scholarly journals Antimalarial Drug Resistance Profiling of Plasmodium falciparum Infections in Ghana Using Molecular Inversion Probes and Next-Generation Sequencing

2020 ◽  
Vol 64 (4) ◽  
Author(s):  
Benedicta A. Mensah ◽  
Ozkan Aydemir ◽  
James L. Myers-Hansen ◽  
Millicent Opoku ◽  
Nicholas J. Hathaway ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Plasmodium Falciparum ◽  
Next Generation Sequencing ◽  
Antimalarial Drug ◽  
Next Generation ◽  
Cape Coast ◽  
Antimalarial Drug Resistance ◽  
Content Type ◽  
Molecular Inversion Probes ◽  
Generation Sequencing

ABSTRACT A key drawback to monitoring the emergence and spread of antimalarial drug resistance in sub-Saharan Africa is early detection and containment. Next-generation sequencing methods offer the resolution, sensitivity, and scale required to fill this gap by surveilling for molecular markers of drug resistance. We performed targeted sequencing using molecular inversion probes to interrogate five Plasmodium falciparum genes (pfcrt, pfmdr1, pfdhps, pfdhfr, and pfk13) implicated in chloroquine, sulfadoxine-pyrimethamine (SP), and artemisinin resistance in two sites in Ghana. A total of 803 dried blood spots from children aged between 6 months and 14 years presenting with uncomplicated P. falciparum malaria at the Begoro District Hospital in Begoro and the Ewim Polyclinic in Cape Coast, Ghana, from 2014 to 2017 were prepared on filter paper. Thirteen years after the removal of drug pressure, chloroquine-sensitive parasite strains with pfcrt K76 have increased nearly to fixation in Begoro, in the forest area (prevalence = 95%), but at a lower rate in Cape Coast, in the coastal region (prevalence = 71%, Z = −3.5, P < 0.001). In addition, pfmdr1 184F-bearing parasites are under strong selection. The pfdhfr/pfdhps quadruple genotype (IRNGK), associated with SP resistance, is near saturation. Our study identified at a 2 to 10% prevalence pfdhps 581G, which is a sulfadoxine resistance marker that correlates with the failure of SP prophylaxis in pregnancy and which has not been observed in Ghana. The differences in the reexpansion of chloroquine-sensitive strains observed at the two study sites, the stronger SP resistance, and the high prevalence of pfmdr1 184F should be further monitored to inform malaria control strategies in Ghana.

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

2019 ◽  
Vol 63 (10) ◽  
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.

scholarly journals Role ofPfmdr1inIn VitroPlasmodium falciparum Susceptibility to Chloroquine, Quinine, Monodesethylamodiaquine, Mefloquine, Lumefantrine, and Dihydroartemisinin

2014 ◽  
Vol 58 (12) ◽  
pp. 7032-7040 ◽  
Author(s):  
Nathalie Wurtz ◽  
Bécaye Fall ◽  
Aurélie Pascual ◽  
Mansour Fall ◽  
Eric Baret ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Plasmodium Falciparum ◽  
Multidrug Resistance ◽  
Antimalarial Drug ◽  
Antimalarial Drugs ◽  
Wild Type ◽  
Antimalarial Drug Resistance ◽  
Content Type ◽  
Increased Susceptibility

ABSTRACTThe involvement ofPfmdr1(Plasmodium falciparummultidrug resistance 1) polymorphisms in antimalarial drug resistance is still debated. Here, we evaluate the association between polymorphisms inPfmdr1(N86Y, Y184F, S1034C, N1042D, and D1246Y) andPfcrt(K76T) andin vitroresponses to chloroquine (CQ), mefloquine (MQ), lumefantrine (LMF), quinine (QN), monodesethylamodiaquine (MDAQ), and dihydroartemisinin (DHA) in 174Plasmodium falciparumisolates from Dakar, Senegal. ThePfmdr186Y mutation was identified in 14.9% of the samples, and the 184F mutation was identified in 71.8% of the isolates. No 1034C, 1042N, or 1246Y mutations were detected. ThePfmdr186Y mutation was significantly associated with increased susceptibility to MDAQ (P= 0.0023), LMF (P= 0.0001), DHA (P= 0.0387), and MQ (P= 0.00002). The N86Y mutation was not associated with CQ (P= 0.214) or QN (P= 0.287) responses. ThePfmdr1184F mutation was not associated with various susceptibility responses to the 6 antimalarial drugs (P= 0.168 for CQ, 0.778 for MDAQ, 0.324 for LMF, 0.961 for DHA, 0.084 for QN, and 0.298 for MQ). ThePfmdr186Y-Y184 haplotype was significantly associated with increased susceptibility to MDAQ (P= 0.0136), LMF (P= 0.0019), and MQ (P= 0.0001). The additionalPfmdr186Y mutation increased significantly thein vitrosusceptibility to MDAQ (P< 0.0001), LMF (P< 0.0001), MQ (P< 0.0001), and QN (P= 0.0026) in wild-typePfcrtK76 parasites. The additionalPfmdr186Y mutation significantly increased thein vitrosusceptibility to CQ (P= 0.0179) inPfcrt76T CQ-resistant parasites.

scholarly journals In Vitroand Molecular Surveillance for Antimalarial Drug Resistance in Plasmodium falciparum Parasites in Western Kenya Reveals Sustained Artemisinin Sensitivity and Increased Chloroquine Sensitivity

2015 ◽  
Vol 59 (12) ◽  
pp. 7540-7547 ◽  
Author(s):  
Naomi W. Lucchi ◽  
Franklin Komino ◽  
Sheila Akinyi Okoth ◽  
Ira Goldman ◽  
Philip Onyona ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Plasmodium Falciparum ◽  
Antimalarial Drug ◽  
Mutant Allele ◽  
Surveillance Program ◽  
Wild Type ◽  
Antimalarial Drug Resistance ◽  
Western Kenya ◽  
Content Type

ABSTRACTMalaria control is hindered by the evolution and spread of resistance to antimalarials, necessitating multiple changes to drug policies over time. A comprehensive antimalarial drug resistance surveillance program is vital for detecting the potential emergence of resistance to antimalarials, including current artemisinin-based combination therapies. An antimalarial drug resistance surveillance study involving 203Plasmodium falciparummalaria-positive children was conducted in western Kenya between 2010 and 2013. Specimens from enrolled children were analyzedin vitrofor sensitivity to chloroquine (CQ), amodiaquine (AQ), mefloquine (MQ), lumefantrine, and artemisinin derivatives (artesunate and dihydroartemisinin) and for drug resistance allele polymorphisms inP. falciparum crt(Pfcrt),Pfmdr-1, and the K13 propeller domain (K13). We observed a significant increase in the proportion of samples with thePfcrtwild-type (CVMNK) genotype, from 61.2% in 2010 to 93.0% in 2013 (P< 0.0001), and higher proportions of parasites with elevated sensitivity to CQin vitro. The majority of isolates harbored the wild-type N allele inPfmdr-1codon 86 (93.5%), with only 7 (3.50%) samples with the N86Ymutant allele (the mutant nucleotide is underlined). Likewise, most isolates harbored the wild-typePfmdr-1D1246 allele (79.8%), with only 12 (6.38%) specimens with the D1246Ymutant allele and 26 (13.8%) with mixed alleles. All the samples had a single copy of thePfmdr-1gene (mean of 0.907 ± 0.141 copies). None of the sequenced parasites had mutations in K13. Our results suggest that artemisinin is likely to remain highly efficacious and that CQ sensitivity appears to be on the rise in western Kenya.

scholarly journals Next-Generation Sequencing for Characterizing Drug Resistance-ConferringMycobacterium tuberculosisGenes from Clinical Isolates in the Ukraine

2018 ◽  
Vol 56 (6) ◽  
Author(s):  
L. T. Daum ◽  
O. S. Konstantynovska ◽  
O. S. Solodiankin ◽  
O. O. Liashenko ◽  
P. I. Poteiko ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Next Generation Sequencing ◽  
Gene Promoter ◽  
Genomic Analysis ◽  
Clinical Isolates ◽  
Next Generation ◽  
Crimean Peninsula ◽  
Content Type ◽  
The World ◽  
Generation Sequencing

ABSTRACTThe Ukraine ranks among the top 20 countries with the highest number of multidrug-resistant (MDR) and extensively drug resistant (XDR)Mycobacterium tuberculosiscases in the world. However, little is known of the genetic diversity, i.e., resistance signatures, in clinical isolates from this region. We analyzed seven of most prevalent MDR/XDR antibiotic resistance-conferring genes from clinical isolates (n= 75) collected from geographically diverse Ukrainian oblasts and the southern Crimean peninsula. Genomic analysis revealed that 6 (8%) were sensitive, 3 (4%) were resistant to at least one antibiotic but were not MDR, 40 (53%) were MDR, and 26 (35%) were XDR. The majority of isolates (81%) were of the Beijing-like lineage. This is the first study to use next-generation sequencing (NGS) of clinical isolates from the Ukraine to characterize mutations in genes conferringM. tuberculosisdrug resistance. Several isolates harbored drug resistance signatures that have not been observed in other countries with high-burden tuberculosis. Most notably, the absence ofinhAgene promoter mutations, a diversity of mutation types in therpoBresistance-determining region, and detection of heteroresistance provide a broader understanding of MDR/XDR from this area of the world.

scholarly journals Mosquitoes as a feasible sentinel group for anti-malarial resistance surveillance by Next Generation Sequencing of Plasmodium falciparum

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Rebecca Smith-Aguasca ◽  
Himanshu Gupta ◽  
Estefania Uberegui ◽  
Mara Maquina ◽  
Francisco Saute ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Plasmodium Falciparum ◽  
Next Generation Sequencing ◽  
Sanger Sequencing ◽  
Anopheles Funestus ◽  
Allele Frequencies ◽  
Mixed Infections ◽  
Next Generation ◽  
Suitable Alternative ◽  
Generation Sequencing

Abstract Background Plasmodium falciparum drug resistance surveillance is key to successful disease control and eradication. Contemporary methods that only allow determination of prevalence of resistance are expensive, time consuming and require ethical considerations. A newer method involving Next Generation Sequencing (NGS) permits obtaining frequency of resistance while allowing to detect minority variants in mixed infections. Here, NGS was tested for P. falciparum resistance marker detection in mosquito samples as a feasible and suitable alternative for molecular resistance surveillance. Anopheles funestus were collected in southern Mozambique using CDC light traps and manual collections. DNA was extracted from either whole mosquito, head-thorax and abdomen separately or pools of five mosquitoes. These samples were screened for P. falciparum and if positive for k13, pfcrt, pfmdr1, pfdhps and pfdhfr mutations related to anti-malarial drug resistance with Sanger sequencing and NGS. Results Among the 846 samples screened for P. falciparum, 122 were positive by 18S ssrDNA qPCR with an infection rate of 23.6%. No mutations were observed for k13 and pfcrt72-76 and almost zero for pfmdr86, but quintuple pfdhfr/pfdhps mutations were near fixation and about half of the isolates contained the pfmdr184F polymorphism. Similar allele frequencies of resistance markers were estimated with NGS in comparison with the prevalence of markers obtained with the gold standard Sanger sequencing. Conclusions Pooled deep sequencing of P. falciparum isolates extracted from mosquitoes is a promising, efficient and cost-effective method to quantify allele frequencies at population level which allows to detect known and unknown markers of resistance in single and mixed infections in a timelier manner. Using mosquitoes as sentinel group and focusing on allele frequency opposed to prevalence, permits active surveillance across a more homogeneous geographical range.

scholarly journals Association between Polymorphisms in the Pfmdr6 Gene and Ex Vivo Susceptibility to Quinine in Plasmodium falciparum Parasites from Dakar, Senegal

2017 ◽  
Vol 61 (3) ◽  
Author(s):  
Mathieu Gendrot ◽  
Silman Diawara ◽  
Marylin Madamet ◽  
Mame Bou Kounta ◽  
Sébastien Briolant ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Plasmodium Falciparum ◽  
Multidrug Resistance ◽  
Antimalarial Drug ◽  
Ex Vivo ◽  
Atp Binding ◽  
Antimalarial Drug Resistance ◽  
Content Type ◽  
Polymorphic Microsatellite ◽  
Atp Binding Cassette

ABSTRACT Polymorphisms and the overexpression of transporter genes, especially of the ATP-binding cassette superfamily, have been involved in antimalarial drug resistance. The objective of this study was to use 77 Senegalese Plasmodium falciparum isolates to evaluate the association between the number of Asn residues in the polymorphic microsatellite region of the Plasmodium falciparum multidrug resistance 6 gene (Pfmdr6) and the ex vivo susceptibility to antimalarials. A significant association was observed between the presence of 7 or 9 Asn repeats and reduced susceptibility to quinine.

scholarly journals HIV Drug Resistance Mutations Detection by Next-Generation Sequencing during Antiretroviral Therapy Interruption in China

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 264
Author(s):  
Miaomiao Li ◽  
Shujia Liang ◽  
Chao Zhou ◽  
Min Chen ◽  
Shu Liang ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Next Generation Sequencing ◽  
Antiretroviral Therapy ◽  
Detection Threshold ◽  
Next Generation ◽  
Resistance Mutations ◽  
Hiv Drug Resistance ◽  
Drug Resistance Mutations ◽  
Therapy Interruption ◽  
Generation Sequencing

Patients with antiretroviral therapy interruption have a high risk of virological failure when re-initiating antiretroviral therapy (ART), especially those with HIV drug resistance. Next-generation sequencing may provide close scrutiny on their minority drug resistance variant. A cross-sectional study was conducted in patients with ART interruption in five regions in China in 2016. Through Sanger and next-generation sequencing in parallel, HIV drug resistance was genotyped on their plasma samples. Rates of HIV drug resistance were compared by the McNemar tests. In total, 174 patients were included in this study, with a median 12 (interquartile range (IQR), 6–24) months of ART interruption. Most (86.2%) of them had received efavirenz (EFV)/nevirapine (NVP)-based first-line therapy for a median 16 (IQR, 7–26) months before ART interruption. Sixty-one (35.1%) patients had CRF07_BC HIV-1 strains, 58 (33.3%) CRF08_BC and 35 (20.1%) CRF01_AE. Thirty-four (19.5%) of the 174 patients were detected to harbor HIV drug-resistant variants on Sanger sequencing. Thirty-six (20.7%), 37 (21.3%), 42 (24.1%), 79 (45.4%) and 139 (79.9) patients were identified to have HIV drug resistance by next-generation sequencing at 20% (v.s. Sanger, p = 0.317), 10% (v.s. Sanger, p = 0.180), 5% (v.s. Sanger, p = 0.011), 2% (v.s. Sanger, p < 0.001) and 1% (v.s. Sanger, p < 0.001) of detection thresholds, respectively. K65R was the most common minority mutation, of 95.1% (58/61) and 93.1% (54/58) in CRF07_BC and CRF08_BC, respectively, when compared with 5.7% (2/35) in CRF01_AE (p < 0.001). In 49 patients that followed-up a median 10 months later, HIV drug resistance mutations at >20% frequency such as K103N, M184VI and P225H still existed, but with decreased frequencies. The prevalence of HIV drug resistance in ART interruption was higher than 15% in the survey. Next-generation sequencing was able to detect more minority drug resistance variants than Sanger. There was a sharp increase in minority drug resistance variants when the detection threshold was below 5%.

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