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Limited genetic diversity in the PvK12 Kelch protein in Plasmodium vivax isolates from Southeast Asia.

ABSTRACT: Artemisinin resistance in Plasmodium falciparum has emerged as a major threat for malaria control and elimination worldwide. Mutations in the Kelch propeller domain of PfK13 are the only known molecular markers for artemisinin resistance in this parasite. Over 100 non-synonymous mutations have been identified in PfK13 from various malaria endemic regions. This study aimed to investigate the genetic diversity of PvK12, the Plasmodium vivax ortholog of PfK13, in parasite populations from Southeast Asia, where artemisinin resistance in P. falciparum has emerged.The PvK12 sequences in 120 P. vivax isolates collected from Thailand (22), Myanmar (32) and China (66) between 2004 and 2008 were obtained and 353 PvK12 sequences from worldwide populations were retrieved for further analysis.These PvK12 sequences revealed a very low level of genetic diversity (? = 0.00003) with only three single nucleotide polymorphisms (SNPs). Of these three SNPs, only G581R is nonsynonymous. The synonymous mutation S88S is present in 3% (1/32) of the Myanmar samples, while G704G and G581R are present in 1.5% (1/66) and 3% (2/66) of the samples from China, respectively. None of the mutations observed in the P. vivax samples were associated with artemisinin resistance in P. falciparum. Furthermore, analysis of 473 PvK12 sequences from twelve worldwide P. vivax populations confirmed the very limited polymorphism in this gene and detected only five distinct haplotypes.The PvK12 sequences from global P. vivax populations displayed very limited genetic diversity indicating low levels of baseline polymorphisms of PvK12 in these areas.

SUBMITTER: Wang M

PROVIDER: S-EPMC5100195 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Limited genetic diversity in the PvK12 Kelch protein in Plasmodium vivax isolates from Southeast Asia.

Wang Meilian M Siddiqui Faiza Amber FA Fan Qi Q Luo Enjie E Cao Yaming Y Cui Liwang L

Malaria journal 20161108 1

<h4>Background</h4>Artemisinin resistance in Plasmodium falciparum has emerged as a major threat for malaria control and elimination worldwide. Mutations in the Kelch propeller domain of PfK13 are the only known molecular markers for artemisinin resistance in this parasite. Over 100 non-synonymous mutations have been identified in PfK13 from various malaria endemic regions. This study aimed to investigate the genetic diversity of PvK12, the Plasmodium vivax ortholog of PfK13, in parasite populat ...[more]

PMID: 27821166

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Project description:BackgroundPlasmodium vivax is the predominant malaria species in northern Mauritania. Molecular data on P. vivax isolates circulating in West Africa are scarce. The present study analysed molecular markers associated with resistance to antifolates (Pvdhfr and Pvdhps), chloroquine (Pvmdr1), and artemisinin (Pvk12) in P. vivax isolates collected in two cities located in the Saharan zone of Mauritania.MethodsBlood samples were obtained from P. vivax-infected patients recruited for chloroquine therapeutic efficacy study in 2013 and febrile patients spontaneously consulting health facilities in Nouakchott and Atar in 2015-2016. Fragments of Pvdhfr (codons 13, 33, 57, 58, 61, 117, and 174), Pvdhps (codons 382, 383, 512, 553, and 585), Pvmdr1 (codons 976 and 1076) and Pvk12 (codon 552) genes were amplified by PCR and sequenced.ResultsMost of the isolates in Nouakchott (126/154, 81.8%) and Atar (44/45, 97.8%) carried the wild-type Pvdhfr allelic variant (IPFSTSI). In Nouakchott, all mutants (28/154; 18.2%) had double Pvdhfr mutations in positions 58 and 61 (allelic variant IPFRMSI), whereas in Atar only 1 isolate was mutant (S117N, allelic variant IPFSTNI). The wild-type Pvdhps allelic variant (SAKAV) was found in all tested isolates (Nouakchott, n = 93; Atar, n = 37). Few isolates in Nouakchott (5/115, 4.3%) and Atar (3/79, 3.8%) had the mutant Pvmdr1 allele 976F or 1076L, but not both, including in pre-treatment isolates obtained from patients treated successfully with chloroquine. All isolates (59 in Nouakchott and 48 in Atar) carried the wild-type V552 allele in Pvk12.ConclusionsPolymorphisms in Pvdhfr, Pvdhps, Pvmdr1, and Pvk12 were limited in P. vivax isolates collected recently in Nouakchott and Atar. Compared to the isolates collected in Nouakchott in 2007-2009, there was no evidence for selection of mutants. The presence of one, but not both, of the two potential markers of chloroquine resistance in Pvmdr1 in pre-treatment isolates did not influence the clinical outcome, putting into question the role of Pvmdr1 mutant alleles 976F and 1076L in treatment failure. Molecular surveillance is an important component of P. vivax malaria control programme in the Saharan zone of Mauritania to predict possible emergence of drug-resistant parasites.

Project description:BACKGROUND:Antigens expressed in sexual stages of the malaria parasites are targets of transmission-blocking vaccines (TBVs). HAP2/GCS1, a TBV candidate, is critical for fertilization in Plasmodium. Here, the genetic diversity of PvHAP2 was studied in Plasmodium vivax parasite populations from the Greater Mekong Subregion (GMS). METHODS:Plasmodium vivax clinical isolates were collected in clinics from the China-Myanmar border region (135 samples), western Thailand (41 samples) and western Myanmar (51 samples). Near full-length Pvhap2 (nucleotides 13-2574) was amplified and sequenced from these isolates. Molecular evolution studies were conducted to evaluate the genetic diversity, selection and population differentiation. RESULTS:Sequencing of the pvhap2 gene for a total of 227 samples from the three P. vivax populations revealed limited genetic diversity of this gene in the GMS (? = 0.00036 ± 0.00003), with the highest ? value observed in Myanmar (0.00053 ± 0.00009). Y133S was the dominant mutation in the China-Myanmar border (99.26%), Myanmar (100%) and Thailand (95.12%). Results of all neutrality tests were negative for all the three populations, suggesting the possible action of purifying selection. Codon-based tests identified specific codons which are under purifying or positive selections. Wright's fixation index showed low to moderate genetic differentiation of P. vivax populations in the GMS, with FST ranging from 0.04077 to 0.24833, whereas high levels of genetic differentiation were detected between the China-Myanmar border and Iran populations (FST = 0.60266), and between Thailand and Iran populations (FST = 0.44161). A total of 20 haplotypes were identified, with H2 being the abundant haplotype in China-Myanmar border, Myanmar and Thailand populations. Epitope mapping prediction of Pvhap2 antigen showed that high-score B-cell epitopes are located in the S307-G324, L429-P453 and V623-D637 regions. The E317K and D637N mutations located within S307-G324 and V623-D637 epitopes slightly reduced the predicted score for potential epitopes. CONCLUSIONS:The present study showed a very low level of genetic diversity of pvhap2 gene among P. vivax populations in the Greater Mekong Subregion. The relative conservation of pvhap2 supports further evaluation of a Pvhap2-based TBV.

Project description:Objectives: In this study, we aimed to analyse the genetic diversity Kelch 13 (K13) propeller allele of the Plasmodium falciparum isolates mainly imported from Southeast Asia and Africa in southern China, including the provinces of Yunnan and Guangxi. Methods: At enrolment, we collected blood samples from patients with confirmed cases of malaria infection between January 2012 and December 2017, for analysis. Individual patient information was obtained via a malaria surveillance system. The malaria infections and P. falciparum K13 mutations were diagnosed by using a nested polymerase chain reaction (PCR) method. Results: The K13 mutations were identified in 283 P. falciparum isolates from 18 counties in Yunnan and 22 counties in Guangxi. Of Forty-six isolates (46/283, 16.3%) that harbored K13 mutant alleles were detected: 26.8% in Yunnan (33/123) and 8.1% in Guangxi (13/160). A total of 18 different K13 mutations were detected. Only the F446I mutation was detected in Yunnan isolates, and F446I was more frequent (20/46, 43.5%) than other alleles. Further, the temporal distribution of the F446I mutation ratio from 2012 to 2015 exhibited no significant difference in Yunnan Province (2012, 2/13, 15.4%; 2013, 7/40, 17.5%; 2014, 7/33, 21.2%; 2015, 4/37, 10.8%, p = 0.121). A578S allele was the main K13 mutation (5/283, 1.8%) from Africa. The K13 mutants were present in 33.3% of indigenous isolates, 27.4% of isolates from Southeast Asia, and 7.9% of isolates from Africa. The analysis of 10 neutral microsatellite loci of 60 isolates showed that at the TAA109 locus, the expected heterozygosity of F446I (H e = 0.112 ± 0.007) was much lower than that of wild type and other mutation types in Myanmar isolates. With respect to geographic distribution, TAA109 also exhibited a significant difference between isolates from Southeast Asia (H e = 0.139 ± 0.012) and those from Africa (H e = 0.603 ± 0.044). Conclusions: The present findings on the geographic diversity of K13 mutant alleles in P. falciparum may provide a basis for routine molecular surveillance and risk assessment, to monitor artemisinin resistance (ART) in China. Our results will be helpful for enriching the artemisinin resistance database in China during the elimination and post-elimination phases.

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.

Dataset Information

Limited genetic diversity in the PvK12 Kelch protein in Plasmodium vivax isolates from Southeast Asia.

Publications

Limited genetic diversity in the PvK12 Kelch protein in Plasmodium vivax isolates from Southeast Asia.

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