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Plasmodium falciparum multidrug resistance protein 1 (pfmrp1) gene and its association with in vitro drug susceptibility of parasite isolates from north-east Myanmar.

ABSTRACT: Plasmodium falciparum multidrug resistance protein 1 (pfmrp1) has recently emerged as an important determinant of drug resistance and mutations in the gene have been associated with several drugs. The aim of this study was to understand the level of genetic diversity in pfmrp1 and to determine the association of different mutations with altered drug susceptibilities of P. falciparum.We analysed 193 sequences of pfmrp1 from South-East Asia, west Asia, Africa, Oceania and South America. We measured the level of genetic diversity and determined signatures of selection on the gene. In vitro susceptibilities of 28 P. falciparum isolates from north-east Myanmar to a panel of seven commonly used antimalarials were determined. Statistical analysis was performed to determine the association of different mutations with in vitro drug susceptibilities.A total of 28 single nucleotide polymorphisms were identified in 193 sequences, of which 22 were non-synonymous. Whereas mutations in the pfmrp1 gene were conserved among different countries within a continent, they were different between continents. Seven non-synonymous mutations were identified in the north-east Myanmar isolates; all were relatively frequent in this region as well as in other neighbouring countries. Molecular evolutionary analysis detected signatures of positive selection on the gene. Moreover, some mutations in this gene were found to be associated with reduced susceptibilities to chloroquine, mefloquine, pyronaridine and lumefantrine.Evidence of the positive selection of pmfrp1 and its association with the susceptibilities of parasites to multiple drugs signifies its potential as an important candidate for monitoring drug resistance.

SUBMITTER: Gupta B

PROVIDER: S-EPMC4100709 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Plasmodium falciparum multidrug resistance protein 1 (pfmrp1) gene and its association with in vitro drug susceptibility of parasite isolates from north-east Myanmar.

Gupta Bhavna B Xu Shuhui S Wang Zenglei Z Sun Ling L Miao Jun J Cui Liwang L Yang Zhaoqing Z

The Journal of antimicrobial chemotherapy 20140522 8

<h4>Objectives</h4>Plasmodium falciparum multidrug resistance protein 1 (pfmrp1) has recently emerged as an important determinant of drug resistance and mutations in the gene have been associated with several drugs. The aim of this study was to understand the level of genetic diversity in pfmrp1 and to determine the association of different mutations with altered drug susceptibilities of P. falciparum.<h4>Methods</h4>We analysed 193 sequences of pfmrp1 from South-East Asia, west Asia, Africa, Oc ...[more]

PMID: 24855124

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Project description:Mutations in the Kelch domain of the K13 gene (PF3D7_1343700) were previously associated with artemisinin resistance in Plasmodium falciparum. This study followed the dynamics of the K13 polymorphisms in P. falciparum parasites from the China-Myanmar border area obtained in 2007-2016, and their in vitro sensitivities to artesunate (AS) and dihydroartemisinin (DHA). The 50% effective concentration (EC5072h) values of 133 culture-adapted field isolates to AS and DHA, measured by the conventional 72 h SYBR Green I-based assay, varied significantly among the parasites from different years; all were significantly higher than that of the reference strain 3D7. Compared with parasites from 2007 to 2008, ring survival rates almost doubled in parasites obtained in later years. Sequencing the full-length K13 genes identified 11 point mutations present in 85 (63.9%) parasite isolates. F446I was the predominant (55/133) variant, and its frequency was increased from 17.6% (3/17) in 2007 to 55.9% (19/34) in 2014-2016. No wild-type (WT) Kelch domain sequences were found in the 34 samples obtained from 2014 to 2016. In the 2014-2016 samples, a new mutation (G533S) appeared and reached 44.1% (15/34). Collectively, parasites with the Kelch domain mutations (after amino acid 440) had significantly higher ring survival rates than the WT parasites. Individually, F446I, G533S and A676D showed significantly higher ring survival rates than the WT. Although the drug sensitivity phenotypes measured by the RSA6h and EC5072h assays may be intrinsically linked to the in vivo clinical efficacy data, the values determined by these two assays were not significantly correlated. This study identified the trend of K13 mutations in parasite populations from the China-Myanmar border area, confirmed an overall correlation of Kelch domain mutations with elevated ring-stage survival rates, and emphasized the importance of monitoring the evolution and spread of parasites with reduced artemisinin sensitivity along the malaria elimination course.

Project description:The emergence and spread in Southeast Asia of Plasmodium falciparum resistance to artemisinin (ART) derivatives, the cornerstone of first-line artemisinin-based combination therapies (ACTs), underscore the urgent need to identify suitable replacement drugs. Discovery and development efforts have identified a series of ozonides with attractive chemical and pharmacological properties that are being touted as suitable replacements. Partial resistance to ART, defined as delayed parasite clearance in malaria patients treated with an ART derivative or an ACT, has been associated with mutations in the P. falciparum K13 gene. In light of reports showing that ART derivatives and ozonides share similar modes of action, we have investigated whether parasites expressing mutant K13 are cross-resistant to the ozonides OZ439 (artefenomel) and OZ227 (arterolane). This work used a panel of culture-adapted clinical isolates from Cambodia that were genetically edited to express variant forms of K13. Phenotypic analyses employed ring-stage survival assays (ring-stage survival assay from 0 to 3 h [RSA0-3h]), whose results have earlier been shown to correlate with parasite clearance rates in patients. Our results document cross-resistance between OZ277 and dihydroartemisinin (DHA), a semisynthetic derivative of ART, in parasites carrying the K13 mutations C580Y, R539T, and I543T. For OZ439, we observed cross-resistance only for parasites that carried the rare K13 I543T mutation, with no evidence of cross-resistance afforded by the prevalent C580Y mutation. Mixed-culture competition experiments with isogenic lines carrying modified K13 revealed variable growth deficits depending on the K13 mutation and parasite strain and provide a rationale for the broad dissemination of the fitness-neutral K13 C580Y mutation throughout strains currently circulating in Southeast Asia.IMPORTANCE ACTs have helped halve the malaria disease burden in recent years; however, emerging resistance to ART derivatives threatens to reverse this substantial progress. Resistance is driven primarily by mutations in the P. falciparum K13 gene. These mutations pose a threat to ozonides, touted as promising alternatives to ARTs that share a similar mode of action. We report that DHA was considerably more potent than OZ439 and OZ277 against ART-sensitive asexual blood-stage parasites cultured in vitro We also document that mutant K13 significantly compromised the activity of the registered drug OZ277. In contrast, OZ439 remained effective against most parasite lines expressing mutant K13, with the exception of I543T that merits further monitoring in field-based OZ439 efficacy studies. K13 mutations differed considerably in their impact on parasite growth rates, in a strain-dependent context, with the most prevalent C580Y mutation being fitness neutral in recently culture-adapted strains from Cambodia, the epicenter of emerging ART resistance.

Project description:Background:Plasmodium lactate dehydrogenase (pLDH) is a major target in diagnosing the erythrocytic stage of malaria parasites because it is highly expressed during blood-stage parasites and is distinguished from human LDH. Rapid diagnostic tests (RDTs) for malaria use pLDH as a target antigen; however, genetic variations in pLDH within the natural population threaten the efficacy of pLDH-based RDTs.Methods:Genetic polymorphisms of Plasmodium vivax LDH (PvLDH) and Plasmodium falciparum LDH (PfLDH) in Myanmar isolates were analysed by nucleotide sequencing analysis. Genetic polymorphisms and the natural selection of PvLDH and PfLDH were analysed using DNASTAR, MEGA6, and DnaSP ver. 5.10.00 programs. The genetic diversity and natural selection of global PvLDH and PfLDH were also analysed. The haplotype network of global PvLDH and PfLDH was constructed using NETWORK ver. 5.0.0.3. Three-dimensional structures of PvLDH and PfLDH were built with YASARA Structure ver. 18.4.24 and the impact of mutations on structural change and stability was evaluated with SDM ver. 2, CUPSAT and MAESTROweb.Results:Forty-nine PvLDH and 52 PfLDH sequences were obtained from Myanmar P. vivax and P. falciparum isolates. Non-synonymous nucleotide substitutions resulting in amino acid changes were identified in both Myanmar PvLDH and PfLDH. Amino acid changes were also identified in the global PvLDH and PfLDH populations, but they did not produce structural alterations in either protein. Low genetic diversity was observed in global PvLDH and PfLDH, which may be maintained by a strong purifying selection.Conclusion:This study extends knowledge for genetic diversity and natural selection of global PvLDH and PfLDH. Although amino acid changes were observed in global PvLDH and PfLDH, they did not alter the conformational structures of the proteins. These suggest that PvLDH and PfLDH are genetically well-conserved in global populations, which indicates that they are suitable antigens for diagnostic purpose and attractive targets for drug development.

Project description:BackgroundPlasmodium falciparum resistance to artemisinins, the first line treatment for malaria worldwide, has been reported in western Cambodia. Resistance is characterized by significantly delayed clearance of parasites following artemisinin treatment. Artemisinin resistance has not previously been reported in Myanmar, which has the highest falciparum malaria burden among Southeast Asian countries.MethodsA non-randomized, single-arm, open-label clinical trial of artesunate monotherapy (4 mg/kg daily for seven days) was conducted in adults with acute blood-smear positive P. falciparum malaria in Kawthaung, southern Myanmar. Parasite density was measured every 12 hours until two consecutive negative smears were obtained. Participants were followed weekly at the study clinic for three additional weeks. Co-primary endpoints included parasite clearance time (the time required for complete clearance of initial parasitemia), parasite clearance half-life (the time required for parasitemia to decrease by 50% based on the linear portion of the parasite clearance slope), and detectable parasitemia 72 hours after commencement of artesunate treatment. Drug pharmacokinetics were measured to rule out delayed clearance due to suboptimal drug levels.ResultsThe median (range) parasite clearance half-life and time were 4.8 (2.1-9.7) and 60 (24-96) hours, respectively. The frequency distributions of parasite clearance half-life and time were bimodal, with very slow parasite clearance characteristic of the slowest-clearing Cambodian parasites (half-life longer than 6.2 hours) in approximately 1/3 of infections. Fourteen of 52 participants (26.9%) had a measurable parasitemia 72 hours after initiating artesunate treatment. Parasite clearance was not associated with drug pharmacokinetics.ConclusionsA subset of P. falciparum infections in southern Myanmar displayed markedly delayed clearance following artemisinin treatment, suggesting either emergence of artemisinin resistance in southern Myanmar or spread to this location from its site of origin in western Cambodia. Resistance containment efforts are underway in Myanmar.Trial registrationAustralian New Zealand Clinical Trials Registry ACTRN12610000896077.

Project description:BackgroundPlasmodium falciparum merozoite surface protein-3 (PfMSP-3) is a target of naturally acquired immunity against P. falciparum infection and is a promising vaccine candidate because of its critical role in the erythrocyte invasion of the parasite. Understanding the genetic diversity of pfmsp-3 is important for recognizing genetic nature and evolutionary aspect of the gene in the natural P. falciparum population and for designing an effective vaccine based on the antigen.MethodsBlood samples collected from P. falciparum-infected patients in Naung Cho and Pyin Oo Lwin, Myanmar, in 2015 were used in this study. The pfmsp-3 was amplified by polymerase chain reaction, cloned, and sequenced. Genetic polymorphism and natural selection of Myanmar pfmsp-3 were analysed using the programs DNASTAR, MEGA6, and DnaSP 5.10.00. Genetic diversity and natural selection of the global pfmsp-3 were also comparatively analysed.ResultsMyanmar pfmsp-3 displayed 2 different alleles, 3D7 and K1. The 3D7 allelic type was predominant in the population, but genetic polymorphism was less diverse than for the K1 allelic type. Polymorphic characters in both allelic types were caused by amino acid substitutions, insertions, and deletions. Amino acid substitutions were mainly occurred at the alanine heptad repeat domains, whereas most insertions and deletions were found at the glutamate rich domain. Overall patterns of amino acid polymorphisms detected in Myanmar pfmsp-3 were similar in the global pfmsp-3 population, but novel amino acid changes were observed in Myanmar pfmsp-3 with low frequencies. Complicated patterns of natural selection and recombination events were predicted in the global pfmsp-3, which may act as major driving forces to maintain and generate genetic diversity of the global pfmsp-3 population.ConclusionGlobal pfmsp-3 revealed genetic polymorphisms, suggesting that the functional and structural consequences of the polymorphisms should be considered in designing a vaccine based on PfMSP-3. Further examination of genetic diversity of pfmsp-3 in the global P. falciparum population is necessary to gain in-depth insight for the population structure and evolutionary aspect of global pfmsp-3.

Project description:The analysis of in vitro anti-malarial drug susceptibility testing is vulnerable to the effects of different statistical approaches and selection biases. These confounding factors were assessed with respect to pfmdr1 gene mutation and amplification in 490 clinical isolates. Two statistical approaches for estimating the drug concentration associated with 50% effect (EC50 ) were compared: the commonly used standard two-stage (STS) method, and nonlinear mixed-effects modelling. The in vitro concentration-effect relationships for, chloroquine, mefloquine, lumefantrine and artesunate, were derived from clinical isolates obtained from patients on the western border of Thailand. All isolates were genotyped for polymorphisms in the pfmdr1 gene. The EC50 estimates were similar for the two statistical approaches but 15-28% of isolates in the STS method had a high coefficient of variation (>15%) for individual estimates of EC50 and these isolates had EC50 values that were 32 to 66% higher than isolates derived with more precision. In total 41% (202/490) of isolates had amplification of pfmdr1 and single nucleotide polymorphisms were found in 50 (10%). Pfmdr1 amplification was associated with an increase in EC50 for mefloquine (139% relative increase in EC50 for 2 copies, 188% for 3+ copies), lumefantrine (82% and 75% for 2 and 3+ copies respectively) and artesunate (63% and 127% for 2 and 3+ copies respectively). In contrast pfmdr1 mutation at codons 86 or 1042 were associated with an increase in chloroquine EC50 (44-48%). Sample size calculations showed that to demonstrate an EC50 shift of 50% or more with 80% power if the prevalence was 10% would require 430 isolates and 245 isolates if the prevalence was 20%. In conclusion, although nonlinear mixed-effects modelling did not demonstrate any major advantage for determining estimates of anti-malarial drug susceptibility, the method includes all isolates, thereby, potentially improving confirmation of candidate molecular markers of anti-malarial drug susceptibility.

Dataset Information

Plasmodium falciparum multidrug resistance protein 1 (pfmrp1) gene and its association with in vitro drug susceptibility of parasite isolates from north-east Myanmar.

Publications

Plasmodium falciparum multidrug resistance protein 1 (pfmrp1) gene and its association with in vitro drug susceptibility of parasite isolates from north-east Myanmar.

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