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Journal of Clinical Medicine Feb 2020Accurate 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...
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 (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.
PubMed: 32046149
DOI: 10.3390/jcm9020465 -
BMC Infectious Diseases Jan 2020Macrophages play a key role in the infection process, and alternatively activated macrophages (M2 polarization) play important roles in persistent infection via the...
BACKGROUND
Macrophages play a key role in the infection process, and alternatively activated macrophages (M2 polarization) play important roles in persistent infection via the immune escape of pathogens. This suggests that immune escape of pathogens from host immunity is an important factor to consider in treatment failure and multidrug-resistant tuberculosis (MDR-TB)/extensively drug-resistant tuberculosis (XDR-TB). In this study, we investigated the association between macrophage polarization and MDR-TB/XDR-TB and the association between macrophage polarization and the anti-TB drugs used.
METHODS
iNOS and arginase-1, a surface marker of polarized macrophages, were quantified by immunohistochemical staining and imaging analysis of lung tissues of patients who underwent surgical treatment for pulmonary TB. Drug susceptibility/resistance and the type and timing of anti-tuberculosis drugs used were investigated.
RESULTS
The M2-like polarization rate and the ratio of the M2-like polarization rate to the M1-like polarization rate were significantly higher in the MDR-TB/XDR-TB group than in the DS-TB group. The association between a high M2-like polarization rate and MDR-TB/XDR-TB was more pronounced in patients with a low M1-like polarization rate. Younger age and a higher M2-like polarization rate were independent associated factors for MDR-TB/XDR-TB. The M2-like polarization rate was significantly higher in patients who received anti-TB drugs containing pyrazinamide continuously for 4 or 6 weeks than in those who received anti-TB drugs not containing pyrazinamide.
CONCLUSIONS
The M2-like polarization of macrophages is associated with MDR-TB/XDR-TB and anti-TB drug regimens including pyrazinamide or a combination of pyrazinamide, prothionamide and cycloserine.
Topics: Adult; Antitubercular Agents; Cycloserine; Extensively Drug-Resistant Tuberculosis; Female; Humans; Lung; Macrophage Activation; Macrophages; Male; Middle Aged; Mycobacterium tuberculosis; Prothionamide; Pyrazinamide; Treatment Failure; Tuberculosis, Multidrug-Resistant; Tuberculosis, Pulmonary
PubMed: 31996142
DOI: 10.1186/s12879-020-4802-9 -
The European Respiratory Journal Mar 2020We sought to compare the effectiveness of two World Health Organization (WHO)-recommended regimens for the treatment of rifampin- or multidrug-resistant (RR/MDR)...
We sought to compare the effectiveness of two World Health Organization (WHO)-recommended regimens for the treatment of rifampin- or multidrug-resistant (RR/MDR) tuberculosis (TB): a standardised regimen of 9-12 months (the "shorter regimen") and individualised regimens of ≥20 months ("longer regimens").We collected individual patient data from observational studies identified through systematic reviews and a public call for data. We included patients meeting WHO eligibility criteria for the shorter regimen: not previously treated with second-line drugs, and with fluoroquinolone- and second-line injectable agent-susceptible RR/MDR-TB. We used propensity score matched, mixed effects meta-regression to calculate adjusted odds ratios and adjusted risk differences (aRDs) for failure or relapse, death within 12 months of treatment initiation and loss to follow-up.We included 2625 out of 3378 (77.7%) individuals from nine studies of shorter regimens and 2717 out of 13 104 (20.7%) individuals from 53 studies of longer regimens. Treatment success was higher with the shorter regimen than with longer regimens (pooled proportions 80.0% 75.3%), due to less loss to follow-up with the former (aRD -0.15, 95% CI -0.17- -0.12). The risk difference for failure or relapse was slightly higher with the shorter regimen overall (aRD 0.02, 95% CI 0-0.05) and greater in magnitude with baseline resistance to pyrazinamide (aRD 0.12, 95% CI 0.07-0.16), prothionamide/ethionamide (aRD 0.07, 95% CI -0.01-0.16) or ethambutol (aRD 0.09, 95% CI 0.04-0.13).In patients meeting WHO criteria for its use, the standardised shorter regimen was associated with substantially less loss to follow-up during treatment compared with individualised longer regimens and with more failure or relapse in the presence of resistance to component medications. Our findings support the need to improve access to reliable drug susceptibility testing.
Topics: Antitubercular Agents; Humans; Microbial Sensitivity Tests; Mycobacterium tuberculosis; Rifampin; Treatment Outcome; Tuberculosis, Multidrug-Resistant
PubMed: 31862767
DOI: 10.1183/13993003.01467-2019 -
Surveillance of adverse events in the treatment of drug-resistant tuberculosis: first global report.The European Respiratory Journal Dec 2019The World Health Organization (WHO) recommends that countries implement pharmacovigilance and collect information on active drug safety monitoring (aDSM) and management...
The World Health Organization (WHO) recommends that countries implement pharmacovigilance and collect information on active drug safety monitoring (aDSM) and management of adverse events.The aim of this prospective study was to evaluate the frequency and severity of adverse events to anti-tuberculosis (TB) drugs in a cohort of consecutive TB patients treated with new ( bedaquiline, delamanid) and repurposed ( clofazimine, linezolid) drugs, based on the WHO aDSM project. Adverse events were collected prospectively after attribution to a specific drug together with demographic, bacteriological, radiological and clinical information at diagnosis and during therapy. This interim analysis included patients who completed or were still on treatment at time of data collection.Globally, 45 centres from 26 countries/regions reported 658 patients (68.7% male, 4.4% HIV co-infected) treated as follows: 87.7% with bedaquiline, 18.4% with delamanid (6.1% with both), 81.5% with linezolid and 32.4% with clofazimine. Overall, 504 adverse event episodes were reported: 447 (88.7%) were classified as minor (grade 1-2) and 57 (11.3%) as serious (grade 3-5). The majority of the 57 serious adverse events reported by 55 patients (51 out of 57, 89.5%) ultimately resolved. Among patients reporting serious adverse events, some drugs held responsible were discontinued: bedaquiline in 0.35% (two out of 577), delamanid in 0.8% (one out of 121), linezolid in 1.9% (10 out of 536) and clofazimine in 1.4% (three out of 213) of patients. Serious adverse events were reported in 6.9% (nine out of 131) of patients treated with amikacin, 0.4% (one out of 221) with ethionamide/prothionamide, 2.8% (15 out of 536) with linezolid and 1.8% (eight out of 498) with cycloserine/terizidone.The aDSM study provided valuable information, but implementation needs scaling-up to support patient-centred care.
Topics: Adult; Aged; Antitubercular Agents; Drug-Related Side Effects and Adverse Reactions; Female; Humans; Male; Middle Aged; Pharmacovigilance; Prospective Studies; Tuberculosis, Multidrug-Resistant
PubMed: 31601711
DOI: 10.1183/13993003.01522-2019 -
Frontiers in Microbiology 2019Whole-genome sequencing (WGS) is a viable and financially feasible tool for timely and comprehensive diagnosis of drug resistance in developed countries. With the...
Evaluation of Whole-Genome Sequence Method to Diagnose Resistance of 13 Anti-tuberculosis Drugs and Characterize Resistance Genes in Clinical Multi-Drug Resistance Isolates From China.
Whole-genome sequencing (WGS) is a viable and financially feasible tool for timely and comprehensive diagnosis of drug resistance in developed countries. With the increase in the incidence of multidrug-resistant tuberculosis (MDR-TB), second-line anti-TB drugs are gaining importance. However, genetic resistance to second-line anti-TB drugs based on WGS has not been fully studied. We randomly selected 100 MDR-TB and 10 non-MDR-TB isolates from a hospital in Zhejiang Province, China. Drug susceptibility tests against 13 anti-TB drugs were performed, and 34 drug resistance-related genes were analyzed using WGS in all isolates. For each drug, the accuracy, sensitivity, specificity, and positive and negative predictive values of WGS were compared with those of the conventional drug susceptibility test. The overall sensitivity and specificity for WGS were respectively, 99.0 and 100.0% for isoniazid (INH), 99.0 and 100.0% for rifampicin (RIF), 94.8 and 65.3% for ethambutol (EMB), 86.2 and 84.4% for pyrazinamide (PZA), 95.6 and 95.6% for levofloxacin (LFX), 89.5 and 65.3% for moxifloxacin (MFX), 91.3 and 95.1% for streptomycin (SM), 90.9 and 99.0% for kanamycin, 90.9 and 100.0% for amikacin, 88.9 and 98.0% for capreomycin, 87.0 and 85.1% for prothionamide (PTO), 85.7 and 99.0% for para-aminosalicylic acid (PAS), and 66.7 and 95.9% for clofazimine (CLO). WGS is a promising approach to predict resistance to INH, RIF, PZA, LFX, SM, second-line injectable drugs (SLIDs), and PTO with satisfactory accuracy, sensitivity, and specificity of over 85.0%. The specificity of WGS in diagnosing resistance to EMB, and high-level resistance to MFX (2.0 mg/L) needs to be improved.
PubMed: 31417530
DOI: 10.3389/fmicb.2019.01741 -
MBio Apr 2019In , recent genome-wide association studies have identified a novel constellation of mutations that are correlated with high-level drug resistances. Interpreting the...
In , recent genome-wide association studies have identified a novel constellation of mutations that are correlated with high-level drug resistances. Interpreting the functional importance of the new resistance-associated mutations has been complicated, however, by a lack of experimental validation and a poor understanding of the epistatic factors influencing these correlations, including strain background and programmatic variation in treatment regimens. Here we perform a genome-wide association analysis in a panel of strains from China to identify variants correlated with resistance to the second-line prodrug ethionamide (ETH). Mutations in a bacterial monooxygenase, Rv0565c, are significantly associated with ETH resistance. We demonstrate that Rv0565c is a novel activator of ETH, independent of the two known activators, EthA and MymA. Clinically prevalent mutations abrogate Rv0565c function, and deletion of Rv0565c confers a consistent fitness benefit on in the presence of partially inhibitory doses of ETH. Interestingly, Rv0565c activity affects susceptibility to prothionamide (PTH), the ETH analog used in China, to a greater degree. Further, clinical isolates vary in their susceptibility to both ETH and PTH, to an extent that correlates with the total expression of ETH/PTH activators (EthA, MymA, and Rv0565c). These results suggest that clinical strains considered susceptible to ETH/PTH are not equally fit during treatment due to both Rv0565c mutations and more global variation in the expression of the prodrug activators. Phenotypic antibiotic susceptibility testing in is slow and cumbersome. Rapid molecular diagnostics promise to help guide therapy, but such assays rely on complete knowledge of the molecular determinants of altered antibiotic susceptibility. Recent genomic studies of antibiotic-resistant have identified several candidate loci beyond those already known to contribute to antibiotic resistance; however, efforts to provide experimental validation have lagged. Our study identifies a gene (Rv0565c) that is associated with resistance to the second-line antibiotic ethionamide at a population level. We then use bacterial genetics to show that the variants found in clinical strains of improve bacterial survival after ethionamide exposure.
Topics: Antitubercular Agents; China; Drug Resistance, Bacterial; Ethionamide; Genome-Wide Association Study; Microbial Sensitivity Tests; Mutation; Mycobacterium tuberculosis; Oxidoreductases; Prothionamide
PubMed: 31015328
DOI: 10.1128/mBio.00616-19 -
Journal of Pharmaceutical and... May 2019Tuberculosis is one of the top concerns in the world and acutely threatens human health. A new potent candidate regimen containing pyrazinamide (PZA), ethambutol (EMB),...
Simultaneous determination of the potent anti-tuberculosis regimen-Pyrazinamide, ethambutol, protionamide, clofazimine in beagle dog plasma using LC-MS/MS method coupled with 96-well format plate.
Tuberculosis is one of the top concerns in the world and acutely threatens human health. A new potent candidate regimen containing pyrazinamide (PZA), ethambutol (EMB), protionamide (PTO) and clofazimine (CFZ) was proposed by Parabolic Response Surface/Feedback System Control (FSC/PRS) system and showed excellent outcomes in vitro and vivo studies. Here, a convenient liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneously determination of four compounds in beagle dog plasma. The plasma samples, 50 μL for each, were pretreated by methanol on 96-well format plates and a further dilution step was designed to reduce predictable matrix effect and lessen the burden of subsequent analysis. The chromatographic separation was achieved on an Agilent SB-Aq column (4.6 mm × 150 mm, 5 μm) at 30 °C by a gradient elution within 6 min. The mobile phase was a mixture of 0.2% formic acid-5 mM ammonium acetate aqueous solution (phase A) and 0.2% formic acid methanol (phase B) with a total flow rate of 1 mL/min. The 30% of post-column eluant was injected into mass spectrometer, equipped with electrospray ionization (ESI) source under positive mode and multiple-reaction monitoring (MRM). This quantification method was proved to be satisfied in selectivity, accuracy, precision, linearity (r > 0.998), recovery, matrix effect and stability. Under the specialized conditions, the calibration curves ranged from 20 to 5000 ng/mL for PZA, 1 to 500 ng/mL for EMB, 1 to 500 ng/mL for PTO, and 1 to 200 ng/mL for CFZ. The quantitative accuracy was further assessed under different degrees of hemolyses in detail. This method was proved to be robust and efficient, and successfully applied to the pharmacokinetic study of the new regimen in Beagle dogs.
Topics: Animals; Antitubercular Agents; Calibration; Chromatography, Liquid; Clofazimine; Dogs; Ethambutol; Prothionamide; Pyrazinamide; Reproducibility of Results; Tandem Mass Spectrometry
PubMed: 30784889
DOI: 10.1016/j.jpba.2019.02.006 -
BMC Bioinformatics Feb 2019It is possible to predict whether a tuberculosis (TB) patient will fail to respond to specific antibiotics by sequencing the genome of the infecting Mycobacterium...
BACKGROUND
It is possible to predict whether a tuberculosis (TB) patient will fail to respond to specific antibiotics by sequencing the genome of the infecting Mycobacterium tuberculosis (Mtb) and observing whether the pathogen carries specific mutations at drug-resistance sites. This advancement has led to the collation of TB databases such as PATRIC and ReSeqTB that possess both whole genome sequences and drug resistance phenotypes of infecting Mtb isolates. Bioinformatics tools have also been developed to predict drug resistance from whole genome sequencing (WGS) data. Here, we evaluate the performance of four popular tools (TBProfiler, MyKrobe, KvarQ, PhyResSE) with 6746 isolates compiled from publicly available databases, and subsequently identify highly probable phenotyping errors in the databases by genetically predicting the drug phenotypes using all four software.
RESULTS
Our results show that these bioinformatics tools generally perform well in predicting the resistance status for two key first-line agents (isoniazid, rifampicin), but the accuracy is lower for second-line injectables and fluoroquinolones. The error rates in the databases are also non-trivial, reaching as high as 31.1% for prothionamide, and that phenotypes from ReSeqTB are more susceptible to errors.
CONCLUSIONS
The good performance of the automated software for drug resistance prediction from TB WGS data shown in this study further substantiates the usefulness and promise of utilising genetic data to accurately profile TB drug resistance, thereby reducing misdiagnoses arising from error-prone culture-based drug susceptibility testing.
Topics: Algorithms; Antitubercular Agents; Benchmarking; Calibration; Databases, Genetic; Drug Resistance, Bacterial; Genomics; Humans; Microbial Sensitivity Tests; Mycobacterium tuberculosis; Sensitivity and Specificity; Software; Tuberculosis
PubMed: 30736750
DOI: 10.1186/s12859-019-2658-z -
Clinical Microbiology and Infection :... Aug 2019Prothionamide, a structural analogue of isoniazid, is used mainly for treating multidrug-resistant tuberculosis (MDR-TB). Both drugs have a common target InhA, so...
OBJECTIVES
Prothionamide, a structural analogue of isoniazid, is used mainly for treating multidrug-resistant tuberculosis (MDR-TB). Both drugs have a common target InhA, so prothionamide can be ineffective against isoniazid-resistant (INH) Mycobacterium tuberculosis. We aimed to investigate the prevalence of mutations in katG, ethA, ndh, ethR, mshA, inhA and/or its promoter associated with independent resistance and cross-resistance to INH and/or prothionamide-resistant (PTO) M. tuberculosis isolates.
METHODS
We sequenced the above genes in 206 M. tuberculosis isolates with susceptibility testing against ten drugs.
RESULTS
Of the 173 INH PTO isolates, 170 (98.3%) harboured mutations in katG, 111 (64.2%) in ethA, 58 (33.5%) in inhA or its promoter, 5 (2.9%) in ndh, 3 (1.7 %) in ethR and 2 (1.2%) in mshA. Among the 18 INH PTO isolates, mutations in katG were found in all of them; one had a mutation in the inhA promoter and another in ndh. Of the five INH PTO isolates, four showed mutations in ethA and two in the inhA promoter. Notably, 55 novel non-synonymous mutations were found in them and 20.2% of the PTOM. tuberculosis isolates harboured no known mutations.
CONCLUSIONS
This is the first report to investigate cross-resistance between INH and/or PTO isolates. Among INH (94.4% MDR-TB) M. tuberculosis isolates, the high diversity of mutations for independent resistance and cross-resistance with prothionamide highlight the importance of both phenotypic susceptibility and genotypic diagnosis when using it to treat patients with INH-TB. The high proportion (one-fifth) of PTOM. tuberculosis isolates showed no known mutation related to PTO genes, so uncovered resistance mechanism(s) of prothionamide exist.
Topics: Antitubercular Agents; Bacterial Proteins; Drug Resistance, Multiple, Bacterial; Genotype; Humans; Isoniazid; Microbial Sensitivity Tests; Mutation; Mycobacterium tuberculosis; Promoter Regions, Genetic; Prothionamide; Sequence Analysis, DNA; Tuberculosis, Multidrug-Resistant
PubMed: 30583053
DOI: 10.1016/j.cmi.2018.12.008 -
Tuberculosis and Respiratory Diseases Apr 2019The purpose of this study was to analyze the relationship between the gene mutation patterns by the GenoType MTBDR (MTBDR) assay and the phenotypic drug susceptibility...
BACKGROUND
The purpose of this study was to analyze the relationship between the gene mutation patterns by the GenoType MTBDR (MTBDR) assay and the phenotypic drug susceptibility test (pDST) results of isoniazid (INH) and prothionamide (Pto).
METHODS
A total of 206 patients whose MTBDR assay results revealed or mutations were enrolled in the study. The pDST results were compared to mutation patterns on the MTBDR assay.
RESULTS
The and mutations were identified in 68.0% and 35.0% of patients, respectively. Among the 134 isolated mutations, three (2.2%), 127 (94.8%) and 11 (8.2%) were phenotypically resistant to low-level INH, high-level INH, and Pto, respectively. Among the 66 isolated mutations, 34 (51.5%), 18 (27.3%) and 21 (31.8%) were phenotypically resistant to low-level INH, high-level INH, and Pto, respectively. Of the 34 phenotypic Pto resistant isolates, 21 (61.8%), 11 (32.4%), and two (5.9%) had , , and both gene mutations.
CONCLUSION
It is noted that Pto may still be selected as one of the appropriate multidrug-resistant tuberculosis regimen, although mutation is detected by the MTBDR assay until pDST confirms a Pto resistance. The reporting of detailed mutation patterns of the MTBDR assay may be important for clinical practice, rather than simply presenting resistance or susceptibility test results.
PubMed: 30302956
DOI: 10.4046/trd.2018.0027