scholarly journals Antibiotic resistance prediction for Mycobacterium tuberculosis from genome sequence data with Mykrobe

2019 ◽  
Vol 4 ◽  
pp. 191 ◽  
Author(s):  
Martin Hunt ◽  
Phelim Bradley ◽  
Simon Grandjean Lapierre ◽  
Simon Heys ◽  
Mark Thomsit ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Sequence Data ◽  
Universal Access ◽  
Independent Set ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Error Rates ◽  
World Health ◽  
Sequencing Data ◽  
Resistance Prediction

Two billion people are infected with Mycobacterium tuberculosis, leading to 10 million new cases of active tuberculosis and 1.5 million deaths annually. Universal access to drug susceptibility testing (DST) has become a World Health Organization priority. We previously developed a software tool, Mykrobe predictor, which provided offline species identification and drug resistance predictions for M. tuberculosis from whole genome sequencing (WGS) data. Performance was insufficient to support the use of WGS as an alternative to conventional phenotype-based DST, due to mutation catalogue limitations.  Here we present a new tool, Mykrobe, which provides the same functionality based on a new software implementation. Improvements include i) an updated mutation catalogue giving greater sensitivity to detect pyrazinamide resistance, ii) support for user-defined resistance catalogues, iii) improved identification of non-tuberculous mycobacterial species, and iv) an updated statistical model for Oxford Nanopore Technologies sequencing data. Mykrobe is released under MIT license at https://github.com/mykrobe-tools/mykrobe. We incorporate mutation catalogues from the CRyPTIC consortium et al. (2018) and from Walker et al. (2015), and make improvements based on performance on an initial set of 3206 and an independent set of 5845 M. tuberculosis Illumina sequences. To give estimates of error rates, we use a prospectively collected dataset of 4362 M. tuberculosis isolates. Using culture based DST as the reference, we estimate Mykrobe to be 100%, 95%, 82%, 99% sensitive and 99%, 100%, 99%, 99% specific for rifampicin, isoniazid, pyrazinamide and ethambutol resistance prediction respectively. We benchmark against four other tools on 10207 (=5845+4362) samples, and also show that Mykrobe gives concordant results with nanopore data.  We measure the ability of Mykrobe-based DST to guide personalized therapeutic regimen design in the context of complex drug susceptibility profiles, showing 94% concordance of implied regimen with that driven by phenotypic DST, higher than all other benchmarked tools.

scholarly journals Quantitative measurement of antibiotic resistance in Mycobacterium tuberculosis reveals genetic determinants of resistance and susceptibility in a target gene approach

2021 ◽  
Author(s):  
◽  
Joshua J Carter
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Quantitative Measurement ◽  
Target Gene ◽  
Microtiter Plate ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
World Health ◽  
Genetic Determinants ◽  
Microtiter Plate Assay ◽  
Health Organization

AbstractThe World Health Organization goal of universal drug susceptibility testing for patients with tuberculosis is most likely to be achieved through molecular diagnostics; however, to date these have focused largely on first-line drugs, and always on predicting binary susceptibilities. Here, we used whole genome sequencing and a quantitative microtiter plate assay to relate genomic mutations to minimum inhibitory concentration in 15,211 Mycobacterium tuberculosis patient isolates from 27 countries across five continents.This work identifies 449 unique MIC-elevating genetic determinants across thirteen drugs, as well as 91 mutations resulting in hypersensitivity for eleven drugs. Our results provide a guide for further implementation of personalized medicine for the treatment of tuberculosis using genetics-based diagnostics and can serve as a training set for novel approaches to predict drug resistance.

scholarly journals Validation of Bedaquiline Phenotypic Drug Susceptibility Testing Methods and Breakpoints: a Multilaboratory, Multicountry Study

2020 ◽  
Vol 58 (4) ◽  
Author(s):  
Koné Kaniga ◽  
Akio Aono ◽  
Emanuele Borroni ◽  
Daniela Maria Cirillo ◽  
Christel Desmaretz ◽  
...  
Keyword(s):  
Susceptibility Testing ◽  
Improve Patient Care ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Error Rates ◽  
World Health ◽  
Health Concern ◽  
Content Type ◽  
Indicator Tube ◽  
Health Organization

ABSTRACT Drug-resistant tuberculosis persists as a major public health concern. Alongside efficacious treatments, validated and standardized drug susceptibility testing (DST) is required to improve patient care. This multicountry, multilaboratory external quality assessment (EQA) study aimed to validate the sensitivity, specificity, and reproducibility of provisional bedaquiline MIC breakpoints and World Health Organization interim critical concentrations (CCs) for categorizing clinical Mycobacterium tuberculosis isolates as susceptible/resistant to the drug. Three methods were used: Middlebrook 7H11 agar proportion (AP) assay, broth microdilution (BMD) assay, and mycobacterial growth indicator tube (MGIT) assay. Each of the five laboratories tested the 40-isolate (20 unique isolates, duplicated) EQA panel at three time points. The study validated the sensitivity and specificity of a bedaquiline MIC susceptibility breakpoint of 0.12 μg/ml for the BMD method and WHO interim CCs of 1 μg/ml for MGIT and 0.25 μg/ml for the 7H11 AP methods. Categorical agreements between observed and expected results and sensitivities/specificities for correctly identifying an isolate as susceptible/resistant were highest at the 0.25, 0.12, and 1 μg/ml bedaquiline concentrations for the AP method, BMD (frozen or dry plates), and MGIT960, respectively. At these concentrations, the very major error rates for erroneously categorizing an isolate as susceptible when it was resistant were the lowest and within CLSI guidelines. The most highly reproducible bedaquiline DST methods were MGIT960 and BMD using dry plates. These findings validate the use of standardized DST methodologies and interpretative criteria to facilitate routine phenotypic bedaquiline DST and to monitor the emergence of bedaquiline resistance.

scholarly journals Nanoluciferase Reporter Mycobacteriophage for Sensitive and Rapid Detection of Mycobacterium tuberculosis Drug Susceptibility

2020 ◽  
Vol 202 (22) ◽  
Author(s):  
Paras Jain ◽  
Spencer Garing ◽  
Deepshikha Verma ◽  
Rajagopalan Saranathan ◽  
Nicholas Clute-Reinig ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Susceptibility Testing ◽  
Limit Of Detection ◽  
Reference Method ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
World Health ◽  
Metabolic Inhibitors ◽  
Health Crisis ◽  
Content Type

ABSTRACT Phenotypic testing for drug susceptibility of Mycobacterium tuberculosis is critical to basic research and managing the evolving problem of antimicrobial resistance in tuberculosis management, but it remains a specialized technique to which access is severely limited. Here, we report on the development and validation of an improved phage-mediated detection system for M. tuberculosis. We incorporated a nanoluciferase (Nluc) reporter gene cassette into the TM4 mycobacteriophage genome to create phage TM4-nluc. We assessed the performance of this reporter phage in the context of cellular limit of detection and drug susceptibility testing using multiple biosafety level 2 drug-sensitive and -resistant auxotrophs as well as virulent M. tuberculosis strains. For both limit of detection and drug susceptibility testing, we developed a standardized method consisting of a 96-hour cell preculture followed by a 72-hour experimental window for M. tuberculosis detection with or without antibiotic exposure. The cellular limit of detection of M. tuberculosis in a 96-well plate batch culture was ≤102 CFU. Consistent with other phenotypic methods for drug susceptibility testing, we found TM4-nluc to be compatible with antibiotics representing multiple classes and mechanisms of action, including inhibition of core central dogma functions, cell wall homeostasis, metabolic inhibitors, compounds currently in clinical trials (SQ109 and Q203), and susceptibility testing for bedaquiline, pretomanid, and linezolid (components of the BPaL regimen for the treatment of multi- and extensively drug-resistant tuberculosis). Using the same method, we accurately identified rifampin-resistant and multidrug-resistant M. tuberculosis strains. IMPORTANCE Mycobacterium tuberculosis, the causative agent of tuberculosis disease, remains a public health crisis on a global scale, and development of new interventions and identification of drug resistance are pillars in the World Health Organization End TB Strategy. Leveraging the tractability of the TM4 mycobacteriophage and the sensitivity of the nanoluciferase reporter enzyme, the present work describes an evolution of phage-mediated detection and drug susceptibility testing of M. tuberculosis, adding a valuable tool in drug discovery and basic biology research. With additional validation, this system may play a role as a quantitative phenotypic reference method and complement to genotypic methods for diagnosis and antibiotic susceptibility testing.

scholarly journals DeepAMR for predicting co-occurrent resistance of Mycobacterium tuberculosis

2019 ◽  
Vol 35 (18) ◽  
pp. 3240-3249 ◽  
Author(s):  
Yang Yang ◽  
Timothy M Walker ◽  
A Sarah Walker ◽  
Daniel J Wilson ◽  
Timothy E A Peto ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
World Health ◽  
Supplementary Information ◽  
Multiple Drug ◽  
First Line ◽  
Baseline Model ◽  
Latent Space ◽  
Mdr Tb

Abstract Motivation Resistance co-occurrence within first-line anti-tuberculosis (TB) drugs is a common phenomenon. Existing methods based on genetic data analysis of Mycobacterium tuberculosis (MTB) have been able to predict resistance of MTB to individual drugs, but have not considered the resistance co-occurrence and cannot capture latent structure of genomic data that corresponds to lineages. Results We used a large cohort of TB patients from 16 countries across six continents where whole-genome sequences for each isolate and associated phenotype to anti-TB drugs were obtained using drug susceptibility testing recommended by the World Health Organization. We then proposed an end-to-end multi-task model with deep denoising auto-encoder (DeepAMR) for multiple drug classification and developed DeepAMR_cluster, a clustering variant based on DeepAMR, for learning clusters in latent space of the data. The results showed that DeepAMR outperformed baseline model and four machine learning models with mean AUROC from 94.4% to 98.7% for predicting resistance to four first-line drugs [i.e. isoniazid (INH), ethambutol (EMB), rifampicin (RIF), pyrazinamide (PZA)], multi-drug resistant TB (MDR-TB) and pan-susceptible TB (PANS-TB: MTB that is susceptible to all four first-line anti-TB drugs). In the case of INH, EMB, PZA and MDR-TB, DeepAMR achieved its best mean sensitivity of 94.3%, 91.5%, 87.3% and 96.3%, respectively. While in the case of RIF and PANS-TB, it generated 94.2% and 92.2% sensitivity, which were lower than baseline model by 0.7% and 1.9%, respectively. t-SNE visualization shows that DeepAMR_cluster captures lineage-related clusters in the latent space. Availability and implementation The details of source code are provided at http://www.robots.ox.ac.uk/∼davidc/code.php. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Comparison of Proportion and Resistance Ratio Methods for Drug Susceptibility Testing of Mycobacterium tuberculosis Isolated from Patients Attending National Tuberculosis Centre, Nepal

2010 ◽  
Vol 5 (1) ◽  
pp. 13-20
Author(s):  
S Acharya ◽  
P Ghimire ◽  
DK Khadka ◽  
S Nepali
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Fluorescence Microscopy ◽  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Resistance Ratio ◽  
National Tuberculosis ◽  
Smear Negative ◽  
Culture Positive ◽  
Good Agreement

Background: Tuberculosis (TB) is among the most serious infectious cause of global morbidity and mortality. Emergence of Multi-drug resistant tuberculosis (MDR-TB) is posing an increased threat to TB control programs. Drug susceptibility testing (DST) of Mycobacterium tuberculosis (M. tuberculosis) isolates is important for tackling such problems. Setting: National Tuberculosis Centre (NTC), Thimi, Bhaktapur, Nepal. Objectives: Comparative evaluation of two in vitro DST methods in determining susceptibility of M. tuberculosis isolates from patients attending NTC, to front-line anti-TB drugs: (Isoniazid-INH, Rifampicin-RFP, Streptomycin-SM, and Ethambutol-EMB). Methodology: This study was conducted from Sep 2006-Jun 2007. A total of 862 sputum samples (diagnosis or follow up cases) collected from patients (type of patients or their categories was not differentiated in this study) attending NTC bacteriology lab for sputum direct smear microscopy were analyzed using fluorescence microscopy. All smear positive samples, smear negative samples requested for culture were cultured. All culture positive samples confirmed as M. tuberculosis by biochemical tests were processed for DST by both proportion (PR) and resistance ratio (RR) methods. Results: Out of 862 sputum samples analyzed, 226 (26.2%) samples were positive for Acid Fast Bacilli (AFB) by fluorescence microscopy. Among 323 samples 226 smear positive samples and 97 smear negative samples requested for culture), 221 (68.4%) were culture positive, 92 (28.5%) were culture negative and 10 (3.1%) were contaminated. Out of 221 isolates of M. tuberculosis, 57.5% were resistant to one or more drugs by the PR method and 56.6% by the RR method. Similarly, MDR isolates were 29.9% and 29% by PR and RR methods respectively. On correlation analysis using Mc Nemar Chi-square test, no significant difference between the two tests were observed (p>0.05). The results showed high agreement between both methods and agreement rates to INH, RFP, SM and EMB were 93.2%, 93.7%, 93.2% and 94.1% respectively. Similarly, the agreement rates between both methods using kappa analysis showed kappa (k) value of 0.86, 0.85, 0.86 and 0.84 for INH, RFP, SM and EMB respectively, which is believed to be good agreement between both methods (k=0.80 to 1.00: Very good agreement). Conclusion: In conclusion, this study showed that both the Proportion and Resistance ratio methods are equally good for determining drug susceptibility of M. tuberculosis. Keywords: Mycobacterium tuberculosis; Drug Susceptibility Testing; Proportion Method; Resistance Ratio Method. DOI: 10.3126/saarctb.v5i1.3078 SAARC J. Tuber. Lung Dis. HIV/AIDS 2008 Vol.5(1) 13-20

scholarly journals Comparison of the Performances of Two In-House Rapid Methods for Antitubercular Drug Susceptibility Testing

2008 ◽  
Vol 53 (2) ◽  
pp. 808-810 ◽  
Author(s):  
Agustina I. de la Iglesia ◽  
Emma J. Stella ◽  
Héctor R. Morbidoni
Keyword(s):  
Drug Resistance ◽  
Mycobacterium Tuberculosis ◽  
Sensitivity And Specificity ◽  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Antitubercular Drug ◽  
Rapid Methods ◽  
Resistance Assessment

ABSTRACT Resistance to rifampin (rifampicin), isoniazid, and streptomycin of 69 Mycobacterium tuberculosis isolates was analyzed by an in-house method based on mycobacteriophage D29 and a colorimetric micromethod. Both methods showed sensitivity and specificity values ranging from 93% to 100%. These simple methods offer an option for drug resistance assessment of M. tuberculosis.

scholarly journals Rapid Mycobacterium tuberculosis spoligotyping from uncorrected long reads using Galru

2020 ◽  
Author(s):  
Andrew J. Page ◽  
Nabil-Fareed Alikhan ◽  
Michael Strinden ◽  
Thanh Le Viet ◽  
Timofey Skvortsov
Keyword(s):  
Mycobacterium Tuberculosis ◽  
State Of The Art ◽  
Sequence Data ◽  
Human Pathogen ◽  
Sequencing Data ◽  
Short Read ◽  
Short Read Sequencing ◽  
Long Reads ◽  
Long Read

AbstractSpoligotyping of Mycobacterium tuberculosis provides a subspecies classification of this major human pathogen. Spoligotypes can be predicted from short read genome sequencing data; however, no methods exist for long read sequence data such as from Nanopore or PacBio. We present a novel software package Galru, which can rapidly detect the spoligotype of a Mycobacterium tuberculosis sample from as little as a single uncorrected long read. It allows for near real-time spoligotyping from long read data as it is being sequenced, giving rapid sample typing. We compare it to the existing state of the art software and find it performs identically to the results obtained from short read sequencing data. Galru is freely available from https://github.com/quadram-institute-bioscience/galru under the GPLv3 open source licence.

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