Whole-genome sequencing in drug susceptibility testing of Mycobacterium tuberculosis in routine practice in Lyon, France

Accurate and timely detection of drug resistance can minimize the risk of further resistance development and lead to effective treatment. The aim of this study was to determine the resistance to first/second-line anti-tuberculosis drugs in rifampicin/multidrug-resistant Mycobacterium tuberculosis (RR/MDR-MTB) isolates. Molecular epidemiology of strains was determined using whole genome sequencing (WGS)-based genotyping. A total of 35 RR/MDR-MTB isolates were subjected to drug susceptibility testing against first/second-line drugs using 7H9 Middlebrook in broth microdilution method. Illumina technology was used for paired-end WGS applying a Maxwell 16 Cell DNA Purification kit and the NextSeq platform. Data analysis and single nucleotide polymorphism calling were performed using MTBseq pipeline. The genome-based resistance to each drug among the resistant phenotypes was as follows: rifampicin (97.1%), isoniazid (96.6%), ethambutol (100%), levofloxacin (83.3%), moxifloxacin (83.3%), amikacin (100%), kanamycin (100%), capreomycin (100%), prothionamide (100%), D-cycloserine (11.1%), clofazimine (20%), bedaquiline (0.0%), and delamanid (44.4%). There was no linezolid-resistant phenotype, and a bedaquiline-resistant strain was wild type for related genes. The Beijing, Euro-American, and Delhi-CAS were the most populated lineage/sublineages. Drug resistance-associated mutations were mostly linked to minimum inhibitory concentration results. However, the role of well-known drug-resistant genes for D-cycloserine, clofazimine, bedaquiline, and delamanid was found to be more controversial.

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Mortality of Drug-resistant Tuberculosis in High-burden Countries: Comparison of Routine Drug Susceptibility Testing with Whole-genome Sequencing

10.1101/2020.08.03.20167213 ◽

2020 ◽

Author(s):

Martina L. Reichmuth ◽

Kathrin Zürcher ◽

Marie Ballif ◽

Chloé Loiseau ◽

Sonia Borrell ◽

...

Keyword(s):

Drug Resistance ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Susceptibility Testing ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Whole Genome ◽

Drug Resistant ◽

Hiv Status ◽

High Burden

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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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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