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

Quantitative Measurement ◽

Target Gene ◽

Microtiter Plate ◽

Drug Susceptibility ◽

World Health ◽

Genetic Determinants ◽

Microtiter Plate Assay ◽

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.

TB Elimination Requires Discovery and Development of Transformational Agents

Applied Sciences ◽

10.3390/app10072605 ◽

2020 ◽

Vol 10 (7) ◽

pp. 2605 ◽

Cited By ~ 1

Author(s):

Christian Lienhardt ◽

Mario C. Raviglione

Keyword(s):

Rapid Detection ◽

Susceptibility Testing ◽

Drug Susceptibility ◽

New Drugs ◽

World Health ◽

Drug Resistant ◽

The World ◽

Health Organization ◽

Combination Regimens

The World Health Organization (WHO) End Tuberculosis (TB) Strategy has set ambitious targets to reduce 2015 TB incidence and deaths by 80% and 90%, respectively, by the year 2030. Given the current rate of TB incidence decline (about 2% per year annually), reaching these targets will require new transformational tools and innovative ways to deliver them. In addition to improved tests for early and rapid detection of TB and universal drug-susceptibility testing, as well as novel vaccines for improved prevention, better, safer, shorter and more efficacious treatments for all forms of TB are needed. Only a handful of new drugs are currently in phase II or III clinical trials, and a few combination regimens are being tested, mainly for drug-resistant TB. In this article, capitalising on an increasingly rich medicine pipeline and taking advantage of new methodological designs with great potential, the main areas where progress is needed for a transformational improvement of treatment of all forms of TB are described.

Guidance for Studies Evaluating the Accuracy of Rapid Tuberculosis Drug-Susceptibility Tests

The Journal of Infectious Diseases ◽

10.1093/infdis/jiz106 ◽

2019 ◽

Vol 220 (Supplement_3) ◽

pp. S126-S135 ◽

Cited By ~ 1

Author(s):

Sophia B Georghiou ◽

Samuel G Schumacher ◽

Timothy C Rodwell ◽

Rebecca E Colman ◽

Paolo Miotto ◽

...

Keyword(s):

Limit Of Detection ◽

Evaluation Studies ◽

Drug Susceptibility ◽

World Health ◽

Policy Recommendations ◽

Operational Characteristics ◽

Susceptibility Tests ◽

Health Organization ◽

Intended Use

Abstract The development and implementation of rapid molecular diagnostics for tuberculosis (TB) drug-susceptibility testing is critical to inform treatment of patients and to prevent the emergence and spread of resistance. Optimal trial planning for existing tests and those in development will be critical to rapidly gather the evidence necessary to inform World Health Organization review and to support potential policy recommendations. The evidence necessary includes an assessment of the performance for TB and resistance detection as well as an assessment of the operational characteristics of these platforms. The performance assessment should include analytical studies to confirm the limit of detection and assay ability to detect mutations conferring resistance across globally representative strains. The analytical evaluation is typically followed by multisite clinical evaluation studies to confirm diagnostic performance in sites and populations of intended use. This paper summarizes the considerations for the design of these analytical and clinical studies.

Sensititre MYCOTB MIC Plate for Testing Mycobacterium tuberculosis Susceptibility to First- and Second-Line Drugs

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01209-13 ◽

2013 ◽

Vol 58 (1) ◽

pp. 11-18 ◽

Cited By ~ 52

Author(s):

Jongseok Lee ◽

Derek T. Armstrong ◽

Willy Ssengooba ◽

Jeong-ae Park ◽

Yeuni Yu ◽

...

Keyword(s):

Susceptibility Testing ◽

Critical Concentration ◽

Microtiter Plate ◽

Drug Susceptibility ◽

Second Line ◽

Content Type ◽

The Republic ◽

Phenotypic Methods

ABSTRACTForMycobacterium tuberculosis, phenotypic methods for drug susceptibility testing of second-line drugs are poorly standardized and technically challenging. The Sensititre MYCOTB MIC plate (MYCOTB) is a microtiter plate containing lyophilized antibiotics and configured for determination of MICs to first- and second-line antituberculosis drugs. To evaluate the performance of MYCOTB forM. tuberculosisdrug susceptibility testing using the Middlebrook 7H10 agar proportion method (APM) as the comparator, we conducted a two-site study using archivedM. tuberculosisisolates from Uganda and the Republic of Korea. Thawed isolates were subcultured, and dilutions were inoculated into MYCOTB wells and onto 7H10 agar. MYCOTB results were read at days 7, 10, 14, and 21; APM results were read at 21 days. A total of 222 isolates provided results on both platforms. By APM, 106/222 (47.7%) of isolates were resistant to at least isoniazid and rifampin. Agreement between MYCOTB and APM with respect to susceptibility or resistance was ≥92% for 7 of 12 drugs when a strict definition was used and ≥96% for 10 of 12 drugs when agreement was defined by allowing a ± one-well range of dilutions around the APM critical concentration. For ethambutol, agreement was 80% to 81%. For moxifloxacin, agreement was 83% to 85%; incorporating existing DNA sequencing information for discrepant analysis raised agreement to 91% to 96%. For MYCOTB, the median time to plate interpretation was 10 days and interreader agreement was ≥95% for all drugs. MYCOTB provided reliable results forM. tuberculosissusceptibility testing of first- and second-line drugs except ethambutol, and results were available sooner than those determined by APM.

Evaluation of the GenoType MTBDRplusand MTBDRslfor the detection of drug-resistantMycobacterium tuberculosison isolates from Beijing, China

10.1101/311944 ◽

2018 ◽

Author(s):

Jiyong Jian ◽

Xinyu Yang ◽

Jun Yang ◽

Liang Chen

Keyword(s):

Drug Susceptibility ◽

Multidrug Resistant ◽

World Health ◽

Adequate Treatment ◽

Extreme Drug Resistance ◽

Mdr Tb ◽

Health Organization ◽

Beijing China ◽

Timely Treatment

ABSTRACTThe incidence of tuberculosis (TB) and especially multidrug-resistant TB (MDR) and extreme drug resistance (XDR-TB) continue to increase alarmingly worldwide and reliable and fast diagnosis of MDR-TB and XDR-TB is essential for the adequate treatment of patients. So molecular line probe assays (LPAs) for detection of MDR-TB and XDR-TB have been endorsed by the World Health Organization (WHO). We analyzed 96 isolates from Beijing comparing culture-based drug susceptibility testing (DST) to LPAs detecting rifampicin (RFP), isoniazid (INH), and levofloxacin (LFX), amikacin (AM), capreomycin (CMP), ethambutol (EMB) resistance. Compared to phenotypic DST, the GenoType MTBDRplusand MTBDRslshowed a sensitivity of 98.7% and a specificity of 88.9% for detection of RFP resistance, 82.1% and 94.4% for INH, 89.7% and 94.4% for LFX, 60.0% and 98.7% for AM/CPM, 57.5% and 98.2% for EMB, respectively. The sensitivity and specificity of LPAs for MDR-TB and XDR-TB were 80.8%, 100% and 50.0%, 97.6%. Mutations in codon S531L of therpoBgene and S315T1 ofKatGgene were dominated in MDR-TB strains. The most frequently observed mutations were in codon A90V of thegyrAgene, A1401G of therrsgene and M306V of theembBgene, according to the MTBDRslresults. Our study showed that, in combination to phenotypic DST, application of the LPAs might be an efficient and reliable supplementary DST assay for rapid susceptibility screening of MDR-TB and XDR-TB. Using LPAs in high MDR/XDR burden countries allows for appropriate and timely treatment, which will reduce transmission rates, morbidity and improve treatment outcomes in patients.

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

Journal of Clinical Microbiology ◽

10.1128/jcm.01677-19 ◽

2020 ◽

Vol 58 (4) ◽

Cited By ~ 5

Author(s):

Koné Kaniga ◽

Akio Aono ◽

Emanuele Borroni ◽

Daniela Maria Cirillo ◽

Christel Desmaretz ◽

...

Keyword(s):

Susceptibility Testing ◽

Improve Patient Care ◽

Drug Susceptibility ◽

Error Rates ◽

World Health ◽

Health Concern ◽

Content Type ◽

Indicator Tube ◽

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.

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

Journal of Bacteriology ◽

10.1128/jb.00411-20 ◽

2020 ◽

Vol 202 (22) ◽

Author(s):

Paras Jain ◽

Spencer Garing ◽

Deepshikha Verma ◽

Rajagopalan Saranathan ◽

Nicholas Clute-Reinig ◽

...

Keyword(s):

Susceptibility Testing ◽

Limit Of Detection ◽

Reference Method ◽

Drug Susceptibility ◽

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.

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

Wellcome Open Research ◽

10.12688/wellcomeopenres.15603.1 ◽

2019 ◽

Vol 4 ◽

pp. 191 ◽

Cited By ~ 7

Author(s):

Martin Hunt ◽

Phelim Bradley ◽

Simon Grandjean Lapierre ◽

Simon Heys ◽

Mark Thomsit ◽

...

Keyword(s):

Sequence Data ◽

Universal Access ◽

Independent Set ◽

Drug Susceptibility ◽

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.

A generalisable approach to drug susceptibility prediction for M. Tuberculosis using machine learning and whole-genome sequencing

10.1101/2021.09.14.458035 ◽

2021 ◽

Author(s):

◽

Alexander S Lachapelle

Keyword(s):

Machine Learning ◽

Drug Susceptibility ◽

Multidrug Resistant ◽

Learning System ◽

World Health ◽

Whole Genome ◽

Resistant Tuberculosis ◽

Mdr Tb ◽

Rapid and up-to-date drug susceptibility testing is urgently needed to address the threat of multidrug resistant tuberculosis. We developed a composite machine learning system to predict susceptibility from whole-genome sequences for 13 anti-tuberculosis drugs. We trained, validated and externally tested the system, and assessed its performance against a previously validated mutation catalogue, existing molecular assays, and World Health Organization Target Product Profiles. 174,492 phenotypes and 26,328 isolates from 15 countries were studied. The sensitivity of the model was greater than 90% for all drugs except ethionamide, clofazimine and linezolid. Specificity was greater than 95% for all drugs except ethambutol, ethionamide, and bedaquiline, delamanid and clofazimine. The machine learning system was more sensitive than the catalogue and assay (all p<0.01), and correctly predicted a pan-susceptible regimen with 98% accuracy in MDR-TB samples. The proposed system can help guide therapy and be updated automatically as new resistance emerges.

Direct detection of resistance to fluoroquinolones/SLIDs in sputum specimen by GenoType MTBDRsl v.2.0 assay A study from Eastern Uttar Pradesh, India

Annals of Clinical Microbiology and Antimicrobials ◽

10.1186/s12941-021-00463-6 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Kamal Singh ◽

Richa Kumari ◽

Smita Gupta ◽

Rajneesh Tripathi ◽

Anjali Srivastava ◽

...

Keyword(s):

Susceptibility Testing ◽

Direct Detection ◽

Uttar Pradesh ◽

Drug Susceptibility ◽

High Rate ◽

World Health ◽

Second Line ◽

Indicator Tube ◽

Abstract Background According to World Health Organization (WHO), drug-resistant tuberculosis (DR-TB) is a major contributor to antimicrobial resistance globally and continues to be a public health threat. Annually, about half a million people fall ill with DR-TB globally. The gradual increase in resistance to fluoroquinolones (FQs) and second-line injectable drugs (SLIDs), poses a serious threat to effective TB control and adequate patient management. Therefore, WHO suggests the use of GenoType MTBDRsl v.2.0 assay for detection of multiple mutations associated with FQs and SLIDs. Hence, the study was conducted to determine the prevalence of resistance to FQs and SLIDs by comparing direct GenoType MTBDRsl v.2.0 assay with phenotypic drug susceptibility testing (DST). Methods The study was conducted on 1320 smear positive sputum samples from a total of 2536 RR-TB, confirmed by GeneXpert MTB/RIF. The smear positive specimens were decontaminated, and DNA extraction was performed. Furthermore, the extracted DNA was used for GenoType MTBDRsl v.2.0 assay. While 20% of the decontaminated specimens were inoculated in Mycobacterium growth indicator tube (MGIT) for drug susceptibility testing (DST). Results Out of 1320 smear positive sputum samples, 1178 were identified as Mycobacterium tuberculosis complex (MTBC) and remaining were negative by GenoType MTBDRsl v.2.0 assay. Of the 1178 MTBC positive, 26.6% were sensitive to both FQs and SLIDs, whereas 57.3% were only FQs resistant and 15.9% were resistant to both FQs and SLIDs. Further DST of 225 isolates by liquid culture showed that 17% were sensitive to both FQs and SLIDs, 61.3% were only FQs resistant and 21.3% were resistant to both. The specificity for FQs and SLIDs was 92.31% and 100% whereas sensitivity was 100% respectively by GenoType MTBDRsl v.2.0 assay in direct sputum samples. Conclusions Our study clearly suggests that GenoType MTBDRsl v.2.0 assay is a reliable test for the rapid detection of resistance to second-line drugs after confirmation by GeneXpert MTB/RIF assay for RR-TB. Though, high rate FQ (ofloxacin) resistance was seen in our setting, moxifloxacin could be used as treatment option owing to very low resistance.

DeepAMR for predicting co-occurrent resistance of Mycobacterium tuberculosis

Bioinformatics ◽

10.1093/bioinformatics/btz067 ◽

2019 ◽

Vol 35 (18) ◽

pp. 3240-3249 ◽

Cited By ~ 3

Author(s):

Yang Yang ◽

Timothy M Walker ◽

A Sarah Walker ◽

Daniel J Wilson ◽

Timothy E A Peto ◽

...

Keyword(s):

Drug Susceptibility ◽

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.