scholarly journals Molecular Epidemiology of Plasmodium falciparum kelch13 Mutations in Senegal Determined by Using Targeted Amplicon Deep Sequencing

2017 ◽  
Vol 61 (3) ◽  
Author(s):  
Eldin Talundzic ◽  
Yaye D. Ndiaye ◽  
Awa B. Deme ◽  
Christian Olsen ◽  
Dhruviben S. Patel ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Plasmodium Falciparum ◽  
Deep Sequencing ◽  
Artemisinin Resistance ◽  
Potential Drug ◽  
Drug Pressure ◽  
Low Frequencies ◽  
Content Type ◽  
Public Health Activity ◽  
Codon Positions

ABSTRACT The emergence of Plasmodium falciparum resistance to artemisinin in Southeast Asia threatens malaria control and elimination activities worldwide. Multiple polymorphisms in the P. falciparum kelch gene found in chromosome 13 (Pfk13) have been associated with artemisinin resistance. Surveillance of potential drug resistance loci within a population that may emerge under increasing drug pressure is an important public health activity. In this context, P. falciparum infections from an observational surveillance study in Senegal were genotyped using targeted amplicon deep sequencing (TADS) for Pfk13 polymorphisms. The results were compared to previously reported Pfk13 polymorphisms from around the world. A total of 22 Pfk13 propeller domain polymorphisms were identified in this study, of which 12 have previously not been reported. Interestingly, of the 10 polymorphisms identified in the present study that were also previously reported, all had a different amino acid substitution at these codon positions. Most of the polymorphisms were present at low frequencies and were confined to single isolates, suggesting they are likely transient polymorphisms that are part of naturally evolving parasite populations. The results of this study underscore the need to identify potential drug resistance loci existing within a population, which may emerge under increasing drug pressure.

scholarly journals Next-Generation Sequencing and Bioinformatics Protocol for Malaria Drug Resistance Marker Surveillance

2018 ◽  
Vol 62 (4) ◽  
pp. e02474-17 ◽  
Author(s):  
Eldin Talundzic ◽  
Shashidhar Ravishankar ◽  
Julia Kelley ◽  
Dhruviben Patel ◽  
Mateusz Plucinski ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Next Generation Sequencing ◽  
Deep Sequencing ◽  
Artemisinin Resistance ◽  
The United States ◽  
Imported Malaria ◽  
Chloroquine Resistance ◽  
Next Generation ◽  
Content Type ◽  
Generation Sequencing

ABSTRACT The recent advances in next-generation sequencing technologies provide a new and effective way of tracking malaria drug-resistant parasites. To take advantage of this technology, an end-to-end Illumina targeted amplicon deep sequencing (TADS) and bioinformatics pipeline for molecular surveillance of drug resistance in P. falciparum, called malaria resistance surveillance (MaRS), was developed. TADS relies on PCR enriching genomic regions, specifically target genes of interest, prior to deep sequencing. MaRS enables researchers to simultaneously collect data on allele frequencies of multiple full-length P. falciparum drug resistance genes (crt, mdr1, k13, dhfr, dhps, and the cytochrome b gene), as well as the mitochondrial genome. Information is captured at the individual patient level for both known and potential new single nucleotide polymorphisms associated with drug resistance. The MaRS pipeline was validated using 245 imported malaria cases that were reported to the Centers for Disease Control and Prevention (CDC). The chloroquine resistance crt CVIET genotype (mutations underlined) was observed in 42% of samples, the highly pyrimethamine-resistant dhps IRN triple mutant in 92% of samples, and the sulfadoxine resistance dhps mutation SGEAA in 26% of samples. The mdr1 NFSND genotype was found in 40% of samples. With the exception of two cases imported from Cambodia, no artemisinin resistance k13 alleles were identified, and 99% of patients carried parasites susceptible to atovaquone-proguanil. Our goal is to implement MaRS at the CDC for routine surveillance of imported malaria cases in the United States and to aid in the adoption of this system at participating state public health laboratories, as well as by global partners.

scholarly journals Effect of Drug Pressure on Promoting the Emergence of Antimalarial-Resistant Parasites among Pregnant Women in Ghana

2020 ◽  
Vol 64 (6) ◽  
Author(s):  
Bernard Tornyigah ◽  
Romain Coppée ◽  
Pascal Houze ◽  
Kwadwo A. Kusi ◽  
Bright Adu ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Pregnant Women ◽  
Marker Gene ◽  
Intermittent Preventive Treatment ◽  
Artemisinin Resistance ◽  
Preventive Treatment ◽  
Drug Pressure ◽  
Gene Markers ◽  
Content Type ◽  
Artemisinin Derivatives

ABSTRACT The continuous spread of antimalarial drug resistance is a threat to current chemotherapy efficacy. Therefore, characterizing the genetic diversity of drug resistance markers is needed to follow treatment effectiveness and further update control strategies. Here, we genotyped Plasmodium falciparum resistance gene markers associated with sulfadoxine-pyrimethamine (SP) and artemisinin-based combination therapy (ACT) in isolates from pregnant women in Ghana. The prevalence of the septuple IRNI-A/FGKGS/T pfdhfr/pfdhps haplotypes, including the pfdhps A581G and A613S/T mutations, was high at delivery among post-SP treatment isolates (18.2%) compared to those of first antenatal care (before initiation of intermittent preventive treatment of malaria in pregnancy with sulfadoxine-pyrimethamine [IPTp-SP]; 6.1%; P = 0.03). Regarding the pfk13 marker gene, two nonsynonymous mutations (N458D and A481C) were detected at positions previously related to artemisinin resistance in isolates from Southeast Asia. These mutations were predicted in silico to alter the stability of the pfk13 propeller-encoding domain. Overall, these findings highlight the need for intensified monitoring and surveillance of additional mutations associated with increased SP resistance as well as emergence of resistance against artemisinin derivatives.

scholarly journals The Malaria TaqMan Array Card Includes 87 Assays for Plasmodium falciparum Drug Resistance, Identification of Species, and Genotyping in a Single Reaction

2017 ◽  
Vol 61 (5) ◽  
Author(s):  
Suporn Pholwat ◽  
Jie Liu ◽  
Suzanne Stroup ◽  
Shevin T. Jacob ◽  
Patrick Banura ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Plasmodium Falciparum ◽  
Large Scale ◽  
Limit Of Detection ◽  
Artemisinin Resistance ◽  
Clinical Samples ◽  
Content Type ◽  
Antimalarial Resistance ◽  
Resistance Markers ◽  
Taqman Array

ABSTRACT Antimalarial drug resistance exacerbates the global disease burden and complicates eradication efforts. To facilitate the surveillance of resistance markers in countries of malaria endemicity, we developed a suite of TaqMan assays for known resistance markers and compartmentalized them into a single array card (TaqMan array card, TAC). We included 87 assays for species identification, for the detection of Plasmodium falciparum mutations associated with chloroquine, atovaquone, pyrimethamine, sulfadoxine, and artemisinin resistance, and for neutral single nucleotide polymorphism (SNP) genotyping. Assay performance was first optimized using DNA from common laboratory parasite lines and plasmid controls. The limit of detection was 0.1 to 10 pg of DNA and yielded 100% accuracy compared to sequencing. The tool was then evaluated on 87 clinical blood samples from around the world, and the malaria TAC once again achieved 100% accuracy compared to sequencing and in addition detected the presence of mixed infections in clinical samples. With its streamlined protocol and high accuracy, this malaria TAC should be a useful tool for large-scale antimalarial resistance surveillance.

scholarly journals Plasmodium falciparum Genetic Diversity in Continental Equatorial Guinea before and after Introduction of Artemisinin-Based Combination Therapy

2016 ◽  
Vol 61 (1) ◽  
Author(s):  
Mónica Guerra ◽  
Rita Neres ◽  
Patrícia Salgueiro ◽  
Cristina Mendes ◽  
Nicolas Ndong-Mabale ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Drug Resistance ◽  
Plasmodium Falciparum ◽  
Combination Therapy ◽  
Microsatellite Loci ◽  
Point Mutations ◽  
Artemisinin Resistance ◽  
Equatorial Guinea ◽  
Content Type ◽  
Before And After

ABSTRACT Efforts to control malaria may affect malaria parasite genetic variability and drug resistance, the latter of which is associated with genetic events that promote mechanisms to escape drug action. The worldwide spread of drug resistance has been a major obstacle to controlling Plasmodium falciparum malaria, and thus the study of the origin and spread of associated mutations may provide some insights into the prevention of its emergence. This study reports an analysis of P. falciparum genetic diversity, focusing on antimalarial resistance-associated molecular markers in two socioeconomically different villages in mainland Equatorial Guinea. The present study took place 8 years after a previous one, allowing the analysis of results before and after the introduction of an artemisinin-based combination therapy (ACT), i.e., artesunate plus amodiaquine. Genetic diversity was assessed by analysis of the Pfmsp2 gene and neutral microsatellite loci. Pfdhps and Pfdhfr alleles associated with sulfadoxine-pyrimethamine (SP) resistance and flanking microsatellite loci were investigated, and the prevalences of drug resistance-associated point mutations of the Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps genes were estimated. Further, to monitor the use of ACT, we provide the baseline prevalences of K13 propeller mutations and Pfmdr1 copy numbers. After 8 years, noticeable differences occurred in the distribution of genotypes conferring resistance to chloroquine and SP, and the spread of mutated genotypes differed according to the setting. Regarding artemisinin resistance, although mutations reported as being linked to artemisinin resistance were not present at the time, several single nucleotide polymorphisms (SNPs) were observed in the K13 gene, suggesting that closer monitoring should be maintained to prevent the possible spread of artemisinin resistance in Africa.

scholarly journals Artemisinin-Resistant Plasmodium falciparum Parasites Exhibit Altered Patterns of Development in Infected Erythrocytes

2015 ◽  
Vol 59 (6) ◽  
pp. 3156-3167 ◽  
Author(s):  
Amanda Hott ◽  
Debora Casandra ◽  
Kansas N. Sparks ◽  
Lindsay C. Morton ◽  
Geocel-Grace Castanares ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Artemisinin Resistance ◽  
Drug Pressure ◽  
Content Type ◽  
Phenotypic Resistance ◽  
Artemisinin Derivatives ◽  
Stage Of Development ◽  
Clinical Resistance

ABSTRACTArtemisinin derivatives are used in combination with other antimalarial drugs for treatment of multidrug-resistant malaria worldwide. Clinical resistance to artemisinin recently emerged in southeast Asia, yetin vitrophenotypes for discerning mechanism(s) of resistance remain elusive. Here, we describe novel phenotypic resistance traits expressed by artemisinin-resistantPlasmodium falciparum. The resistant parasites exhibit altered patterns of development that result in reduced exposure to drug at the most susceptible stage of development in erythrocytes (trophozoites) and increased exposure in the most resistant stage (rings). In addition, a novelin vitrodelayed clearance assay (DCA) that assesses drug effects on asexual stages was found to correlate with parasite clearance half-lifein vivoas well as with mutations in the Kelch domain gene associated with resistance (Pf3D7_1343700). Importantly, all of the resistance phenotypes were stable in cloned parasites for more than 2 years without drug pressure. The results demonstrate artemisinin-resistantP. falciparumhas evolved a novel mechanism of phenotypic resistance to artemisinin drugs linked to abnormal cell cycle regulation. These results offer insights into a novel mechanism of drug resistance inP. falciparumand new tools for monitoring the spread of artemisinin resistance.

scholarly journals Molecular surveillance of drug resistance: Plasmodium falciparum artemisinin resistance single nucleotide polymorphisms in Kelch protein propeller (K13) domain from Southern Pakistan

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Najia Karim Ghanchi ◽  
Bushra Qurashi ◽  
Hadiqa Raees ◽  
Mohammad Asim Beg
Keyword(s):  
Drug Resistance ◽  
Plasmodium Falciparum ◽  
Molecular Markers ◽  
Single Nucleotide Polymorphisms ◽  
Low Frequency ◽  
Artemisinin Resistance ◽  
University Hospital ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Low Frequencies

Abstract Background K13 propeller (k13) polymorphism are useful molecular markers for tracking the emergence and spread of artemisinin resistance in Plasmodium falciparum. Polymorphisms are reported from Cambodia with rapid invasion of the population and almost near fixation in south East Asia. The study describes single nucleotide polymorphisms in Kelch protein propeller domain of P. falciparum associated with artemisinin resistance from Southern Pakistan. Methods Two hundred and forty-nine samples were collected from patients with microscopy confirmed P. falciparum malaria attending Aga Khan University Hospital during September 2015-April 2018. DNA was isolated using the whole blood protocol for the QIAmp DNA Blood Kit. The k13 propeller gene (k13) was amplified using nested PCR. Double-strand sequencing of PCR products was performed using Sanger sequencing methodology. Sequences were analysed with MEGA 6 and Bio edit software to identify specific SNP combinations. Results All isolates analysed for k13 propeller allele were observed as wild-type in samples collected post implementation of ACT in Pakistan. C580Y, A675V, Y493H and R539T variants associated with reduced susceptibility to artemisinin-based combination therapy (ACT) were not found. Low frequency of M476I and C469Y polymorphisms was found, which is significantly associated with artemisinin resistance. Conclusion Low frequencies of both nonsynonymous and synonymous polymorphisms were observed in P. falciparum isolates circulating in Southern Pakistan. The absence of known molecular markers of artemisinin resistance in this region is favourable for anti-malarial efficacy of ACT. Surveillance of anti-malarial drug resistance to detect its emergence and spread need to be strengthened in Pakistan.

scholarly journals Phenotypic and Genotypic Analysis ofIn Vitro-Selected Artemisinin-Resistant Progeny of Plasmodium falciparum

2011 ◽  
Vol 56 (1) ◽  
pp. 302-314 ◽  
Author(s):  
Matthew S. Tucker ◽  
Tina Mutka ◽  
Kansas Sparks ◽  
Janus Patel ◽  
Dennis E. Kyle
Keyword(s):  
Plasmodium Falciparum ◽  
Artemisinin Resistance ◽  
Drug Pressure ◽  
Green Method ◽  
Drug Induced ◽  
Content Type ◽  
Genotypic Analysis ◽  
Resistant Progeny ◽  
Method Analysis

ABSTRACTEmergence of artemisinin resistance in Cambodia highlights the importance of characterizing resistance to this class of drugs. Previously, intermediate levels of resistance inPlasmodium falciparumwere generatedin vitrofor artelinic acid (AL) and artemisinin (QHS). Here we expanded on earlier selection efforts to produce levels of clinically relevant concentrations, and the resulting lines were characterized genotypically and phenotypically. Recrudescence assays determined the ability of resistant and parent lines to recover following exposure to clinically relevant levels of drugs. Interestingly, the parent clone (D6) tolerated up to 1,500 ng/ml QHS, but the resistant parasite, D6.QHS340×3, recovered following exposure to 2,400 ng/ml QHS. Resistant D6, W2, and TM91c235 parasites all exhibited elevated 50% inhibitory concentrations (IC50s) to multiple artemisinin drugs, with >3-fold resistance to QHS and AL; however, the degree of resistance obtained with standard methods was remarkably less than expected for parasite lines that recovered from 2,400-ng/ml drug pressure. A novel assay format with radiolabeled hypoxanthine demonstrated a greater degree of resistancein vitrothan the standard SYBR green method. Analysis of merozoite number in resistant parasites found D6 and TM91c235 resistant progeny had significantly fewer merozoites than parent strains, whereas W2 resistant progeny had significantly more. Amplification ofpfmdr1increased proportionately to the increased drug levels tolerated by W2 and TM91c235, but not in resistant D6. In summary, we define the artemisinin resistance phenotype as a decrease in susceptibility to artemisinins along with the ability to recover from drug-induced dormancy following supraclinical concentrations of the drug.

scholarly journals Ex VivoDrug Susceptibility Testing and Molecular Profiling of Clinical Plasmodium falciparum Isolates from Cambodia from 2008 to 2013 Suggest Emerging Piperaquine Resistance

2015 ◽  
Vol 59 (8) ◽  
pp. 4631-4643 ◽  
Author(s):  
Suwanna Chaorattanakawee ◽  
David L. Saunders ◽  
Darapiseth Sea ◽  
Nitima Chanarat ◽  
Kritsanai Yingyuen ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Plasmodium Falciparum ◽  
Antimalarial Activity ◽  
Ex Vivo ◽  
Artemisinin Combination Therapy ◽  
Artemisinin Resistance ◽  
Drug Susceptibility ◽  
Chloroquine Resistance ◽  
First Line ◽  
Content Type

ABSTRACTCambodia's first-line artemisinin combination therapy, dihydroartemisinin-piperaquine (DHA-PPQ), is no longer sufficiently curative against multidrug-resistantPlasmodium falciparummalaria at some Thai-Cambodian border regions. We report recent (2008 to 2013) drug resistance trends in 753 isolates from northern, western, and southern Cambodia by surveying forex vivodrug susceptibility and molecular drug resistance markers to guide the selection of an effective alternative to DHA-PPQ. Over the last 3 study years, PPQ susceptibility declined dramatically (geomean 50% inhibitory concentration [IC50] increased from 12.8 to 29.6 nM), while mefloquine (MQ) sensitivity doubled (67.1 to 26 nM) in northern Cambodia. These changes in drug susceptibility were significantly associated with a decreased prevalence ofP. falciparummultidrug resistance 1 gene (Pfmdr1) multiple copy isolates and coincided with the timing of replacing artesunate-mefloquine (AS-MQ) with DHA-PPQ as the first-line therapy. Widespread chloroquine resistance was suggested by all isolates being of theP. falciparumchloroquine resistance transporter gene CVIET haplotype. Nearly all isolates collected from the most recent years hadP. falciparumkelch13mutations, indicative of artemisinin resistance.Ex vivobioassay measurements of antimalarial activity in plasma indicated 20% of patients recently took antimalarials, and their plasma had activity (median of 49.8 nM DHA equivalents) suggestive of substantialin vivodrug pressure. Overall, our findings suggest DHA-PPQ failures are associated with emerging PPQ resistance in a background of artemisinin resistance. The observed connection between drug policy changes and significant reduction in PPQ susceptibility with mitigation of MQ resistance supports reintroduction of AS-MQ, in conjunction with monitoring of theP. falciparummdr1copy number, as a stop-gap measure in areas of DHA-PPQ failure.

scholarly journals Emergence and Spread of kelch13 Mutations Associated with Artemisinin Resistance in Plasmodium falciparum Parasites in 12 Thai Provinces from 2007 to 2016

2018 ◽  
Vol 62 (4) ◽  
Author(s):  
Theerayot Kobasa ◽  
Eldin Talundzic ◽  
Rungniran Sug-aram ◽  
Patcharida Boondat ◽  
Ira F. Goldman ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Gene Mutations ◽  
Artemisinin Resistance ◽  
Parasite Clearance ◽  
Resistance Mutations ◽  
Content Type ◽  
Artemisinin Derivatives ◽  
Reduced Ring ◽  
Control And Prevention ◽  
Greater Mekong Subregion

ABSTRACT Artemisinin-based combination therapy (ACT) is the most effective and widely used treatment for uncomplicated Plasmodium falciparum malaria and is a cornerstone for malaria control and prevention globally. Resistance to artemisinin derivatives has been confirmed in the Greater Mekong Subregion (GMS) and manifests as slow parasite clearance in patients and reduced ring stage susceptibility to artemisinins in survival assays. The P. falciparum kelch13 gene mutations associated with artemisinin-resistant parasites are now widespread in the GMS. We genotyped 277 samples collected during an observational study from 2012 to 2016 from eight provinces in Thailand to identify P. falciparum kelch13 mutations. The results were combined with previously reported genotyping results from Thailand to construct a map illustrating the evolution of P. falciparum kelch13 mutations from 2007 to 2016 in that country. Different mutant alleles were found in strains with different geographical origins. The artemisinin resistance-conferring Y493H and R539T mutations were detected mainly in eastern Thailand (bordering Cambodia), while P574L was found only in western Thailand and R561H only in northwestern Thailand. The C580Y mutation was found across the entire country and was nearing fixation along the Thai-Cambodia border. Overall, the prevalence of artemisinin resistance mutations increased over the last 10 years across Thailand, especially along the Thai-Cambodia border. Molecular surveillance and therapeutic efficacy monitoring should be intensified in the region to further assess the extent and spread of artemisinin resistance.

scholarly journals Mutation in Plasmodium falciparum BTB/POZ domain of K13 protein confers artemisinin resistance

2021 ◽  
Author(s):  
Lucie Paloque ◽  
Romain Coppée ◽  
Barbara H. Stokes ◽  
Nina F. Gnädig ◽  
Karamoko Niaré ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Artemisinin Resistance ◽  
Parasite Clearance ◽  
West African ◽  
Whole Genome Sequence ◽  
Drug Pressure ◽  
Synonymous Mutations ◽  
Survival Assay ◽  
One Year

Partial artemisinin resistance, defined in patients as a delayed parasite clearance following artemisinin-based treatment, is conferred by non-synonymous mutations in the Kelch beta-propeller domain of the Plasmodium falciparum k13 ( pfk13 ) gene. Here, we carried out in vitro selection over a one-year period on a West African P. falciparum strain isolated from Kolle (Mali) under a dose-escalating artemisinin regimen. After 18 cycles of sequential drug pressure, the selected parasites exhibited enhanced survival to dihydroartemisinin in the ring-stage survival assay (RSA 0-3h = 9.2%). Sanger and whole-genome sequence analyses identified the PfK13 P413A mutation, localized in the BTB/POZ domain, upstream of the propeller domain. This mutation was sufficient to confer in vitro artemisinin resistance when introduced into the PfK13 coding sequence of the parasite strain Dd2 by CRISPR/Cas9 gene editing. These results together with structural studies of the protein demonstrate that the propeller domain is not the sole in vitro mediator of PfK13-mediated artemisinin resistance, and highlight the importance of monitoring for mutations throughout PfK13.

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