scholarly journals Detection of Minority Variants and Mixed Infections in Mycobacterium tuberculosis by Direct Whole-Genome Sequencing on Noncultured Specimens Using a Specific-DNA Capture Strategy

mSphere ◽  
2021 ◽  
Author(s):  
Nuria Lozano ◽  
Val F. Lanza ◽  
Julia Suárez-González ◽  
Marta Herranz ◽  
Pedro J. Sola-Campoy ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Direct Detection ◽  
Mixed Infections ◽  
Whole Genome ◽  
Content Type ◽  
New Strategy ◽  
Minority Variants ◽  
First Moment

We present a new strategy to identify mixed infections and minority variants in Mycobacterium tuberculosis by whole-genome sequencing. The objective of the strategy is the direct detection in patient sputum; in this way, minority populations of resistant strains can be identified at the time of diagnosis, facilitating identification of the most appropriate treatment for the patient from the first moment.

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 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 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 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.

scholarly journals Revisiting Activation of and Mechanism of Resistance to Compound IQG-607 in Mycobacterium tuberculosis

2017 ◽  
Vol 62 (2) ◽  
Author(s):  
Bruno L. Abbadi ◽  
Anne D. Villela ◽  
Valnês S. Rodrigues-Junior ◽  
Fernanda T. Subtil ◽  
Pedro F. Dalberto ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Metal Complex ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Clinical Isolates ◽  
Whole Genome ◽  
Content Type ◽  
Mechanism Of Resistance ◽  
Resistant Strains ◽  
Spontaneous Mutants

ABSTRACT IQG-607 is a metal complex previously reported as a promising anti-tuberculosis (TB) drug against isoniazid (INH)-resistant strains of Mycobacterium tuberculosis. Unexpectedly, we found that INH-resistant clinical isolates were resistant to IQG-607. Spontaneous mutants resistant to IQG-607 were subjected to whole-genome sequencing, and all sequenced colonies carried alterations in the katG gene. The katG(S315T) mutation was sufficient to confer resistance to IQG-607 in both MIC assays and inside macrophages. Moreover, overexpression of the InhA(S94A) protein caused IQG-607's resistance.

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 Genetic Diversity of Multi- and Extensively Drug-Resistant Mycobacterium tuberculosis Isolates in the Capital of Iran, Revealed by Whole-Genome Sequencing

2018 ◽  
Vol 57 (1) ◽  
Author(s):  
Farzam Vaziri ◽  
Thomas A. Kohl ◽  
Hasan Ghajavand ◽  
Mansour Kargarpour Kamakoli ◽  
Matthias Merker ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Drug Resistant ◽  
Content Type ◽  
Extensively Drug Resistant ◽  
Recent Transmission ◽  
Mdr Tb ◽  
Lineage 2

ABSTRACT The emergence and spread of multidrug resistant (MDR) Mycobacterium tuberculosis complex (MTBC) strains is a critical global health problem. Between 2014 and 2018, 606 MTBC strains were isolated from 13,892 suspected pulmonary tuberculosis (TB) patients in Tehran, Iran, including 16 (2.6%) MDR-TB cases. A combination of phenotypic and genotypic methods (whole-genome sequencing) was employed for the identification of additional drug resistances and strain-to-strain genetic distances as a marker for recent transmission events. MDR and extensively drug-resistant (XDR) TB cases were almost exclusively infected by lineage 2/Beijing strains (14/16, P < 0.001). We further showed that recent transmission and/or recent introduction of lineage 2/Beijing strains contribute to high XDR-TB rates among all MDR-TB cases and should be considered an emerging threat for TB control in Tehran. In addition, the extensive pre-existing drug resistance profiles of MDR/XDR strains will further challenge TB diagnostics in the region.

scholarly journals Whole-Genome Sequencing of a Mycobacterium tuberculosis Strain Belonging to Lineage 1 (Indo-Oceanic) and the East African Indian Spoligotype, Isolated in Jazan, Saudi Arabia

2020 ◽  
Vol 9 (33) ◽  
Author(s):  
Aymen Abdelhaleem ◽  
Almonther Hershan ◽  
Pradeep Agarwal ◽  
Abdullah Farasani ◽  
Shaheed V. Omar ◽  
...  
Keyword(s):  
Saudi Arabia ◽  
Mycobacterium Tuberculosis ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Draft Genome ◽  
Whole Genome ◽  
East African ◽  
Content Type ◽  
Tuberculosis Strain ◽  
Mycobacterium Tuberculosis Strain

ABSTRACT We describe here the draft genome sequence of AY1MRC, a Mycobacterium tuberculosis strain belonging to lineage 1 (Indo-Oceanic) and the East African Indian spoligotype, isolated from a patient with tuberculosis in Jazan, Saudi Arabia.

scholarly journals Whole-Genome Sequencing and Annotation of a Drug-Resistant Extrapulmonary Clinical Isolate of Beijing Genotype Mycobacterium tuberculosis from Pune, India

2018 ◽  
Vol 6 (25) ◽  
Author(s):  
Swarupa M. Hatolkar ◽  
Rabindra N. Misra ◽  
Rajesh Mahato ◽  
Savita Jadhav
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Disease Transmission ◽  
Immunocompetent Patient ◽  
Beijing Genotype ◽  
Whole Genome ◽  
Drug Resistant ◽  
Content Type ◽  
Genomic Signatures

ABSTRACT Whole-genome sequencing has emerged as a powerful tool to map genetic diversity among Mycobacterium tuberculosis isolates and identify the genomic signatures associated with drug resistance, pathogenesis, and disease transmission. Isolate LJ319 of the Mycobacterium tuberculosis complex (MTC)-Beijing genotype circulating in Maharashtra, India, which was obtained from the cerebrospinal fluid (CSF) of an immunocompetent patient, was subjected to whole-genome sequencing.

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