Whole-Genome Sequencing to Identify Missed Rifampicin and Isoniazid Resistance Among Tuberculosis Isolates—Chennai, India, 2013–2016

India has a high burden of drug-resistant tuberculosis (DR TB) and many cases go undetected by current drug susceptibility tests (DSTs). This study was conducted to identify rifampicin (RIF) and isoniazid (INH) resistance associated genetic mutations undetected by current clinical diagnostics amongst persons with DR TB in Chennai, India. Retrospectively stored 166 DR TB isolates during 2013–2016 were retrieved and cultured in Löwenstein-Jensen medium. Whole genome sequencing (WGS) and MGIT DST for RIF and INH were performed. Discordant genotypic and phenotypic sensitivity results were repeated for confirmation and the discrepant results considered final. Further, drug resistance-conferring mutations identified through WGS were analyzed for their presence as targets in current WHO-recommended molecular diagnostics. WGS detected additional mutations for rifampicin and isoniazid resistance than WHO-endorsed line probe assays. For RIF, WGS was able to identify an additional 10% (15/146) of rpoB mutant isolates associated with borderline rifampicin resistance compared to MGIT DST. WGS could detect additional DR TB cases than commercially available and WHO-endorsed molecular DST tests. WGS results reiterate the importance of the recent WHO revised critical concentrations of current MGIT DST to detect low-level resistance to rifampicin. WGS may help inform effective treatment selection for persons at risk of, or diagnosed with, DR TB.

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Individualized Treatment of Multidrug-resistant Tuberculosis Using Whole-Genome Sequencing and Expanded Drug-Susceptibility Testing

Clinical Infectious Diseases ◽

10.1093/cid/ciaa526 ◽

2020 ◽

Vol 71 (11) ◽

pp. 2981-2985

Author(s):

Navisha Dookie ◽

Nesri Padayatchi ◽

Richard J Lessells ◽

Cherise L Naicker ◽

Sunitha Chotoo ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Susceptibility Testing ◽

Drug Susceptibility ◽

Multidrug Resistant ◽

Drug Susceptibility Testing ◽

Individualized Treatment ◽

Whole Genome ◽

Resistant Tuberculosis ◽

Multidrug Resistant Tuberculosis

Abstract A case of multidrug-resistant tuberculosis is presented. It highlights the role of whole-genome sequencing, expanded phenotypic drug susceptibility testing, and enhanced case management, offering a more complete understanding of drug susceptibility to Mycobacterium tuberculosis. This approach guides an effective individualized treatment strategy that results in rapid sustained culture conversion.

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Isoniazid resistance in Mycobacterium tuberculosis is a heterogeneous phenotype comprised of overlapping MIC distributions with different underlying resistance mechanisms

10.1101/524157 ◽

2019 ◽

Author(s):

Arash Ghodousi ◽

Elisa Tagliani ◽

Eranga Karunaratne ◽

Stefan Niemann ◽

Jennifer Perera ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Clinical Laboratory ◽

Resistance Mechanisms ◽

Whole Genome ◽

Isoniazid Resistance ◽

Low Level ◽

Promoter Mutations ◽

Level Resistance

AbstractMIC testing using the BACTEC 960 MGIT system of 70 phylogenetically diverse, isoniazid-resistant clinical strains of Mycobacterium tuberculosis revealed a complex pattern of overlapping MIC distributions. Whole-genome sequencing could explain most of the level of resistance observed. The MIC distribution of strains with only inhA promoter mutations was split by the current concentration that is endorsed by the Clinical Laboratory Standards Institute to detect low-level resistance to isoniazid and is therefore likely not optimally set.

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Setup, Validation, and Quality Control of a Centralized Whole-Genome-Sequencing Laboratory: Lessons Learned

Journal of Clinical Microbiology ◽

10.1128/jcm.00261-18 ◽

2018 ◽

Vol 56 (8) ◽

Cited By ~ 6

Author(s):

Cath Arnold ◽

Kirstin Edwards ◽

Meeta Desai ◽

Steve Platt ◽

Jonathan Green ◽

...

Keyword(s):

Public Health ◽

Quality Control ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Food Poisoning ◽

Drug Susceptibility ◽

Lessons Learned ◽

Microbial Pathogens ◽

Whole Genome ◽

Whole Genome Analysis

ABSTRACT Routine use of whole-genome analysis for infectious diseases can be used to enlighten various scenarios pertaining to public health, including identification of microbial pathogens, relating individual cases to an outbreak of infectious disease, establishing an association between an outbreak of food poisoning and a specific food vehicle, inferring drug susceptibility, source tracing of contaminants, and study of variations in the genome that affect pathogenicity/virulence. We describe the setup, validation, and ongoing verification of a centralized whole-genome-sequencing (WGS) laboratory to carry out sequencing for these public health functions for the National Infection Services, Public Health England, in the United Kingdom. The performance characteristics and quality control metrics measured during validation and verification of the entire end-to-end process (accuracy, precision, reproducibility, and repeatability) are described and include information regarding the automated pass and release of data to service users without intervention.

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Low-level rifampin resistance and rpoB mutations in Mycobacterium tuberculosis: An analysis of whole-genome sequencing and drug susceptibility test data in New York

Journal of Clinical Microbiology ◽

10.1128/jcm.01885-20 ◽

2020 ◽

Author(s):

Joseph Shea ◽

Tanya A. Halse ◽

Donna Kohlerschmidt ◽

Pascal Lapierre ◽

Herns A. Modestil ◽

...

Keyword(s):

New York ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Critical Concentration ◽

Gene Sequencing ◽

Drug Susceptibility ◽

Whole Genome ◽

Drug Resistant ◽

Low Level ◽

Indicator Tube

Rapid and reliable detection of rifampin (RIF) resistance is critical for the diagnosis and treatment of drug-resistant and multi-drug resistant (MDR) tuberculosis. Discordant RIF phenotype/genotype susceptibility results remain a challenge due to the presence of rpoB mutations which do not confer high levels of RIF resistance as have been exhibited in strains with mutations such as Ser450Leu. These strains, termed low-level RIF resistant, exhibit elevated RIF minimum inhibitory concentrations (MICs) compared to fully susceptible strains, however remain phenotypically susceptible by mycobacteria growth indicator tube (MGIT) testing and have been associated with poor patient outcomes. Here we assess RIF resistance prediction by whole-genome sequencing (WGS) among a set of 1779 prospectively tested strains by both prevalence of rpoB gene mutation and phenotype as part of routine clinical testing during a 21/2-year period. During this time, 139 strains were found to have nonsynonymous rpoB mutations, 53 of which were associated with RIF resistance, including both low-level and high-level resistance. Resistance to RIF (1.0 μg/mL in MGIT) was identified in 43 (81.1%) isolates. The remaining 10 (18.9%) strains were susceptible by MGIT, however were confirmed to be low-level RIF resistant by MIC testing. Full rpoB gene sequencing overcame the limitations of critical concentration phenotyping, probe-based genotyping, and partial-gene sequencing methods. Universal clinical WGS with concurrent phenotypic testing provided a more complete understanding of the prevalence and type of rpoB mutations and their association with RIF resistance in New York.

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Whole genome sequencing for drug-resistant tuberculosis management in South Africa: What gaps would this address and what are the challenges to implementation?

Journal of Clinical Tuberculosis and Other Mycobacterial Diseases ◽

10.1016/j.jctube.2019.100115 ◽

2019 ◽

Vol 16 ◽

pp. 100115 ◽

Cited By ~ 1

Author(s):

Mlungisi Thabiso Dlamini ◽

Richard Lessells ◽

Thato Iketleng ◽

Tulio de Oliveira

Keyword(s):

South Africa ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Whole Genome ◽

Drug Resistant ◽

Drug Resistant Tuberculosis ◽

Resistant Tuberculosis

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799. Genetic Diversity of Mycobacterium tuberculosis Strains Causing Drug-Resistant Tuberculosis in Central Region of Mozambique: The Whole-Genome Sequencing

Open Forum Infectious Diseases ◽

10.1093/ofid/ofy210.806 ◽

2018 ◽

Vol 5 (suppl_1) ◽

pp. S286-S287

Author(s):

Evangelina Namburete

Keyword(s):

Genetic Diversity ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Central Region ◽

Disease Spread ◽

Whole Genome ◽

Drug Resistant ◽

Illumina Hiseq ◽

Drug Resistant Tuberculosis ◽

Resistant Tuberculosis

Abstract Background Knowing the genetic diversity of M. tuberculosis strains causing drug-resistant tuberculosis (DR-TB) in high burden TB and low resources countries such as Mozambique is a key factor to TB disease spread control and world TB epidemic control. Whole-genome sequencing (WGS) better describes molecular diversity, lineages and sub lineages, relationship between strains, underline mutations conferring drug-resistant TB, which may not be shown by molecular and phenotypic tests. As far as we know this is the first study that describes genetic diversity of M. tuberculosis strains causing DR-TB and using WGS in central region of Mozambique.We aim to describe genetic diversity of M. tuberculosis strains causing DR-TB in central Mozambique. Methods A total of 35 strains from Beira Mozambique were evaluated with genotypic tests (Genotype MTBDRplus™, and MTBDRsl™); phenotypic (MGIT-SIRE™) and DST. All isolates resistant to isoniazid (H) or rifampicin (R) or both were submitted to WGS Illumina HiSeq 2000 and analyzed with TB profiler database and phylogenetic tree was done using Figtree tool. This was a descriptive cross-sectional study. Results WGS shown that strains analyzed, belongs to three of six major lineages, with Lineage 4: 25(71.4%); Lineage 1: 5(14.3%); and Lineage 2 Beijing family: 5(14.3%)]. All pre-XDR strains 3(8.6%) were from lineage 4.3. By WGS, all 35 strains had any mutations conferring DR-TB while in one strain, mutation was not shown by genotypic neither phenotypic DST. Compared with genotypic tests, WGS had best performance in showing mutation conferring resistance to etambutol 12/35 (34.3%) and 7/35 (20%). Conclusion The DR-TB disease in Beira Mozambique is mainly caused by M. tuberculosis strains of Lineage 4, sub-lineage 3 although lineage 1 and 2 are also present. WGS shows underline mutations causing DR–TB that are not detected by genotypic and phenotypic DST test. Disclosures All authors: No reported disclosures.

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