Determining Genotypic Drug Resistance by Ion Semiconductor Sequencing With the Ion AmpliSeq™ TB Panel in Multidrug-Resistant Mycobacterium tuberculosis Isolates

Park, Joonhong; Shin, So Youn; Kim, Kyungjong; Park, Kuhn; Shin, Soyoung; Ihm, Chunhwa

doi:10.3343/alm.2018.38.4.316

Cited by 18 publications

(17 citation statements)

References 39 publications

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“…Our study indicates high percentage of agreement between both methods used for RIF resistance testing (93.4%) with high sensitivity (100%) and specificity (80%). This is concordant with published literature [5,10,13] which reported very good agreement between genotypic and phenotypic DST. Zaw et al [15] showed that mutations in rpoB gene (specifically mutations within 81-bp RIF-resistance determining region; RRDR) are responsible for approximately 95% of all RIF resistance cases in TB strains.…”

Section: Resultssupporting

confidence: 93%

“…Similarly, we also noticed high percent of agreement between phenotypic and genotypic DST results for EMB (93.3%) with high sensitivity (100%) and specificity (88.2%). These results are concordant with some of the published studies [9,10], which showed large overlap in the estimated prevalence of EMB resistance by genetic sequencing and the estimated prevalence by phenotypic testing. However, several other studies observed discrepancies between the presence of common mutations at codon 306 of embB gene and phenotypic EMB resistance [5,8].…”

Section: Resultssupporting

confidence: 91%

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Next-generation sequencing of drug resistant Mycobacterium tuberculosis clinical isolates in low-incidence countries

Содья

Toplak

Koren

et al. 2020

Russian Journal of Infection and Immunity

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Drug resistant tuberculosis (TB), especially multidrug (MDR) and extensively drug-resistant (XDR) TB, is still a serious problem in global TB control. Slovenia and North Macedonia are low-incidence countries with TB incidence rates of 5.4 and 10.4 in 2017, respectively. In both countries, the percentage of drug resistant TB is very low with sporadic cases of MDR-TB. However, global burden of drug-resistant TB continues to increase imposing huge impact on public health systems and strongly stimulating the detection of gene variants related with drug resistance in TB. Next-generation sequencing (NGS) can provide comprehensive analysis of gene variants linked to drug resistance in Mycobacterium tuberculosis. Therefore, the aim of our study was to examine the feasibility of a full-length gene analysis for the drug resistance related genes (inhA, katG, rpoB, embB) using Ion Torrent technology and to compare the NGS results with those obtained from conventional phenotypic drug susceptibility testing (DST) in TB isolates. Between 1996 and 2017, we retrospectively selected 56 TB strains from our National mycobacterial culture collection. Of those, 33 TB isolates from Slovenian patients were isolated from various clinical samples and subjected to phenotypic DST testing in Laboratory for Mycobacteria (University Clinic Golnik, Slovenia). The remaining 23 TB isolates were isolated from Macedonian patients and sent to our laboratory for assistance in phenotypic DST testing. TB strains included were either mono-, poly- or multidrug resistant. For control purposes, we also randomly selected five TB strains susceptible to first-line anti-TB drugs. High concordance between genetic (Ion Torrent technology) and standard phenotypic DST testing for isoniazid, rifampicin and ethambutol was observed, with percent of agreement of 77%, 93.4% and 93.3%, sensitivities of 68.2%, 100% and 100%, and specificities of 100%, 80% and 88.2%, respectively. In conclusion, the genotypic DST using Ion Torrent semiconductor NGS successfully predicted drug resistance with significant shortening of time needed to obtain the resistance profiles from several weeks to just a few days.

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Section: Resultssupporting

confidence: 93%

Section: Resultssupporting

confidence: 91%

Next-generation sequencing of drug resistant Mycobacterium tuberculosis clinical isolates in low-incidence countries

Содья

Toplak

Koren

et al. 2020

Russian Journal of Infection and Immunity

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“…In contrast, targeted NGS (tNGS) can be performed on direct clinical samples and has emerged as a feasible option for comprehensive, fast, and clinically relevant sequencing from patient sputum samples ( 11 – 15 ). However, the complexity, cost, and laboratory requirements of many sequencing platforms have limited the broad uptake of culture-free tNGS for DST in the routine, low-resource settings that carry the highest diagnostic burden.…”

Section: Introductionmentioning

confidence: 99%

Application of Targeted Next-Generation Sequencing Assay on a Portable Sequencing Platform for Culture-Free Detection of Drug-Resistant Tuberculosis from Clinical Samples

et al. 2020

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Targeted Next Generation Sequencing (tNGS) has emerged as a comprehensive alternative to existing methods for drug susceptibility testing (DST) of Mycobacterium tuberculosis from patient sputum samples for clinical diagnosis of drug resistant TB (DR-TB). However, the complexity of sequencing platforms has limited their uptake in low-resource settings. The goal of this study was to evaluate the use of tNGS-based DST solution: Genoscreen Deeplex Myc-TB, for use on the compact, low-cost Oxford Nanopore Technologies MinION sequencer. One hundred four DNA samples extracted from smear-positive sputum sediments, previously sequenced using the Deeplex assay on an Illumina MiniSeq, were re-sequenced on MinION after applying a custom library preparation. MinION read quality, mapping statistics and variant calling were computed using an in-house pipeline and compared to the reference Miniseq data. The average percentage of MinION reads mapped to an H37RV reference genome was 90.8%, versus 99.5% on MiniSeq. The mean depth of coverage was 4151x and 4177x on MinION and MiniSeq, respectively, with heterogeneous distribution across targeted genes. Composite reference coverage breadth was >99% for both platforms. We observed full concordance between technologies in reporting the clinically relevant drug resistant markers, including full gene deletions. We demonstrated that the workflow and sequencing data obtained from Deeplex on MinION are comparable to the MiniSeq, despite the higher raw error rates on MinION, with the added advantage of MinION's portability, versatility and low capital costs. Targeted NGS on MinION is a promising DST solution for rapidly providing clinically relevant data to manage complex DR-TB cases.

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“…Simultaneous PCR-based detection of multiple pathogen species or genotypes is certainly common 28 , but multiplexable primer panels are rarely designed for subsequent sequencing and polymorphism analysis. The Ion AmpliSeq brand currently offers pre-designed panels for studies on ebola 29 and tuberculosis 30 but the use of custom panels for other pathogen species (e.g., Bifidobacterium 31 or human papilloma virus 32 ) remains surprisingly rare in the literature.…”

Section: Introductionmentioning

confidence: 99%

Genome-wide locus sequence typing (GLST) of eukaryotic pathogens

Schwabl¹,

et al. 2020

Preprint

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20Analysis of genetic polymorphism is a powerful tool for epidemiological surveillance and research. Powerful 21 inference from pathogen genetic variation, however, is often restrained by limited access to representative 22 target DNA, especially in the study of obligate parasitic species for which ex vivo culture is resource-intensive 23 or bias-prone. Modern sequence capture methods enable pathogen genetic variation to be analyzed directly 24 from vector/host material but are often too complex and expensive for resource-poor settings where infectious 25 diseases prevail. This study proposes a simple, cost-effective 'genome-wide locus sequence typing' (GLST) 26 tool based on massive parallel amplification of information hotspots throughout the target pathogen genome. 27 The multiplexed polymerase chain reaction amplifies hundreds of different, user-defined genetic targets in a 28 single reaction tube, and subsequent agarose gel-based clean-up and barcoding completes library preparation 29 at under 4 USD per sample. Approximately 100 libraries can be sequenced together in one Illumina MiSeq 30 run. Our study generates a flexible GLST primer panel design workflow for Trypanosoma cruzi, the parasitic 31 agent of Chagas disease. We successfully apply our 203-target GLST panel to direct, culture-free 32 metagenomic extracts from triatomine vectors containing a minimum of 3.69 pg/µl T. cruzi DNA and further 33 elaborate on method performance by sequencing GLST libraries from T. cruzi reference clones representing 34 discrete typing units (DTUs) TcI, TcIII, TcIV, and TcVI. The 780 SNP sites we identify in the sample set 35 repeatably distinguish parasites infecting sympatric vectors and detect correlations between genetic and 36 geographic distances at regional (< 150 km) as well as continental scales. The markers also clearly separate 37 DTUs. We discuss the advantages, limitations and prospects of our method across a spectrum of 38 epidemiological research. 40Genome-wide single nucleotide polymorphism (SNP) analysis is a powerful and increasingly common 41 approach in the study and surveillance of infectious disease. Understanding patterns of SNP diversity within 42 pathogen genomes and across pathogen populations can resolve fundamental biological questions (e.g., 43 reproductive mechanisms in T. cruzi 1 , reconstruct past 2 and present transmission networks (e.g., 44 Staphylococcus infections within hospitals) 3 or identify the genetic bases of virulence 4,5 and resistance to drugs 45 (see examples from Plasmodium spp. 6,7 ). A number of obstacles, however, complicate access to 46 representative, genome-wide SNP information using modern sequencing tools. Micro-pathogens are often 47 sampled in low quantities and together with large amounts of host/vector tissue, microbiota, or environmental 48 DNA. Sequencing is rarely viable directly from the infection source and studies have often found it necessary 49 to isolate and culture the target organism to higher densities before extracting D...

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Determining Genotypic Drug Resistance by Ion Semiconductor Sequencing With the Ion AmpliSeq™ TB Panel in Multidrug-Resistant Mycobacterium tuberculosis Isolates

Cited by 18 publications

References 39 publications

Next-generation sequencing of drug resistant <i>Mycobacterium tuberculosis</i> clinical isolates in low-incidence countries

Next-generation sequencing of drug resistant <i>Mycobacterium tuberculosis</i> clinical isolates in low-incidence countries

Application of Targeted Next-Generation Sequencing Assay on a Portable Sequencing Platform for Culture-Free Detection of Drug-Resistant Tuberculosis from Clinical Samples

Genome-wide locus sequence typing (GLST) of eukaryotic pathogens

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