Molecular Profile of Malaria Drug Resistance Markers of Plasmodium falciparum in Suriname

ABSTRACT In Suriname, an artesunate monotherapy therapeutic efficacy trial was recently conducted to evaluate partial artemisinin resistance emerging in Plasmodium falciparum. We genotyped the PfK13 propeller domain of P. falciparum in 40 samples as well as other mutations proposed to be associated with artemisinin-resistant mutants. We did not find any mutations previously associated with artemisinin resistance in Southeast Asia, but we found fixed resistance mutations for chloroquine (CQ) and sulfadoxine-pyrimethamine. Additionally, the PfCRT C350R mutation, associated with reversal of CQ resistance and piperaquine-selective pressure, was present in 62% of the samples. Our results from neutral microsatellite data also confirmed a high parasite gene flow in the Guiana Shield. Although recruiting participants for therapeutic efficacy studies is challenging in areas where malaria endemicity is very low due to the low number of malaria cases reported, conducting these studies along with molecular surveillance remains essential for the monitoring of artemisinin-resistant alleles and for the characterization of the population structure of P. falciparum in areas targeted for malaria elimination.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02141-17 ◽

2018 ◽

Vol 62 (4) ◽

Cited By ~ 10

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.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00110-17 ◽

2017 ◽

Vol 61 (5) ◽

Cited By ~ 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.

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K13 Propeller Mutations in Plasmodium falciparum Populations in Regions of Malaria Endemicity in Vietnam from 2009 to 2016

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01578-16 ◽

2017 ◽

Vol 61 (4) ◽

Cited By ~ 23

Author(s):

Nguyen Thuy-Nhien ◽

Nguyen Kim Tuyen ◽

Nguyen Thanh Tong ◽

Nguyen Tuong Vy ◽

Ngo Viet Thanh ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Artemisinin Resistance ◽

Parasite Clearance ◽

Single Strain ◽

Content Type ◽

Reduced Ring ◽

Resistance Markers ◽

Multiple Strains

ABSTRACT The spread of artemisinin-resistant Plasmodium falciparum compromises the therapeutic efficacy of artemisinin combination therapies (ACTs) and is considered the greatest threat to current global initiatives to control and eliminate malaria. This is particularly relevant in Vietnam, where dihydroartemisinin-piperaquine (DP) is the recommended ACT for P. falciparum infection. The propeller domain gene of K13, a molecular marker of artemisinin resistance, was successfully sequenced in 1,060 P. falciparum isolates collected at 3 malaria hot spots in Vietnam between 2009 and 2016. Eight K13 propeller mutations (Thr474Ile, Tyr493His, Arg539Thr, Ile543Thr, Pro553Leu, Val568Gly, Pro574Leu, and Cys580Tyr), including several that have been validated to be artemisinin resistance markers, were found. The prevalences of K13 mutations were 29% (222/767), 6% (11/188), and 43% (45/105) in the Binh Phuoc, Ninh Thuan, and Gia Lai Provinces of Vietnam, respectively. Cys580Tyr became the dominant genotype in recent years, with 79.1% (34/43) of isolates in Binh Phuoc Province and 63% (17/27) of isolates in Gia Lai Province carrying this mutation. K13 mutations were associated with reduced ring-stage susceptibility to dihydroartemisinin (DHA) in vitro and prolonged parasite clearance in vivo. An analysis of haplotypes flanking K13 suggested the presence of multiple strains with the Cys580Tyr mutation rather than a single strain expanding across the three sites.

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Historical trends and new surveillance of Plasmodium falciparum drug resistance markers in Angola

Malaria Journal ◽

10.1186/s12936-021-03713-2 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Emily R. Ebel ◽

Fátima Reis ◽

Dmitri A. Petrov ◽

Sandra Beleza

Keyword(s):

Plasmodium Falciparum ◽

Genetic Markers ◽

High Frequency ◽

Artemisinin Resistance ◽

Resistance Mutations ◽

Historical Trends ◽

Public And Private ◽

Haplotype Data ◽

Hospital Patients ◽

Resistance Markers

Abstract Background Plasmodium falciparum resistance to chloroquine (CQ) and sulfadoxine-pyrimethamine (SP) has historically posed a major threat to malaria control throughout the world. The country of Angola officially replaced CQ with artemisinin-based combination therapy (ACT) as a first-line treatment in 2006, but malaria cases and deaths have recently been rising. Many classic resistance mutations are relevant for the efficacy of currently available drugs, making it important to continue monitoring their frequency in Angola. Methods Plasmodium falciparum DNA was sampled from the blood of 50 hospital patients in Cabinda, Angola from October-December of 2018. Each infection was genotyped for 13 alleles in the genes crt, mdr1, dhps, dhfr, and kelch13, which are collectively involved in resistance to six common anti-malarials. To compare frequency patterns over time, P. falciparum genotype data were also collated from studies published from across Angola in the last two decades. Results The two most important alleles for CQ resistance, crt 76T and mdr1 86Y, were found at respective frequencies of 71.4% and 6.5%. Historical data suggest that mdr1 N86 has been steadily replacing 86Y throughout Angola in the last decade, while the frequency of crt 76T has been more variable across studies. Over a third of new samples from Cabinda were ‘quintuple mutants’ for SP resistance in dhfr/dhps, with a sixth mutation at dhps A581G present at 9.6% frequency. The markers dhfr 51I, dhfr 108N, and dhps 437G have been nearly fixed in Angola since the early 2000s, whereas dhfr 59R may have risen to high frequency more recently. Finally, no non-synonymous polymorphisms were detected in kelch13, which is involved in artemisinin resistance in Southeast Asia. Conclusions Genetic markers of P. falciparum resistance to CQ are likely declining in frequency in Angola, consistent with the official discontinuation of CQ in 2006. The high frequency of multiple genetic markers of SP resistance is consistent with the continued public and private use of SP. In the future, more complete haplotype data from mdr1, dhfr, and dhps will be critical for understanding the changing efficacy of multiple anti-malarial drugs. These data can be used to support effective drug policy decisions in Angola.

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Molecular Characterization of the CytochromebGene andIn VitroAtovaquone Susceptibility of Plasmodium falciparum Isolates from Kenya

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03956-14 ◽

2015 ◽

Vol 59 (3) ◽

pp. 1818-1821 ◽

Cited By ~ 6

Author(s):

Luicer A. Ingasia ◽

Hoseah M. Akala ◽

Mabel O. Imbuga ◽

Benjamin H. Opot ◽

Fredrick L. Eyase ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Genetic Polymorphism ◽

Molecular Characterization ◽

Mutant Allele ◽

Inhibitory Concentration ◽

Wild Type Allele ◽

Wild Type ◽

Western Kenya ◽

Content Type

ABSTRACTThe prevalence of a genetic polymorphism(s) at codon 268 in the cytochromebgene, which is associated with failure of atovaquone-proguanil treatment, was analyzed in 227Plasmodium falciparumparasites from western Kenya. The prevalence of the wild-type allele was 63%, and that of the Y268S (denoting a Y-to-S change at position 268) mutant allele was 2%. There were no pure Y268C or Y268N mutant alleles, only mixtures of a mutant allele(s) with the wild type. There was a correlation between parasite 50% inhibitory concentration (IC50) and parasite genetic polymorphism; mutant alleles had higher IC50s than the wild type.

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Molecular Surveillance of Plasmodium falciparum Drug Resistance Markers in Clinical Samples from Botswana

American Journal of Tropical Medicine and Hygiene ◽

10.4269/ajtmh.18-0440 ◽

2018 ◽

Vol 99 (6) ◽

pp. 1499-1503 ◽

Cited By ~ 2

Author(s):

Leabaneng Tawe ◽

Michela Menegon ◽

Pleasure Ramatlho ◽

Charles W. Muthoga ◽

Naledi Mutukwa ◽

...

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Clinical Samples ◽

Molecular Surveillance ◽

Resistance Markers

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Molecular Surveillance of Drug Resistance of Plasmodium falciparum Isolates Imported from Angola in Henan Province, China

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00552-19 ◽

2019 ◽

Vol 63 (10) ◽

Cited By ~ 1

Author(s):

Ruimin Zhou ◽

Chengyun Yang ◽

Suhua Li ◽

Yuling Zhao ◽

Ying Liu ◽

...

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Antimalarial Drug ◽

Henan Province ◽

Nucleotide Polymorphisms ◽

Combination Therapies ◽

Antimalarial Drug Resistance ◽

Single Nucleotide ◽

Molecular Surveillance ◽

Content Type

ABSTRACT Angola was the main origin country for the imported malaria in Henan Province, China. Antimalarial drug resistance has posed a threat to the control and elimination of malaria. Several molecular markers were confirmed to be associated with the antimalarial drug resistance, such as pfcrt, pfmdr1, pfdhfr, pfdhps, and K13. This study evaluated the drug resistance of the 180 imported Plasmodium falciparum isolates from Angola via nested PCR using Sanger sequencing. The prevalences of pfcrt C72V73M74N75K76, pfmdr1 N86Y184S1034N1042D1246, pfdhfr A16N51C59S108D139I164, and pfdhps S436A437A476K540A581 were 69.4%, 59.9%, 1.3% and 6.3%, respectively. Three nonsynonymous (A578S, M579I, and Q613E) and one synonymous (R471R) mutation of K13 were found, the prevalences of which were 2.5% and 1.3%, respectively. The single nucleotide polymorphisms (SNPs) in pfcrt, pfmdr1, pfdhfr, and pfdhps were generally shown as multiple mutations. The mutant prevalence of pfcrt reduced gradually, but pfdhfr and pfdhps still showed high mutant prevalence, while pfmdr1 was relatively low. The mutation of the K13 gene was rare. Molecular surveillance of artemisinin (ART) resistance will be used as a tool to evaluate the real-time efficacy of the artemisinin-based combination therapies (ACTs) and the ART resistance situation.

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Molecular detection of drug resistant polymorphisms in Plasmodium falciparum isolates from Southwest, Nigeria

BMC Research Notes ◽

10.1186/s13104-020-05334-5 ◽

2020 ◽

Vol 13 (1) ◽

Author(s):

Monday Tola ◽

Olumide Ajibola ◽

Emmanuel Taiwo Idowu ◽

Olusesan Omidiji ◽

Samson Taiwo Awolola ◽

...

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Antimalarial Drug Resistance ◽

Allelic Discrimination ◽

Molecular Surveillance ◽

Malaria Burden ◽

Two Samples ◽

Southwest Nigeria ◽

Resistance Markers ◽

Drug Interventions

Abstract Objective Nigeria bears 25% of global malaria burden despite concerted efforts towards its control and elimination. The emergence of drug resistance to first line drugs, artemisinin combination therapies (ACTs), indicates an urgent need for continuous molecular surveillance of drug resistance especially in high burden countries where drug interventions are heavily relied on. This study describes mutations in Plasmodium falciparum genes associated with drug resistance in malaria; Pfk13, Pfmdr1, PfATPase6 and Pfcrt in isolates obtained from 83 symptomatic malaria patients collected in August 2014, aged 1–61 years old from South-west Nigeria. Results Two Pfmdr1, N86 and Y184 variants were present at a prevalence of 56% and 13.25% of isolates respectively. There was one synonymous (S679S) and two non-synonymous (M699V, S769M) mutations in the PATPase6 gene, while Pfcrt genotype (CVIET), had a prevalence of 45%. The Pfk13 C580Y mutant allele was suspected by allelic discrimination in two samples with mixed genotypes although this could not be validated with independent isolation or additional methods. Our findings call for robust molecular surveillance of antimalarial drug resistance markers in west Africa especially with increased use of antimalarial drugs as prophylaxis for Covid-19.

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Efficacy of dihydroartemisinin/piperaquine and artesunate monotherapy for the treatment of uncomplicated Plasmodium falciparum malaria in Central Vietnam

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkaa172 ◽

2020 ◽

Author(s):

Eduard Rovira-Vallbona ◽

Nguyen Van Hong ◽

Johanna H Kattenberg ◽

Ro Mah Huan ◽

Nguyen Thi Thu Hien ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Falciparum Malaria ◽

Ex Vivo ◽

Survival Rates ◽

Artemisinin Resistance ◽

Plasmodium Falciparum Malaria ◽

Parasite Clearance ◽

Resistance Mutations ◽

Malaria Burden ◽

Central Vietnam

Abstract Background Artemisinin-based combination therapies (ACTs) have significantly contributed to reduce Plasmodium falciparum malaria burden in Vietnam, but their efficacy is challenged by treatment failure of dihydroartemisinin/piperaquine ACT in Southern provinces. Objectives To assess the efficacy of dihydroartemisinin/piperaquine for uncomplicated P. falciparum malaria in Gia Lai, Central Vietnam, and determine parasite resistance to artemisinin (ClinicalTrials.gov identifier NCT02604966). Methods Sixty patients received either dihydroartemisinin/piperaquine (4 mg/kg/day, 3 days; n = 33) or artesunate monotherapy (4 mg/kg/day, 3 days; n = 27) followed by dihydroartemisinin/piperaquine (AS + DHA/PPQ). Clinical phenotypes were determined during a 42 day follow-up and analysed together with ex vivo susceptibility to antimalarials and molecular markers of drug resistance. Results Day 3 positivity rate was significantly higher in the AS + DHA/PPQ arm compared with dihydroartemisinin/piperaquine (70.4% versus 39.4%, P = 0.016). Parasite clearance time was 95.2 h (AS + DHA/PPQ) versus 71.9 h (dihydroartemisinin/piperaquine, P = 0.063) and parasite clearance half-life was 7.4 h (AS + DHA/PPQ) versus 7.0 h (dihydroartemisinin/piperaquine, P = 0.140). Adequate clinical and parasitological response at Day 42 was 100% in both arms. By RT–qPCR, 36% (19/53) patients remained positive until Day 7. No recurrences were detected. kelch13 artemisinin resistance mutations were found in 87% (39/45) of isolates and 50% (20/40) were KEL1/C580Y. The piperaquine resistance marker plasmepsin-2 was duplicated in 10.4% (5/48). Isolates from Day 3-positive patients (n = 18) had higher ex vivo survival rates to artemisinin compounds (P < 0.048) and prevalence of kelch13 mutations (P = 0.005) than Day 3-negative patients (n = 5). The WHO definition of artemisinin resistance was fulfilled in 60% (24/40) of cases. Conclusions Although dihydroartemisinin/piperaquine remained effective to treat P. falciparum, the high Day 3 positivity rate and prevalence of KEL1 strains calls for continuous monitoring of dihydroartemisinin/piperaquine efficacy in Central Vietnam.

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Delayed Parasite Clearance after Treatment with Dihydroartemisinin-Piperaquine in Plasmodium falciparum Malaria Patients in Central Vietnam

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02746-14 ◽

2014 ◽

Vol 58 (12) ◽

pp. 7049-7055 ◽

Cited By ~ 50

Author(s):

Kamala Thriemer ◽

Nguyen Van Hong ◽

Anna Rosanas-Urgell ◽

Bui Quang Phuc ◽

Do Manh Ha ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Clearance Rate ◽

Artemisinin Resistance ◽

Parasite Clearance ◽

Clearance Time ◽

Sensitivity Testing ◽

Content Type ◽

Parasite Clearance Time ◽

Central Vietnam

ABSTRACTReduced susceptibility ofPlasmodium falciparumtoward artemisinin derivatives has been reported from the Thai-Cambodian and Thai-Myanmar borders. Following increasing reports from central Vietnam of delayed parasite clearance after treatment with dihydroartemisinin-piperaquine (DHA-PPQ), the current first-line treatment, we carried out a study on the efficacy of this treatment. Between September 2012 and February 2013, we conducted a 42-dayin vivoandin vitroefficacy study in Quang Nam Province. Treatment was directly observed, and blood samples were collected twice daily until parasite clearance. In addition, genotyping, quantitative PCR (qPCR), andin vitrosensitivity testing of isolates was performed. The primary endpoints were parasite clearance rate and time. The secondary endpoints included PCR-corrected and uncorrected cure rates, qPCR clearance profiles,in vitrosensitivity results (for chloroquine, dihydroartemisinin, and piperaquine), and genotyping for mutations in the Kelch 13 propeller domain. Out of 672 screened patients, 95 were recruited and 89 available for primary endpoint analyses. The median parasite clearance time (PCT) was 61.7 h (interquartile range [IQR], 47.6 to 83.2 h), and the median parasite clearance rate had a slope half-life of 6.2 h (IQR, 4.4 to 7.5 h). The PCR-corrected efficacy rates were estimated at 100% at day 28 and 97.7% (95% confidence interval, 91.2% to 99.4%) at day 42. At day 3, theP. falciparumprevalence by qPCR was 2.5 times higher than that by microscopy. The 50% inhibitory concentrations (IC50s) of isolates with delayed clearance times (≥72 h) were significantly higher than those with normal clearance times for all three drugs. Delayed parasite clearance (PCT, ≥72 h) was significantly higher among day 0 samples carrying the 543 mutant allele (47.8%) than those carrying the wild-type allele (1.8%;P= 0.048). In central Vietnam, the efficacy of DHA-PPQ is still satisfactory, but the parasite clearance time and rate are indicative of emerging artemisinin resistance. (This study has been registered at ClinicalTrials.gov under registration no. NCT01775592.)

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