Dataset Information

Evidence for linkage of pfmdr1, pfcrt, and pfk13 polymorphisms to lumefantrine and mefloquine susceptibilities in a Plasmodium falciparum cross.

ABSTRACT: BACKGROUND:Lumefantrine 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. METHODS:Drug 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. RESULTS:Lumefantrine 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. CONCLUSIONS:Correlations 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.

SUBMITTER: Windle ST

PROVIDER: S-EPMC7677662 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Evidence for linkage of pfmdr1, pfcrt, and pfk13 polymorphisms to lumefantrine and mefloquine susceptibilities in a Plasmodium falciparum cross.

Windle Sean T ST Lane Kristin D KD Gadalla Nahla B NB Liu Anna A Mu Jianbing J Caleon Ramoncito L RL Rahman Rifat S RS Sá Juliana M JM Wellems Thomas E TE

International journal for parasitology. Drugs and drug resistance 20201027

<h4>Background</h4>Lumefantrine 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.<h4>Methods</h4>Drug response phenotypes of a cross between Plasmodium falciparum lines 803 (Cambodia) and GB4 (Ghana) were obtained as half-maximal effective concentrations (EC<sub>50</sub>s) and days to recovery (DTR) after 24 h exposure to 500 nM lume ...[more]

PMID: 33197753

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Project description:BackgroundImported malaria parasites with anti-malarial drug resistance (ADR) from Africa is a serious public health challenge in non-malarial regions, including Wuhan, China. It is crucial to assess the ADR status in African Plasmodium falciparum isolates from imported malaria cases, as this will provide valuable information for rational medication and malaria control.MethodsDuring 2017-2019, a cross-sectional study was carried out in Wuhan, China. Peripheral blood 3 ml of returned migrant workers from Africa was collected. The target fragments from pfcrt, pfmdr1, and k13 propeller (pfk13) genes were amplified, sequenced, and analysed.ResultsIn total, 106 samples were collected. Subsequently, 98.11% (104/106), 100% (106/106), and 86.79% (92/106) of these samples were successfully amplified and sequenced for the pfcrt (72-76), pfmdr1, and pfk13 genes, respectively. The prevalence of the pfcrt 76 T, pfmdr1 86Y, and pfmdr1 184F mutations was 9.62, 4.72, and 47.17%, respectively. At codons 72-76, the pfcrt locus displayed three haplotypes, CVMNK (wild-type), CVIET (mutation type), CV M/I N/E K/T (mixed type), with 87.50%, 9.62%, and 2.88% prevalence, respectively. For the pfmdr1 gene, NY (wild type), NF and YF (mutant type), N Y/F, Y Y/F, and N/Y Y/F (mixed type) accounted for 34.91, 43.40, 3.77, 15.09, 0.94, and 1.89% of the haplotypes, respectively. A total of 83 isolates with six unique haplotypes were found in pfcrt and pfmdr1 combined haplotypes, of which NY-CVMNK and NF-CVMNK accounted for 40.96% (34/83) and 43.37% (36/83), respectively. Furthermore, 90 cases were successfully sequenced (84.91%, 90/106) at loci 93, 97, 101, and 145, and 78 cases were successfully sequenced (73.58%, 78/106) at loci 343, 353, and 356 for pfcrt. However, the mutation was observed only in locus 356 with 6.41%. For pfk13, mutations reported in Southeast Asia (at loci 474, 476, 493, 508, 527, 533, 537, 539, 543, 553, 568, 574, 578, and 580) and Africa (at loci 550, 561, 575, 579, and 589) were not observed.ConclusionsThe present data from pfcrt and pfmdr1 demonstrate that anti-malarial drugs including chloroquine, amodiaquine, and mefloquine, remain effective against malaria treatment in Africa. The new mutations in pfcrt related to piperaquine resistance remain at relatively low levels. Another source of concern is the artemether-lumefantrine resistance-related profiles of N86 and 184F of pfmdr1. Although no mutation in pfk13 is detected, molecular surveillance must continue.

Project description:BackgroundThe emergence and spread of multidrug resistance poses a significant risk to malaria control and eradication goals in the world. There has been no indigenous malaria cases reported in China since 2017, and China is approaching national malaria elimination. Therefore, anti-malarial drug resistance surveillance and tracking the emergence and spread of imported drug-resistant malaria cases will be necessary in a post-elimination phase in China.MethodsDried blood spots were obtained from Plasmodium falciparum-infected cases returned from Africa to China between 2012 and 2015, prior to anti-malarial drug treatment. Whole DNA were extracted and known polymorphisms relating to drug resistance of pfcrt, pfmdr1 gene, and the propeller domain of pfk13 were evaluated by nested PCR and sequencing. The haplotypes and prevalence of these three genes were evaluated separately. Chi-squared test and Fisher's exact test were used to evaluate differences among the different sub-regions of Africa. A P value < 0.05 was used to evaluate differences with statistical significance. The maps were created using ArcGIS.ResultsA total of 731 P. falciparum isolates were sequenced at the pfcrt locus. The wild type CVMNK was the most prevalent haplotype with prevalence of 62.8% and 29.8% of the isolates showed the triple mutant haplotype CVIET. A total of 434 P. falciparum isolates were successfully sequenced and pfmdr1 allelic variants were observed in only codons 86, 184 and 1246. Twelve haplotypes were identified and the prevalence of the wild type pfmdr1 NYD was 44.1%. The single mutant pfmdr1 in codons 86 and 184 was predominant but the haplotype NYY with single mutation in codon 1246 was not observed. The double mutant haplotype YFD was common in Africa. About 1,357 isolates were successfully sequenced of pfk13-propeller domain, the wild type was found in 1,308 samples (96.4%) whereby 49 samples (3.6%) had mutation in pfk13. Of 49 samples with pfk13 mutations, 22 non-synonymous and 4 synonymous polymorphic sites were confirmed. The A578S was the most common mutation in pfk13-propeller domain and three mutations associated with artemisinin resistance (M476I, R539T, P553L) were identified in three isolates.ConclusionThis study provides evidence that could give insight into potential issues with anti-malarial drug resistance to inform national drug policy in China in order to treat imported cases.

Project description:BackgroundAnti-malarial drugs are the major focus in the prevention and treatment of malaria. Artemisinin-based combination therapy (ACT) is the WHO recommended first-line treatment for Plasmodium falciparum malaria across the endemic world. Also ACT is increasingly relied upon in treating Plasmodium vivax malaria where chloroquine is failing. The emergence of artemisinin drug-resistant parasites is a serious threat faced by global malaria control programmes. Therefore, the success of treatment and intervention strategies is highly pegged on understanding the genetic basis of resistance.MethodsHere, resistance in P. falciparum was generated in vitro for artemisinin to produce levels above clinically relevant concentrations in vivo, and the molecular haplotypes investigated. Genomic DNA was extracted using the QIAamp mini DNA kit. DNA sequences of Pfk13, Pfcrt and Pfmdr1 genes were amplified by PCR and the amplicons were successfully sequenced. Single nucleotide polymorphisms were traced by standard bidirectional sequencing and reading the transcripts against wild-type sequences in Codon code Aligner Version 5.1 and NCBI blast.ResultsExposure of parasite strains D6 and W2 to artemisinin resulted in a decrease in parasite susceptibility to artemisinin (W2 and D6) and lumefantrine (D6 only). The parasites exhibited elevated IC50s to multiple artemisinins, with >twofold resistance to artemisinin; however, the resistance index obtained with standard methods was noticeably less than expected for parasite lines recovered from 50 µg/ml 48 h drug pressure. The change in parasite susceptibility was associated with Pfmdr-185K mutation, a mutation never reported before. The Pfcrt-CVMNK genotype (Pfcrt codons 72-76) was retained and notably, the study did not detect any polymorphisms reported to reduce P. falciparum susceptibility in vivo in the coding sequences of the Pfk13 gene.DiscussionThis data demonstrate that P. falciparum has the capacity to develop resistance to artemisinin derivatives in vitro and that this phenotype is achieved by mutations in Pfmdr1, the genetic changes that are also underpinning lumefantrine resistance. This finding is of practical importance, because artemisinin drugs in Kenya are used in combination with lumefantrine for the treatment of malaria.ConclusionArtemisinin resistance phenotype as has been shown in this work, is a decrease in parasites susceptibility to artemisinin derivatives together with the parasite's ability to recover from drug-induced dormancy after exposure to drug dosage above the in vivo clinical concentrations. The study surmises that Pfmdr1 may play a role in the anti-malarial activity of artemisinin.

Dataset Information

Evidence for linkage of pfmdr1, pfcrt, and pfk13 polymorphisms to lumefantrine and mefloquine susceptibilities in a Plasmodium falciparum cross.

Publications

Evidence for linkage of pfmdr1, pfcrt, and pfk13 polymorphisms to lumefantrine and mefloquine susceptibilities in a Plasmodium falciparum cross.

Similar Datasets

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