokinetic parameters through Day 56 of three commercial formulations of MQ (Lariam, Mephaquin, and Mefloquina-AC Farma) given in combination with artesunate.MethodsThirty-nine non-pregnant adults with P. falciparum mono-infection were randomly assigned to receive artesunate in combination with either (1) Lariam, (2) Mephaquin, or (3) Mefloquina AC. Patients were assessed on Day 0 (with blood samples for pharmacokinetics at 0, 2, 4, and 8 hours), 1, 2, 3, 7, and then weekly until day 56. Clinical and parasitological outcomes were based on the standardized WHO protocol.Whole blood mefloquine concentrations were determined by high-performance liquid chromatography and pharmacokinetic parameters were determined using non-compartmental analysis of concentration versus time data.ResultsBy day 3, all patients had cleared parasitaemia except for one patient in the AC Farma arm; this patient cleared by day 4. No recurrences of parasitaemia were seen in any of the 34 patients. All three MQ formulations had a terminal half-life of 14-15 days and time to maximum plasma concentration of 45-52 hours. The maximal concentration (Cmax) and interquartile range was 2,820 ng/ml (2,614-3,108) for Lariam, 2,500 ng/ml (2,363-2,713) for Mephaquin, and 2,750 ng/ml (2,550-3,000) for Mefloquina AC Farma. The pharmacokinetics of the three formulations were generally similar, with the exception of the Cmax of Mephaquin which was significantly different to that of Lariam (p = 0.04).ConclusionAll three formulations had similar pharmacokinetics; in addition, the pharmacokinetics seen in this Peruvian population were similar to reports from other ethnic groups. All patients rapidly cleared their parasitaemia with no evidence of recrudescence by Day 56. Continued surveillance is needed to ensure that patients continue to receive optimal therapy.
| S-EPMC2674465 | biostudies-literature
Project description:Molecular markers for surveillance of Plasmodium falciparum resistance to current antimalarials are sorely needed. A 28-day efficacy study of artemether-lumefantrine in eastern Sudan identified 5 treatment failures among 100 evaluable patients; 9 further individuals were parasite positive by PCR during follow-up. Polymorphisms in pfatpase6 and pfmdr1 were evaluated by DNA sequencing. One individual carried parasites with a novel pfmdr1 polymorphism (F1044L). pfmdr1 gene amplification in parasites prior to treatment occurred in three individuals who had recurrent infection during follow-up.
| S-EPMC3195064 | biostudies-literature
Project description:On the western border of Thailand, Plasmodium falciparum has become resistant to almost all antimalarial agents. The molecular basis of resistance in these parasite populations has not been well characterized. This study assessed genetic polymorphisms in the pfmdr1 gene in 54 parasites collected from the western border of Thailand to determine the relationship of pfmdr1 copy number and codon mutations with parasite sensitivities to mefloquine, chloroquine, halofantrine, quinine, and artesunate assessed in vitro. A point mutation at codon 86 (resulting in a change of Asn to Tyr) was associated with a significantly lower 50% inhibitory concentration (IC(50)) of mefloquine (median, 9 ng/ml versus 52.4 ng/ml; P = 0.003). Overall 35% of the isolates (19 of 54) had an increase in pfmdr1 copy number, and all 19 carried the wild-type allele at codon 86. Increased pfmdr1 copy number was associated with higher IC(50)s of mefloquine (P = 0.04) and artesunate (P = 0.005), independent of polymorphism at codon 86. The relationship between pfmdr1 and resistance to structurally distinct antimalarial agents confirms the presence of a true multidrug-resistant phenotype.
| S-EPMC89592 | biostudies-literature