Turnaround time of whole genome sequencing for mycobacterial identification and drug susceptibility testing in routine practice

AbstractBackgroundDrug-resistant Mycobacterium tuberculosis (Mtb) strains threaten tuberculosis (TB) control. We compared data on drug resistance obtained at clinics in seven high TB burden countries during routine care with whole-genome sequencing (WGS) carried out centrally.MethodsWe collected pulmonary Mtb isolates and clinical data from adult TB patients in Africa, Latin America, and Asia, stratified by HIV status and drug resistance, from 2013 to 2016. Participating sites performed drug susceptibility testing (DST) locally, using routinely available methods. WGS was done using Illumina HiSeq 2500 at laboratories in the USA and Switzerland. We used TBprofiler to analyse the genomes. We used multivariable logistic regression adjusted for sex, age, HIV-status, history of TB, sputum positivity, and Mtb-lineage to analyse mortality.FindingsWe included 582 TB patients. The median age was 32 years (interquartile range: 27-43 years), 225 (39%) were female, and 247 (42%) were HIV-positive. Based on WGS, 339 (58%) isolates were pan-susceptible, 35 (6%) monoresistant, 146 (25%) multidrug-resistant, and 24 (4%) pre-/ extensively drug-resistant (pre-XDR/XDR-TB). The local DST results were discordant compared to WGS results in 130/582 (22%) of patients. All testing methods identified isoniazid and rifampicin resistance with relatively high agreement (kappa 0.69 for isoniazid and 0.88 rifampicin). Resistance to ethambutol, pyrazinamide, and second-line drugs was rarely tested locally. Of 576 patients with known treatment, 86 (15%) patients received inadequate treatment according to WGS results and the World Health Organization treatment guidelines. The analysis of mortality was based on 530 patients; 63 patients (12%) died and 77 patients (15%) received inadequate treatment. Mortality ranged from 6% in patients with pan-susceptible Mtb (18/310) to 39% in patients with pre-XDR/XDR-TB (9/23). The adjusted odds ratio for mortality was 4.82 (95% CI 2.43-9.44) for under-treatment and 0.52 (95% CI 0.03-2.73) for over-treatment.InterpretationIn seven high-burden TB countries, we observed discrepancies between drug resistance patterns from local DST and WGS, which resulted in inadequate treatment and higher mortality. WGS can provide accurate and detailed drug resistance information, which is required to improve the outcomes of drug-resistant TB in high burden settings. Our results support the WHO’s call for point-of-care tests based on WGS.

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Drug susceptibility testing of Mycobacterium tuberculosis using next generation sequencing and Mykrobe software

Journal of microbiology epidemiology immunobiology ◽

10.36233/0372-9311-191 ◽

2022 ◽

Vol 98 (6) ◽

pp. 697-705

Author(s):

V. Tolchkov ◽

Y. Hodzhev ◽

B. Tsafarova ◽

E. Bachiyska ◽

Yu. Atanasova ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Susceptibility Testing ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Whole Genome ◽

Drug Resistant ◽

Molecular Tests ◽

Bactec System ◽

Probe Assay

Introduction. Mycobacterium tuberculosis is the causative agent of tuberculosis. Drug susceptibility testing is performed by phenotypic and molecular tests. Commonly used for phenotypic drug susceptibility testing is the automated BACTEC system in a liquid culture medium. Drug susceptibility by line probe molecular tests was introduced almost 15 years ago. Recently whole genome sequencing (WGS) analysis of M. tuberculosis strains demonstrated that genotyping of drug-resistance could be accurately performed. Several software tools were developed.Our study aimed to perform whole-genome sequencing on phenotypically confirmed multi-drug resistant (MDR) M. tuberculosis strains, to identify drug-resistant mutations and to compare whole-genome sequencing profiles with line probe assay and phenotypic results.Materials and methods. We performed analysis on 34 MDR M. tuberculosis Bulgarian strains. Phenotypic drug susceptibility testing was performed on the BACTEC system. For molecular testing of drug susceptibility to first- and second-line tuberculostatics, we applied line probe assay Geno Type MTBDR plus v.1.0 и Geno Type MTBDR sl v.1.0. Sequencing was performed on MiSeq. Generated FASTQ files were analyzed for known drugresistant mutations with the software platform Mykrobe v.0.8.1.Results. All three methods — phenotypic analysis using the BACTEC system, genetic analysis of strains applying the Geno Type test and Mykrobe software gave comparable sensitivity/resistance results for the studied strains. All phenotypically proven rifampicin and isoniazid-resistant strains were 100% confirmed using Mykrobe software. The C-15T mutation is a marker for isoniazid resistance in strains of the SIT41 spoligotype. We observed a 75% (21/28) agreement between BACTEC and Mykrobe for ethambutol resistance. Phenotypically, 87% (n = 27) of the strains are resistant to streptomycin, but only 59% (n = 19) are proven by Mykrobe software. Comparing phenotypic and genotypic resistance to ofloxacin, amikacin and kanamycin, we observed 100% coincidence of results.Conclusions. Whole-genome sequencing approach is relatively expensive and laborious but useful for detailed analysis such as epidemiological genotyping and molecular drug susceptibility testing.

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Systematic evaluation of clarithromycin (CLA) resistance associated mutations in Mycobacterium abscessus complex based on whole-genome sequencing analysis and drug susceptibility testing

10.1183/13993003.congress-2020.2330 ◽

2020 ◽

Author(s):

Mitsunori Yoshida ◽

Hanako Fukano ◽

Kentaro Yamamoto ◽

Masato Suzuki ◽

Kozo Morimoto ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Susceptibility Testing ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Mycobacterium Abscessus ◽

Systematic Evaluation ◽

Whole Genome ◽

Sequencing Analysis ◽

Mycobacterium Abscessus Complex

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Comparisons of whole-genome sequencing and phenotypic drug susceptibility testing for Mycobacterium tuberculosis causing MDR-TB and XDR-TB in Thailand

International Journal of Antimicrobial Agents ◽

10.1016/j.ijantimicag.2019.04.004 ◽

2019 ◽

Vol 54 (2) ◽

pp. 109-116 ◽

Cited By ~ 5

Author(s):

Kiatichai Faksri ◽

Orawee Kaewprasert ◽

Rick Twee-Hee Ong ◽

Prapat Suriyaphol ◽

Therdsak Prammananan ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Susceptibility Testing ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Whole Genome ◽

Mdr 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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Evaluation of whole genome sequencing and software tools for drug susceptibility testing of Mycobacterium tuberculosis

Clinical Microbiology and Infection ◽

10.1016/j.cmi.2018.03.041 ◽

2019 ◽

Vol 25 (1) ◽

pp. 82-86 ◽

Cited By ~ 15

Author(s):

J. van Beek ◽

M. Haanperä ◽

P.W. Smit ◽

S. Mentula ◽

H. Soini

Keyword(s):

Mycobacterium Tuberculosis ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Susceptibility Testing ◽

Drug Susceptibility ◽

Software Tools ◽

Drug Susceptibility Testing ◽

Whole Genome

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Whole genome sequencing for drug resistance profile prediction in Mycobacterium tuberculosis

10.1101/401703 ◽

2018 ◽

Author(s):

Sebastian M. Gygli ◽

Peter M. Keller ◽

Marie Ballif ◽

Nicolas Blöchliger ◽

Rico Hömke ◽

...

Keyword(s):

Drug Resistance ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Susceptibility Testing ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Quality Data ◽

Whole Genome ◽

Genome Sequences ◽

Minimal Inhibitory Concentrations

AbstractWhole genome sequencing allows rapid detection of drug-resistant M. tuberculosis isolates. However, high-quality data linking quantitative phenotypic drug susceptibility testing (DST) and genomic data have thus far been lacking.We determined drug resistance profiles of 176 genetically diverse clinical M. tuberculosis isolates from 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 phenotypic drug susceptibility results with predicted drug resistance profiles inferred by whole genome sequencing.Both phenotypic DST methods identically classified the strains into resistant/susceptible in 73-99% of the cases, depending on the drug. Changes in minimal inhibitory concentrations were readily explained by mutations identified by whole genome sequencing. Using the whole genome sequences we were able to predict quantitative drug resistance levels where wild type and mutant MIC distributions did not overlap. The utility of genome sequences to predict quantitative levels of drug resistance was partially limited due to incompletely understood mechanisms influencing the expression of phenotypic drug resistance. The overall sensitivity and specificity of whole genome-based DST were 86.8% and 94.5%, respectively.Despite some limitations, whole genome sequencing has high predictive power to infer resistance profiles without the need for time-consuming phenotypic methods.One sentence summaryWhole genome sequencing of clinical M. tuberculosis isolates accurately predicts drug resistance profiles and may replace culture-based drug susceptibility testing in the future.

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