scholarly journals Rapid Whole-Genome Sequencing of Mycobacterium tuberculosis Isolates Directly from Clinical Samples

2015 ◽  
Vol 53 (7) ◽  
pp. 2230-2237 ◽  
Author(s):  
Amanda C. Brown ◽  
Josephine M. Bryant ◽  
Katja Einer-Jensen ◽  
Jolyon Holdstock ◽  
Darren T. Houniet ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Positive Culture ◽  
Clinical Samples ◽  
Whole Genome ◽  
Resistance Mutations ◽  
High Quality ◽  
Content Type ◽  
Negative Case

The rapid identification of antimicrobial resistance is essential for effective treatment of highly resistantMycobacterium tuberculosis. Whole-genome sequencing provides comprehensive data on resistance mutations and strain typing for monitoring transmission, but unlike for conventional molecular tests, this has previously been achievable only from cultures ofM. tuberculosis. Here we describe a method utilizing biotinylated RNA baits designed specifically forM. tuberculosisDNA to capture fullM. tuberculosisgenomes directly from infected sputum samples, allowing whole-genome sequencing without the requirement of culture. This was carried out on 24 smear-positive sputum samples, collected from the United Kingdom and Lithuania where a matched culture sample was available, and 2 samples that had failed to grow in culture.M. tuberculosissequencing data were obtained directly from all 24 smear-positive culture-positive sputa, of which 20 were of high quality (>20× depth and >90% of the genome covered). Results were compared with those of conventional molecular and culture-based methods, and high levels of concordance between phenotypical resistance and predicted resistance based on genotype were observed. High-quality sequence data were obtained from one smear-positive culture-negative case. This study demonstrated for the first time the successful and accurate sequencing ofM. tuberculosisgenomes directly from uncultured sputa. Identification of known resistance mutations within a week of sample receipt offers the prospect for personalized rather than empirical treatment of drug-resistant tuberculosis, including the use of antimicrobial-sparing regimens, leading to improved outcomes.

scholarly journals Genome Sequencing of Polydrug-, Multidrug-, and Extensively Drug-Resistant Mycobacterium tuberculosis Strains from South India

2019 ◽  
Vol 8 (12) ◽  
Author(s):  
Sivakumar Shanmugam ◽  
Narender Kumar ◽  
Dina Nair ◽  
Mohan Natrajan ◽  
Srikanth Prasad Tripathy ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
South India ◽  
Sequence Data ◽  
Whole Genome ◽  
Drug Resistant ◽  
Resistance Mutations ◽  
Content Type ◽  
Extensively Drug Resistant

The genomes of 16 clinical Mycobacterium tuberculosis isolates were subjected to whole-genome sequencing to identify mutations related to resistance to one or more anti-Mycobacterium drugs. The sequence data will help in understanding the genomic characteristics of M. tuberculosis isolates and their resistance mutations prevalent in South India.

scholarly journals Comprehensive Whole-Genome Sequencing and Reporting of Drug Resistance Profiles on Clinical Cases of Mycobacterium tuberculosis in New York State

2017 ◽  
Vol 55 (6) ◽  
pp. 1871-1882 ◽  
Author(s):  
Joseph Shea ◽  
Tanya A. Halse ◽  
Pascal Lapierre ◽  
Matthew Shudt ◽  
Donna Kohlerschmidt ◽  
...  
Keyword(s):  
New York ◽  
Drug Resistance ◽  
Mycobacterium Tuberculosis ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Predictive Value ◽  
New York State ◽  
Whole Genome ◽  
Content Type ◽  
York State

ABSTRACTWhole-genome sequencing (WGS) is a newer alternative for tuberculosis (TB) diagnostics and is capable of providing rapid drug resistance profiles while performing species identification and capturing the data necessary for genotyping. Our laboratory developed and validated a comprehensive and sensitive WGS assay to characterizeMycobacterium tuberculosisand otherM. tuberculosiscomplex (MTBC) strains, composed of a novel DNA extraction, optimized library preparation, paired-end WGS, and an in-house-developed bioinformatics pipeline. This new assay was assessed using 608 MTBC isolates, with 146 isolates during the validation portion of this study and 462 samples received prospectively. In February 2016, this assay was implemented to test all clinical cases of MTBC in New York State, including isolates and early positive Bactec mycobacterial growth indicator tube (MGIT) 960 cultures from primary specimens. Since the inception of the assay, we have assessed the accuracy of identification of MTBC strains to the species level, concordance with culture-based drug susceptibility testing (DST), and turnaround time. Species identification by WGS was determined to be 99% accurate. Concordance between drug resistance profiles generated by WGS and culture-based DST methods was 96% for eight drugs, with an average resistance-predictive value of 93% and susceptible-predictive value of 96%. This single comprehensive WGS assay has replaced seven molecular assays and has resulted in resistance profiles being reported to physicians an average of 9 days sooner than with culture-based DST for first-line drugs and 32 days sooner for second-line drugs.

scholarly journals Whole-Genome Sequencing for Drug Resistance Profile Prediction inMycobacterium tuberculosis

2019 ◽  
Vol 63 (4) ◽  
Author(s):  
Sebastian M. Gygli ◽  
Peter M. Keller ◽  
Marie Ballif ◽  
Nicolas Blöchliger ◽  
Rico Hömke ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Critical Concentration ◽  
Drug Susceptibility Testing ◽  
Quality Data ◽  
Whole Genome ◽  
Resistance Mutations ◽  
Content Type ◽  
Agar Dilution

ABSTRACTWhole-genome sequencing allows rapid detection of drug-resistantMycobacterium tuberculosisisolates. However, the availability of high-quality data linking quantitative phenotypic drug susceptibility testing (DST) and genomic data have thus far been limited. We determined drug resistance profiles of 176 genetically diverse clinicalM. tuberculosisisolates from the Democratic Republic of the Congo, Ivory Coast, Peru, Thailand, and Switzerland by quantitative phenotypic DST for 11 antituberculous drugs using the BD Bactec MGIT 960 system and 7H10 agar dilution to generate a cross-validated phenotypic DST readout. We compared DST results with predicted drug resistance profiles inferred by whole-genome sequencing. Classification of strains by the two phenotypic DST methods into resistotype/wild-type populations was concordant in 73 to 99% of cases, depending on the drug. Our data suggest that the established critical concentration (5 mg/liter) for ethambutol resistance (MGIT 960 system) is too high and misclassifies strains as susceptible, unlike 7H10 agar dilution. Increased minimal inhibitory concentrations were explained by mutations identified by whole-genome sequencing. Using whole-genome sequences, we were able to predict quantitative drug resistance levels for the majority of drug resistance mutations. Predicting quantitative levels of drug resistance by whole-genome sequencing was partially limited due to incompletely understood drug resistance mechanisms. The overall sensitivity and specificity of whole-genome-based DST were 86.8% and 94.5%, respectively. Despite some limitations, whole-genome sequencing has the potential to infer resistance profiles without the need for time-consuming phenotypic methods.

scholarly journals Mycobacterial DNA Extraction for Whole-Genome Sequencing from Early Positive Liquid (MGIT) Cultures

2015 ◽  
Vol 53 (4) ◽  
pp. 1137-1143 ◽  
Author(s):  
Antonina A. Votintseva ◽  
Louise J. Pankhurst ◽  
Luke W. Anson ◽  
Marcus R. Morgan ◽  
Deborah Gascoyne-Binzi ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Dna Extraction ◽  
Genome Sequencing ◽  
Solid Phase ◽  
Clinical Samples ◽  
Reference Laboratory ◽  
Whole Genome ◽  
Mycobacterial Species ◽  
Accurate Identification ◽  
Content Type

We developed a low-cost and reliable method of DNA extraction from as little as 1 ml of early positive mycobacterial growth indicator tube (MGIT) cultures that is suitable for whole-genome sequencing to identify mycobacterial species and predict antibiotic resistance in clinical samples. The DNA extraction method is based on ethanol precipitation supplemented by pretreatment steps with a MolYsis kit or saline wash for the removal of human DNA and a final DNA cleanup step with solid-phase reversible immobilization beads. The protocol yielded ≥0.2 ng/μl of DNA for 90% (MolYsis kit) and 83% (saline wash) of positive MGIT cultures. A total of 144 (94%) of the 154 samples sequenced on the MiSeq platform (Illumina) achieved the target of 1 million reads, with <5% of reads derived from human or nasopharyngeal flora for 88% and 91% of samples, respectively. A total of 59 (98%) of 60 samples that were identified by the national mycobacterial reference laboratory (NMRL) asMycobacterium tuberculosiswere successfully mapped to the H37Rv reference, with >90% coverage achieved. The DNA extraction protocol, therefore, will facilitate fast and accurate identification of mycobacterial species and resistance using a range of bioinformatics tools.

scholarly journals Whole-Genome Sequencing of Streptomycin-Resistant Mycobacterium tuberculosis Strain SBH145 from Sabah, Malaysia

2022 ◽  
Author(s):  
Zainal Arifin Mustapha ◽  
Jaeyres Jani ◽  
Cheronie Shely Stanis ◽  
Dg Syahidah Nadiah Abdull Majid ◽  
Chin Kai Ling ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Pulmonary Tuberculosis ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
The Philippines ◽  
Whole Genome ◽  
Content Type ◽  
Tuberculosis Strain ◽  
Mycobacterium Tuberculosis Strain ◽  
Malaysian Borneo

This paper reports on the whole-genome sequencing of a streptomycin-resistant Mycobacterium tuberculosis strain that was isolated from a patient with pulmonary tuberculosis in Sabah state of Malaysian Borneo. The strain belongs to the EAI2-Manila family of lineage 1 and is clustered with M. tuberculosis strains from the Philippines, India, and Taiwan.

scholarly journals Direct DNA Extraction from Mycobacterium tuberculosis Frozen Stocks as a Reculture-Independent Approach to Whole-Genome Sequencing

2015 ◽  
Vol 53 (8) ◽  
pp. 2716-2719 ◽  
Author(s):  
K. Bjorn-Mortensen ◽  
J. Zallet ◽  
T. Lillebaek ◽  
A. B. Andersen ◽  
S. Niemann ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Whole Genome Sequencing ◽  
Dna Extraction ◽  
Genome Sequencing ◽  
Sequence Data ◽  
Whole Genome ◽  
Content Type ◽  
Direct Dna Extraction ◽  
Slow Growing

Culturing before DNA extraction represents a major time-consuming step in whole-genome sequencing of slow-growing bacteria, such asMycobacterium tuberculosis. We report a workflow to extract DNA from frozen isolates without reculturing. Prepared libraries and sequence data were comparable with results from recultured aliquots of the same stocks.

scholarly journals Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples

mBio ◽  
2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Annie N. Cowell ◽  
Dorothy E. Loy ◽  
Sesh A. Sundararaman ◽  
Hugo Valdivia ◽  
Kathleen Fisch ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Whole Genome Sequencing ◽  
Plasmodium Vivax ◽  
Genome Sequencing ◽  
Whole Genome Amplification ◽  
Microbial Pathogens ◽  
Clinical Samples ◽  
Whole Genome ◽  
High Quality ◽  
Genome Amplification

ABSTRACT Whole-genome sequencing (WGS) of microbial pathogens from clinical samples is a highly sensitive tool used to gain a deeper understanding of the biology, epidemiology, and drug resistance mechanisms of many infections. However, WGS of organisms which exhibit low densities in their hosts is challenging due to high levels of host genomic DNA (gDNA), which leads to very low coverage of the microbial genome. WGS of Plasmodium vivax , the most widely distributed form of malaria, is especially difficult because of low parasite densities and the lack of an ex vivo culture system. Current techniques used to enrich P. vivax DNA from clinical samples require significant resources or are not consistently effective. Here, we demonstrate that selective whole-genome amplification (SWGA) can enrich P. vivax gDNA from unprocessed human blood samples and dried blood spots for high-quality WGS, allowing genetic characterization of isolates that would otherwise have been prohibitively expensive or impossible to sequence. We achieved an average genome coverage of 24×, with up to 95% of the P. vivax core genome covered by ≥5 reads. The single-nucleotide polymorphism (SNP) characteristics and drug resistance mutations seen were consistent with those of other P. vivax sequences from a similar region in Peru, demonstrating that SWGA produces high-quality sequences for downstream analysis. SWGA is a robust tool that will enable efficient, cost-effective WGS of P. vivax isolates from clinical samples that can be applied to other neglected microbial pathogens. IMPORTANCE Malaria is a disease caused by Plasmodium parasites that caused 214 million symptomatic cases and 438,000 deaths in 2015. Plasmodium vivax is the most widely distributed species, causing the majority of malaria infections outside sub-Saharan Africa. Whole-genome sequencing (WGS) of Plasmodium parasites from clinical samples has revealed important insights into the epidemiology and mechanisms of drug resistance of malaria. However, WGS of P. vivax is challenging due to low parasite levels in humans and the lack of a routine system to culture the parasites. Selective whole-genome amplification (SWGA) preferentially amplifies the genomes of pathogens from mixtures of target and host gDNA. Here, we demonstrate that SWGA is a simple, robust method that can be used to enrich P. vivax genomic DNA (gDNA) from unprocessed human blood samples and dried blood spots for cost-effective, high-quality WGS.

scholarly journals Genomic Signatures of Pre-Resistance in Mycobacterium tuberculosis

2021 ◽  
Author(s):  
Arturo Torres Ortiz ◽  
Jorge Coronel ◽  
Julia Rios Vidal ◽  
Cesar Bonilla ◽  
David Moore ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Mycobacterium Tuberculosis ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Phylogenetic Trees ◽  
P Value ◽  
Whole Genome ◽  
Resistance Mutations ◽  
Genomic Signatures ◽  
Future Drug

Abstract Recent advances in bacterial whole-genome sequencing have resulted in the identification of a comprehensive catalogue of genomic signatures of antibiotic resistance in Mycobacterium tuberculosis. With a view to pre-empting the emergence of drug-resistance, we hypothesized that pre-existing balanced polymorphisms in drug susceptible genotypes (“pre-resistance mutations”) could increase the risk of acquiring antimicrobial resistance in the future. In order to identify a pathogen genomic signature of future drug resistance we undertook whole-genome sequencing on 3135 culture positive isolates from different patients sampled over a 17-year period in Lima, Peru. Reconstructing ancestral whole genomes on time-calibrated phylogenetic trees we identified no single drug resistance in Peru predating 1940. Moving forward in evolutionary time through the phylogenetic tree from 1940, we apply a novel genome-wide survival analysis to determine the hazard of drug resistance acquisition at the level of lineage, mono-resistance state, and single-nucleotide polymorphism. We demonstrate that lineage 2 has a significantly higher incidence of drug resistance acquisition than lineage 4 (HR 3.36, 95% CI 2.10 - 5.38,p-value =4.25×10-7) and estimate that the hazard of evolving rifampicin following isoniazid resistance acquisition is 14 times that of genomes with a susceptible background (HR 14.45,95% CI 8.46 - 15.50, p-value<10−15). Our findings are validated in a separate publicly available dataset from Samara, Russia. After controlling for population structure, we also show that a deletion in a gene coding for the cell surface protein lppP predisposes to the acquisition of drug resistance in susceptible genotypes (HR 6.71, 95% CI4.82-11.22, p-value =1.17×10−9). Prediction of future drug resistance in susceptible pathogens together with targeted expanded therapy has the potential to prevent drug resistance emergence in Mycobacterium tuberculosis and other pathogens. Prospective cohort studies of participants with and with-out these polymorphisms should be undertaken with a view to implementing personalized pathogen genomic therapy. This approach could be employed to preempt and prevent the emergence of drug resistance and other important traits in other organisms.

scholarly journals Direct Whole-Genome Sequencing of Sputum Accurately Identifies Drug-Resistant Mycobacterium tuberculosis Faster than MGIT Culture Sequencing

2018 ◽  
Vol 56 (8) ◽  
Author(s):  
Ronan M. Doyle ◽  
Carrie Burgess ◽  
Rachel Williams ◽  
Rebecca Gorton ◽  
Helen Booth ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Antimicrobial Resistance ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Resistance Testing ◽  
Whole Genome ◽  
Accurate Identification ◽  
Content Type ◽  
Resistance Profile ◽  
Mgit Culture

ABSTRACT The current methods available to diagnose antimicrobial-resistant Mycobacterium tuberculosis infections require a positive culture or only test a limited number of resistance-associated mutations. A rapid accurate identification of antimicrobial resistance enables the prompt initiation of effective treatment. Here, we determine the utility of whole-genome sequencing (WGS) of M. tuberculosis directly from routinely obtained diagnostic sputum samples to provide a comprehensive resistance profile compared to that from mycobacterial growth indicator tube (MGIT) WGS. We sequenced M. tuberculosis from 43 sputum samples by targeted DNA enrichment using the Agilent SureSelectXT kit, and 43 MGIT positive samples from each participant. Thirty two (74%) sputum samples and 43 (100%) MGIT samples generated whole genomes. The times to antimicrobial resistance profiles and concordance were compared with Xpert MTB/RIF and phenotypic resistance testing from cultures of the same samples. Antibiotic susceptibility could be predicted from WGS of sputum within 5 days of sample receipt and up to 24 days earlier than WGS from MGIT culture and up to 31 days earlier than phenotypic testing. Direct sputum results could be reduced to 3 days with faster hybridization and if only regions encoding drug resistance are sequenced. We show that direct sputum sequencing has the potential to provide comprehensive resistance detection significantly faster than MGIT whole-genome sequencing or phenotypic testing of resistance from cultures in a clinical setting. This improved turnaround time enables prompt appropriate treatment with associated patient and health service benefits. Improvements in sample preparation are necessary to ensure comparable sensitivities and complete resistance profile predictions in all cases.

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