scholarly journals A Method for Amplicon Deep Sequencing of Drug Resistance Genes in Plasmodium falciparum Clinical Isolates from India

2016 ◽  
Vol 54 (6) ◽  
pp. 1500-1511 ◽  
Author(s):  
Pavitra N. Rao ◽  
Swapna Uplekar ◽  
Sriti Kayal ◽  
Prashant K. Mallick ◽  
Nabamita Bandyopadhyay ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Plasmodium Falciparum ◽  
Artemisinin Combination Therapy ◽  
Amplicon Sequencing ◽  
Clinical Isolates ◽  
Clinical Samples ◽  
Ion Torrent ◽  
Content Type ◽  
Ion Torrent Pgm ◽  
Reference Strains

A major challenge to global malaria control and elimination is early detection and containment of emerging drug resistance. Next-generation sequencing (NGS) methods provide the resolution, scalability, and sensitivity required for high-throughput surveillance of molecular markers of drug resistance. We have developed an amplicon sequencing method on the Ion Torrent PGM platform for targeted resequencing of a panel of sixPlasmodium falciparumgenes implicated in resistance to first-line antimalarial therapy, including artemisinin combination therapy, chloroquine, and sulfadoxine-pyrimethamine. The protocol was optimized using 12 geographically diverseP. falciparumreference strains and successfully applied to multiplexed sequencing of 16 clinical isolates from India. The sequencing results from the reference strains showed 100% concordance with previously reported drug resistance-associated mutations. Single-nucleotide polymorphisms (SNPs) in clinical isolates revealed a number of known resistance-associated mutations and other nonsynonymous mutations that have not been implicated in drug resistance. SNP positions containing multiple allelic variants were used to identify three clinical samples containing mixed genotypes indicative of multiclonal infections. The amplicon sequencing protocol has been designed for the benchtop Ion Torrent PGM platform and can be operated with minimal bioinformatics infrastructure, making it ideal for use in countries that are endemic for the disease to facilitate routine large-scale surveillance of the emergence of drug resistance and to ensure continued success of the malaria treatment policy.

scholarly journals Decreasingpfmdr1Copy Number Suggests that Plasmodium falciparum in Western Cambodia Is RegainingIn VitroSusceptibility to Mefloquine

2015 ◽  
Vol 59 (5) ◽  
pp. 2934-2937 ◽  
Author(s):  
Pharath Lim ◽  
Dalin Dek ◽  
Vorleak Try ◽  
Sokunthea Sreng ◽  
Seila Suon ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Combination Therapy ◽  
Molecular Marker ◽  
Copy Number ◽  
Artemisinin Combination Therapy ◽  
Clinical Isolates ◽  
Content Type

ABSTRACTDihydroartemisinin-piperaquine is the current frontline artemisinin combination therapy (ACT) forPlasmodium falciparummalaria in Cambodia but is now failing in several western provinces. To investigate artesunate plus mefloquine (AS+MQ) as a replacement ACT, we measured the prevalence of multiplepfmdr1copies—a molecular marker for MQ resistance—in 844P. falciparumclinical isolates collected in 2008 to 2013. Thepfmdr1copy number is decreasing in Western Cambodia, suggesting thatP. falciparumis regainingin vitrosusceptibility to MQ.

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 The MSPDBL2 Codon 591 Polymorphism Is Associated with LumefantrineIn VitroDrug Responses in Plasmodium falciparum Isolates from Kilifi, Kenya

2014 ◽  
Vol 59 (3) ◽  
pp. 1770-1775 ◽  
Author(s):  
Lynette Isabella Ochola-Oyier ◽  
John Okombo ◽  
Leah Mwai ◽  
Steven M. Kiara ◽  
Lewa Pole ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Plasmodium Falciparum ◽  
Combination Therapy ◽  
High Frequency ◽  
Artemisinin Combination Therapy ◽  
Resistance Development ◽  
Content Type

ABSTRACTThe mechanisms of drug resistance development in thePlasmodium falciparumparasite to lumefantrine (LUM), commonly used in combination with artemisinin, are still unclear. We assessed the polymorphisms ofPfmspdbl2for associations with LUM activity in a Kenyan population. MSPDBL2 codon 591S was associated with reduced susceptibility to LUM (P= 0.04). The high frequency ofPfmspdbl2codon 591S in Kenya may be driven by the widespread use of lumefantrine in artemisinin combination therapy (Coartem).

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 Rapid Drug Susceptibility Testing of Drug-Resistant Mycobacterium tuberculosis Isolates Directly from Clinical Samples by Use of Amplicon Sequencing: a Proof-of-Concept Study

2016 ◽  
Vol 54 (8) ◽  
pp. 2058-2067 ◽  
Author(s):  
Rebecca E. Colman ◽  
Julia Anderson ◽  
Darrin Lemmer ◽  
Erik Lehmkuhl ◽  
Sophia B. Georghiou ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Mycobacterium Tuberculosis ◽  
Susceptibility Testing ◽  
Amplicon Sequencing ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Health Concern ◽  
Clinical Samples ◽  
Drug Resistant ◽  
Content Type

Increasingly complex drug-resistant tuberculosis (DR-TB) is a major global health concern and one of the primary reasons why TB is now the leading infectious cause of death worldwide. Rapid characterization of a DR-TB patient's complete drug resistance profile would facilitate individualized treatment in place of empirical treatment, improve treatment outcomes, prevent amplification of resistance, and reduce the transmission of DR-TB. The use of targeted next-generation sequencing (NGS) to obtain drug resistance profiles directly from patient sputum samples has the potential to enable comprehensive evidence-based treatment plans to be implemented quickly, rather than in weeks to months, which is currently needed for phenotypic drug susceptibility testing (DST) results. In this pilot study, we evaluated the performance of amplicon sequencing ofMycobacterium tuberculosisDNA from patient sputum samples using a tabletop NGS technology and automated data analysis to provide a rapid DST solution (the Next Gen-RDST assay). One hundred sixty-six out of 176 (94.3%) sputum samples from the Republic of Moldova yielded complete Next Gen-RDST assay profiles for 7 drugs of interest. We found a high level of concordance of our Next Gen-RDST assay results with phenotypic DST (97.0%) and pyrosequencing (97.8%) results from the same clinical samples. Our Next Gen-RDST assay was also able to estimate the proportion of resistant-to-wild-type alleles down to mixtures of ≤1%, which demonstrates the ability to detect very low levels of resistant variants not detected by pyrosequencing and possibly below the threshold for phenotypic growth methods. The assay as described here could be used as a clinical or surveillance tool.

scholarly journals Environmental Isolates of Azole-Resistant Aspergillus fumigatus in Germany

2015 ◽  
Vol 59 (7) ◽  
pp. 4356-4359 ◽  
Author(s):  
Oliver Bader ◽  
Jana Tünnermann ◽  
Anna Dudakova ◽  
Marut Tangwattanachuleeporn ◽  
Michael Weig ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Aspergillus Fumigatus ◽  
Antifungal Drug ◽  
Clinical Samples ◽  
Environmental Isolates ◽  
Northern Germany ◽  
Content Type ◽  
Antifungal Drug Resistance ◽  
The World

ABSTRACTAzole antifungal drug resistance inAspergillus fumigatusis an emerging problem in several parts of the world. Here we investigated the distribution of such strains in soils from Germany. At a general positivity rate of 12%, most prevalently, we found strains with the TR34/L98H and TR46/Y121F/T289A alleles, dispersed along a corridor across northern Germany. Comparison of the distributions of resistance alleles and genotypes between environment and clinical samples suggests the presence of local clinical clusters.

scholarly journals Effect of Fluorescent Dyes onIn Vitro-Differentiated, Late-Stage Plasmodium falciparum Gametocytes

2014 ◽  
Vol 58 (12) ◽  
pp. 7398-7404 ◽  
Author(s):  
Tamirat Gebru ◽  
Benjamin Mordmüller ◽  
Jana Held
Keyword(s):  
Plasmodium Falciparum ◽  
Late Stage ◽  
Fluorescent Dyes ◽  
Clinical Symptoms ◽  
Laboratory Strain ◽  
Clinical Isolates ◽  
Synthetic Dyes ◽  
Content Type ◽  
Highly Active

ABSTRACTPlasmodium falciparumgametocytes are not associated with clinical symptoms, but they are responsible for transmitting the pathogen to mosquitoes. Therefore, gametocytocidal interventions are important for malaria control and resistance containment. Currently available drugs and vaccines are not well suited for that purpose. Several dyes have potent antimicrobial activity, but their use against gametocytes has not been investigated systematically. The gametocytocidal activity of nine synthetic dyes and four control compounds was tested against stage V gametocytes of the laboratory strain 3D7 and three clinical isolates ofP. falciparumwith a bioluminescence assay. Five of the fluorescent dyes had submicromolar 50% inhibitory concentration (IC50) values against mature gametocytes. Three mitochondrial dyes, MitoRed, dihexyloxacarbocyanine iodide (DiOC6), and rhodamine B, were highly active (IC50s < 200 nM). MitoRed showed the highest activity against gametocytes, with IC50s of 70 nM against 3D7 and 120 to 210 nM against clinical isolates. All compounds were more active against the laboratory strain 3D7 than against clinical isolates. In particular, the endoperoxides artesunate and dihydroartemisinin showed a 10-fold higher activity against 3D7 than against clinical isolates. In contrast to all clinically used antimalarials, several fluorescent dyes had surprisingly highin vitroactivity against late-stage gametocytes. Since they also act against asexual blood stages, they shall be considered starting points for the development of new antimalarial lead compounds.

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

2018 ◽  
Vol 99 (6) ◽  
pp. 1499-1503 ◽  
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

Simultaneous determination of Mycobacterium leprae drug resistance and single nucleotide polymorphism genotype using nested multiplex PCR with amplicon sequencing

2021 ◽  
Author(s):  
Yasuhisa Iwao ◽  
Shuichi Mori ◽  
Manabu Ato ◽  
Noboru Nakata
Keyword(s):  
Drug Resistance ◽  
Single Nucleotide Polymorphism ◽  
Multiplex Pcr ◽  
Simultaneous Determination ◽  
Mycobacterium Leprae ◽  
Amplicon Sequencing ◽  
Clinical Samples ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide

Mycobacterium leprae is the predominant cause of leprosy worldwide, and its genotypes can be classified into four single nucleotide polymorphism (SNP) types and 16 subtypes. Determining M. leprae drug resistance and genotype is typically done by PCR and Sanger DNA sequencing, which require substantial effort. Here we describe a rapid method involving multiplex PCR in combination with nested amplification and next generation sequence analysis that allows simultaneous determination of M. leprae drug resistance and SNP genotype directly from clinical specimens. We used this method to analyze clinical samples from two paucibacillary, nine multibacillary, and six type-undetermined leprosy patients. Regions in folP1 , rpoB , gyrA , and gyrB that determine drug resistance and those for 84 SNP-InDels in the M. leprae genome were amplified from clinical samples and their sequences were determined. The results showed that seven samples were subtype 1A, three were 1D, and seven were 3K. Three samples of the subtype 3K had folp1 mutation. The method may allow more rapid genetic analyses of M. leprae in clinical samples.

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