Author Correction: Molecular surveillance of Plasmodium falciparum drug-resistance markers in Vietnam using multiplex amplicon sequencing (2000-2016).
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ABSTRACT:
SUBMITTER: Rovira-Vallbona E
PROVIDER: S-EPMC10567743 | biostudies-literature | 2023 Oct
REPOSITORIES: biostudies-literature
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Author Correction: Molecular surveillance of Plasmodium falciparum drug-resistance markers in Vietnam using multiplex amplicon sequencing (2000-2016).
Rovira-Vallbona Eduard E Kattenberg Johanna Helena JH Van Hong Nguyen N Guetens Pieter P Imamura Hideo H Monsieurs Pieter P Chiheb Driss D Erhart Annette A Phuc Bui Quang BQ Xa Nguyen Xuan NX Rosanas-Urgell Anna A
Scientific reports 20231011 1
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Project description:Emergence and spread of Plasmodium falciparum resistance to artemisinin-based combination therapies (ACT) is a major challenge for Greater Mekong Subregion countries in their goal to eliminate malaria by 2030. Tools to efficiently monitor drug resistance beyond resource-demanding therapeutic efficacy studies are necessary. A custom multiplex amplicon sequencing assay based on Illumina technology was designed to target the marker of partial resistance to artemisinin (K13), five candidate modulators of artemisinin resistance, the marker of resistance to chloroquine (crt), and four neutral microsatellite loci. The assay was used to genotype 635 P. falciparum-positive blood samples collected across seven provinces of Vietnam and one of Cambodia between 2000 and 2016. Markers of resistance to artemisinin partner-drugs piperaquine (copy number of plasmepsin-2) and mefloquine (copy number of multidrug-resistance 1) were determined by qPCR. Parasite population structure was further assessed using a 101-SNP barcode. Validated mutations of artemisinin partial resistance in K13 were found in 48.1% of samples, first detection was in 2000, and by 2015 prevalence overcame > 50% in Central Highlands and Binh Phuoc province. K13-C580Y variant became predominant country-wide, quickly replacing an outbreak of K13-I543T in Central Highlands. Mutations in candidate artemisinin resistance modulator genes paralleled the trends of K13 mutants, whereas resistance to piperaquine and mefloquine remained low (≈ 10%) by 2015-2016. Genomic tools applied to malaria surveillance generate comprehensive information on dynamics of drug resistance and population structure and reflect drug efficacy profiles from in vivo studies.
| S-EPMC10457381 | biostudies-literature
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| S-EPMC8924219 | biostudies-literature
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| S-EPMC9151790 | biostudies-literature
Project description:BackgroundUnderstanding the dynamics of gametocyte production in polyclonal Plasmodium falciparum infections requires a genotyping method that detects distinct gametocyte clones and estimates their relative frequencies. Here, a marker was identified and evaluated to genotype P. falciparum mature gametocytes using amplicon deep sequencing.MethodsA data set of polymorphic regions of the P. falciparum genome was mined to identify a gametocyte genotyping marker. To assess marker resolution, the number of unique haplotypes in the marker region was estimated from 95 Malawian P. falciparum whole genome sequences. Specificity of the marker for detection of mature gametocytes was evaluated using reverse transcription-polymerase chain reaction of RNA extracted from NF54 mature gametocytes and rings from a non-gametocyte-producing strain of P. falciparum. Amplicon deep sequencing was performed on experimental mixtures of mature gametocytes from two distinct parasite clones, as well as gametocyte-positive P. falciparum field isolates to evaluate the quantitative ability and determine the limit of detection of the genotyping approach.ResultsA 400 bp region of the pfs230 gene was identified as a gametocyte genotyping marker. A larger number of unique haplotypes was observed at the pfs230 marker (34) compared to the sera-2 (18) and ama-1 (14) markers in field isolates from Malawi. RNA and DNA genotyping accurately estimated gametocyte and total parasite clone frequencies when evaluating agreement between expected and observed haplotype frequencies in gametocyte mixtures, with concordance correlation coefficients of 0.97 [95% CI: 0.92-0.99] and 0.92 [95% CI: 0.83-0.97], respectively. The detection limit of the genotyping method for male gametocytes was 0.41 pfmget transcripts/µl [95% CI: 0.28-0.72] and for female gametocytes was 1.98 ccp4 transcripts/µl [95% CI: 1.35-3.68].ConclusionsA region of the pfs230 gene was identified as a marker to genotype P. falciparum gametocytes. Amplicon deep sequencing of this marker can be used to estimate the number and relative frequency of parasite clones among mature gametocytes within P. falciparum infections. This gametocyte genotyping marker will be an important tool for studies aimed at understanding dynamics of gametocyte production in polyclonal P. falciparum infections.
| S-EPMC10999092 | biostudies-literature
Project description:BackgroundMalaria rapid diagnostic tests (RDTs) remain the main point-of-care tests for diagnosis of symptomatic Plasmodium falciparum malaria in endemic areas. However, parasites with gene deletions in the most common RDT target, histidine rich protein 2 (pfhrp2/HRP2), can produce false-negative RDT results leading to inadequate case management. The objective of this study was to determine the prevalence of hrp2/3 deletions causing false-negative RDT results in Vietnam (Gia Lai and Dak Lak provinces).MethodsIndividuals presenting with malaria symptoms at health facilities were screened for P. falciparum infection using light microscopy and HRP2-RDT (SD Bioline Malaria Antigen Pf/Pv RDT, Abbott). Microscopically confirmed P. falciparum infections were analysed for parasite species by 18S rRNA qPCR, and pfhrp2 and pfhrp3 exon2 deletions were investigated by nested PCR. pfhrp2 amplicons were sequenced by the Sanger method and HRP2 plasma levels were determined by enzyme-linked immunosorbent assay (ELISA).ResultsThe prevalence of false-negative RDT results among symptomatic cases was 5.6% (15/270). No pfhrp2 and pfhrp3 deletions were identified. False-negative RDT results were associated with lower parasite density (p = 0.005) and lower HRP2 plasma concentrations (p < 0.001), as compared to positive RDT.ConclusionsThe absence of hrp2/3 deletions detected in this survey suggests that HRP2-based malaria RDTs remain effective for the diagnosis of symptomatic P. falciparum malaria in Central Vietnam.
| S-EPMC9724378 | biostudies-literature
Project description:BackgroundResistance to anti-malarials is a serious threat to the efforts to control and eliminate malaria. Surveillance based on simple field protocols with centralized testing to detect molecular markers associated with anti-malarial drug resistance can be used to identify locations where further investigations are needed.MethodsDried blood spots were collected from 398 patients (age range 5-59 years, 99% male) with Plasmodium falciparum infections detected using rapid diagnostic tests over two rounds of sample collection conducted in 2016 and 2017 in Komé, South-West Chad. Specimens were genotyped using amplicon sequencing or qPCR for validated markers of anti-malarial resistance including partner drugs used in artemisinin-based combination therapy (ACT).ResultsNo mutations in the pfk13 gene known to be associated with artemisinin resistance were found but a high proportion of parasites carried other mutations, specifically K189T (190/349, 54.4%, 95%CI 49.0-59.8%). Of 331 specimens successfully genotyped for pfmdr1 and pfcrt, 52% (95%CI 46.4-57.5%) carried the NFD-K haplotype, known to be associated with reduced susceptibility to lumefantrine. Only 20 of 336 (6.0%, 95%CI 3.7-9.0%) had parasites with the pfmdr1-N86Y polymorphism associated with increased treatment failures with amodiaquine. Nearly all parasites carried at least one mutation in pfdhfr and/or pfdhps genes but 'sextuple' mutations in pfdhfr-pfdhps including pfdhps -A581G were rare (8/336 overall, 2.4%, 95%CI 1.2-4.6%). Only one specimen containing parasites with pfmdr1 gene amplification was detected.ConclusionsThese results provide information on the likely high efficacy of artemisinin-based combinations commonly used in Chad, but suggest decreasing levels of sensitivity to lumefantrine and high levels of resistance to sulfadoxine-pyrimethamine used for seasonal malaria chemoprevention and intermittent preventive therapy in pregnancy. A majority of parasites had mutations in the pfk13 gene, none of which are known to be associated with artemisinin resistance. A therapeutic efficacy study needs to be conducted to confirm the efficacy of artemether-lumefantrine.
| S-EPMC8917628 | biostudies-literature
Project description: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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Project description:An amendment to this paper has been published and can be accessed via a link at the top of the paper.
| S-EPMC7538963 | biostudies-literature
Project description:BackgroundGenotyping Plasmodium falciparum subpopulations in malaria infections is an important aspect of malaria molecular epidemiology to understand within-host diversity and the frequency of drug resistance markers.MethodsWe characterized P. falciparum genetic diversity in asymptomatic infections and subsequent first febrile infections using amplicon sequencing (AmpSeq) of ama1 in Coastal Kenya. We also examined temporal changes in haplotype frequencies of mdr1, a drug-resistant marker.ResultsWe found >60% of the infections were polyclonal (complexity of infection [COI] >1) and there was a reduction in COI over time. Asymptomatic infections had a significantly higher mean COI than febrile infections based on ama1 sequences (2.7 [95% confidence interval {CI}, 2.65-2.77] vs 2.22 [95% CI, 2.17-2.29], respectively). Moreover, an analysis of 30 paired asymptomatic and first febrile infections revealed that many first febrile infections (91%) were due to the presence of new ama1 haplotypes. The mdr1-YY haplotype, associated with chloroquine and amodiaquine resistance, decreased over time, while the NY (wild type) and the NF (modulates response to lumefantrine) haplotypes increased.ConclusionsThis study emphasizes the utility of AmpSeq in characterizing parasite diversity as it can determine relative proportions of clones and detect minority clones. The usefulness of AmpSeq in antimalarial drug resistance surveillance is also highlighted.
| S-EPMC7613600 | biostudies-literature
Project description:BackgroundIn 2006, the Senegalese National Malaria Control Programme recommended artemisinin-based combination therapy (ACT) with artemether-lumefantrine as the first-line treatment for uncomplicated Plasmodium falciparum malaria. To date, multiple mutations associated with artemisinin delayed parasite clearance have been described in Southeast Asia in the Pfk13 gene, such as Y493H, R539T, I543T and C580Y. Even though ACT remains clinically and parasitologically efficacious in Senegal, the spread of resistance is possible as shown by the earlier emergence of resistance to chloroquine in Southeast Asia that subsequently spread to Africa. Therefore, surveillance of artemisinin resistance in malaria endemic regions is crucial and requires the implementation of sensitive tools, such as next-generation sequencing (NGS) which can detect novel mutations at low frequency.MethodsHere, an amplicon sequencing approach was used to identify mutations in the Pfk13 gene in eighty-one P. falciparum isolates collected from three different regions of Senegal.ResultsIn total, 10 SNPs around the propeller domain were identified; one synonymous SNP and nine non-synonymous SNPs, and two insertions. Three of these SNPs (T478T, A578S and V637I) were located in the propeller domain. A578S, is the most frequent mutation observed in Africa, but has not previously been reported in Senegal. A previous study has suggested that A578S could disrupt the function of the Pfk13 propeller region.ConclusionAs the genetic basis of possible artemisinin resistance may be distinct in Africa and Southeast Asia, further studies are necessary to assess the new SNPs reported in this study.
| S-EPMC7106636 | biostudies-literature