Estimation of country-specific tuberculosis resistance antibiograms using pathogen genomics and machine learning.

BACKGROUND: Global tuberculosis (TB) drug resistance (DR) surveillance focuses on rifampicin. We examined the potential of public and surveillance Mycobacterium tuberculosis (Mtb) whole-genome sequencing (WGS) data, to generate expanded country-level resistance prevalence estimates (antibiograms) using in silico resistance prediction. METHODS: We curated and quality-controlled Mtb WGS data. We used a validated random forest model to predict phenotypic resistance to 12 drugs and bias-corrected for model performance, outbreak sampling and rifampicin resistance oversampling. Validation leveraged a national DR survey conducted in South Africa. RESULTS: Mtb isolates from 29 countries (n=19 149) met sequence quality criteria. Global marginal genotypic resistance among mono-resistant TB estimates overlapped with the South African DR survey, except for isoniazid, ethionamide and second-line injectables, which were underestimated (n=3134). Among multidrug resistant (MDR) TB (n=268), estimates overlapped for the fluoroquinolones but overestimated other drugs. Globally pooled mono-resistance to isoniazid was 10.9% (95% CI: 10.2-11.7%, n=14 012). Mono-levofloxacin resistance rates were highest in South Asia (Pakistan 3.4% (0.1-11%), n=111 and India 2.8% (0.08-9.4%), n=114). Given the recent interest in drugs enhancing ethionamide activity and their expected activity against isolates with resistance discordance between isoniazid and ethionamide, we measured this rate and found it to be high at 74.4% (IQR: 64.5-79.7%) of isoniazid-resistant isolates predicted to be ethionamide susceptible. The global susceptibility rate to pyrazinamide and levofloxacin among MDR was 15.1% (95% CI: 10.2-19.9%, n=3964). CONCLUSIONS: This is the first attempt at global Mtb antibiogram estimation. DR prevalence in Mtb can be reliably estimated using public WGS and phenotypic resistance prediction for key antibiotics, but public WGS data demonstrates oversampling of isolates with higher resistance levels than MDR. Nevertheless, our results raise concerns about the empiric use of short-course fluoroquinolone regimens for drug-susceptible TB in South Asia and indicate underutilisation of ethionamide in MDR treatment.

Discovery of dual-active ethionamide boosters inhibiting the Mycobacterium tuberculosis ESX-1 secretion system.

Drug-resistant Mycobacterium tuberculosis (Mtb) remains a major public health concern requiring complementary approaches to standard anti-tuberculous regimens. Anti-virulence molecules or compounds that enhance the activity of antimicrobial prodrugs are promising alternatives to conventional antibiotics. Exploiting host cell-based drug discovery, we identified an oxadiazole compound (S3) that blocks the ESX-1 secretion system, a major virulence factor of Mtb. S3-treated mycobacteria showed impaired intracellular growth and a reduced ability to lyse macrophages. RNA sequencing experiments of drug-exposed bacteria revealed strong upregulation of a distinct set of genes including ethA, encoding a monooxygenase activating the anti-tuberculous prodrug ethionamide. Accordingly, we found a strong ethionamide boosting effect in S3-treated Mtb. Extensive structure-activity relationship experiments revealed that anti-virulence and ethionamide-boosting activity can be uncoupled by chemical modification of the primary hit molecule. To conclude, this series of dual-active oxadiazole compounds targets Mtb via two distinct mechanisms of action.

Loss-of-function mutations in ndh do not confer delamanid, ethionamide, isoniazid, or pretomanid resistance in Mycobacterium tuberculosis.

Results from clinical strains and knockouts of the H37Rv and CDC1551 laboratory strains demonstrated that ndh (Rv1854c) is not a resistance-conferring gene for isoniazid, ethionamide, delamanid, or pretomanid in Mycobacterium tuberculosis. This difference in the susceptibility to NAD-adduct-forming drugs compared with other mycobacteria may be driven by differences in the absolute intrabacterial NADH concentration.

Genotypic and phenotypic comparison of drug resistance profiles of clinical multidrug-resistant Mycobacterium tuberculosis isolates using whole genome sequencing in Latvia.

BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB) remains a major public health problem in many high tuberculosis (TB) burden countries. Phenotypic drug susceptibility testing (DST) take several weeks or months to result, but line probe assays and Xpert/Rif Ultra assay detect a limited number of resistance conferring gene mutations. Whole genome sequencing (WGS) is an advanced molecular testing method which theoretically can predict the resistance of M. tuberculosis (Mtb) isolates to all anti-TB agents through a single analysis. METHODS: Here, we aimed to identify the level of concordance between the phenotypic and WGS-based genotypic drug susceptibility (DS) patterns of MDR-TB isolates. Overall, data for 12 anti-TB medications were analyzed. RESULTS: In total, 63 MDR-TB Mtb isolates were included in the analysis, representing 27.4% of the total number of MDR-TB cases in Latvia in 2012-2014. Among them, five different sublineages were detected, and 2.2.1 (Beijing group) and 4.3.3 (Latin American-Mediterranean group) were the most abundant. There were 100% agreement between phenotypic and genotypic DS pattern for isoniazid, rifampicin, and linezolid. High concordance rate (> 90%) between phenotypic and genotypic DST results was detected for ofloxacin (93.7%), pyrazinamide (93.7%) and streptomycin (95.4%). Phenotypic and genotypic DS patterns were poorly correlated for ethionamide (agreement 56.4%), ethambutol (85.7%), amikacin (82.5%), capreomycin (81.0%), kanamycin (85.4%), and moxifloxacin (77.8%). For capreomycin, resistance conferring mutations were not identified in several phenotypically resistant isolates, and, in contrary, for ethionamide, ethambutol, amikacin, kanamycin, and moxifloxacin the resistance-related mutations were identified in several phenotypically sensitive isolates. CONCLUSIONS: WGS is a valuable tool for rapid genotypic DST for all anti-TB agents. For isoniazid and rifampicin phenotypic DST potentially can be replaced by genotypic DST based on 100% agreement between the tests. However, discrepant results for other anti-TB agents limit their prescription based solely on WGS data. For clinical decision, at the current level of knowledge, there is a need for combination of genotypic DST with modern, validated phenotypic DST methodologies for those medications which did not showed 100% agreement between the methods.

Metabolomic analysis of Mycobacterium tuberculosis reveals metabolic profiles for identification of drug-resistant tuberculosis.

The detection of pre-extensively (pre-XDR) and extensively drug-resistant tuberculosis (XDR-TB) is challenging. Drug-susceptibility tests for some anti-TB drugs, especially ethambutol (ETH) and ethionamide (ETO), are problematic due to overlapping thresholds to differentiate between susceptible and resistant phenotypes. We aimed to identify possible metabolomic markers to detect Mycobacterium tuberculosis (Mtb) strains causing pre-XDR and XDR-TB. The metabolic patterns of ETH- and ETO-resistant Mtb isolates were also investigated. Metabolomics of 150 Mtb isolates (54 pre-XDR, 63 XDR-TB and 33 pan-susceptible; pan-S) were investigated. Metabolomics of ETH and ETO phenotypically resistant subgroups were analyzed using UHPLC-ESI-QTOF-MS/MS. Orthogonal partial least-squares discriminant analysis revealed distinct separation in all pairwise comparisons among groups. Two metabolites (meso-hydroxyheme and itaconic anhydride) were able to differentiate the pre-XDR and XDR-TB groups from the pan-S group with 100% sensitivity and 100% specificity. In comparisons of the ETH and ETO phenotypically resistant subsets, sets of increased (ETH = 15, ETO = 7) and decreased (ETH = 1, ETO = 6) metabolites specific for the resistance phenotype of each drug were found. We demonstrated the potential for metabolomics of Mtb to differentiate among types of DR-TB as well as between isolates that were phenotypically resistant to ETO and ETH. Thus, metabolomics might be further applied for DR-TB diagnosis and patient management.

MarR-Dependent Transcriptional Regulation of mmpSL5 Induces Ethionamide Resistance in Mycobacterium abscessus.

Mycobacterium abscessus (Mabs) is an emerging nontuberculosis mycobacterial (NTM) pathogen responsible for a wide variety of respiratory and cutaneous infections that are difficult to treat with standard antibacterial therapy. Mabs has a high degree of both innate and acquired antibiotic resistance to most clinically relevant drugs, including standard anti-mycobacterial agents. Ethionamide (ETH), an inhibitor of mycolic acid biosynthesis, is currently utilized as a second-line agent for treating multidrug-resistant tuberculosis infections. Here, we show that ETH displays activity against clinical strains of Mabs in vitro at concentrations that are >100× lower than other mycolic acid targeting drugs. Using transposon mutagenesis followed by transposon sequencing (Tn-Seq) and whole-genome sequencing of spontaneous ETH-resistant mutants, we identified MAB_2648c as a genetic determinant of ETH sensitivity in Mabs. MAB_2648c encodes a MarR family transcriptional regulator of the TetR class of regulators. We show that MAB_2648c represses expression of MAB_2649 (mmpS5) and MAB_2650 (mmpL5). Further, we show that derepression of these genes in MAB_2648c mutants confers resistance to ETH, but not other antibiotics. To identify determinants of resistance that may be shared across antibiotics with distinct mechanisms of action, we also performed Tn-Seq during treatment with amikacin and clarithromycin, drugs currently used clinically to treat Mabs. We found very little overlap in genes that modulate the sensitivity of Mabs to all three antibiotics, suggesting a high degree of specificity for resistance mechanisms in this emerging pathogen.

Omics analysis of Mycobacterium tuberculosis isolates uncovers Rv3094c, an ethionamide metabolism-associated gene.

Global control of the tuberculosis epidemic is threatened by increasing prevalence of drug resistant M. tuberculosis isolates. Many genome-wide studies focus on SNP-associated drug resistance mechanisms, but drug resistance in 5-30% of M. tuberculosis isolates (varying with antibiotic) appears unrelated to reported SNPs, and alternative drug resistance mechanisms involving variation in gene/protein expression are not well-studied. Here, using an omics approach, we identify 388 genes with lineage-related differential expression and 68 candidate drug resistance-associated gene pairs/clusters in 11 M. tuberculosis isolates (variable lineage/drug resistance profiles). Structural, mutagenesis, biochemical and bioinformatic studies on Rv3094c from the Rv3093c-Rv3095 gene cluster, a gene cluster selected for further investigation as it contains a putative monooxygenase/repressor pair and is associated with ethionamide resistance, provide insights on its involvement in ethionamide sulfoxidation, the initial step in its activation. Analysis of the structure of Rv3094c and its complex with ethionamide and flavin mononucleotide, to the best of our knowledge the first structures of an enzyme involved in ethionamide activation, identify key residues in the flavin mononucleotide and ethionamide binding pockets of Rv3094c, and F221, a gate between flavin mononucleotide and ethionamide allowing their interaction to complete the sulfoxidation reaction. Our work broadens understanding of both lineage- and drug resistance-associated gene/protein expression perturbations and identifies another player in mycobacterial ethionamide metabolism.

Variation in missed doses and reasons for discontinuation of anti-tuberculosis drugs during hospital treatment for drug-resistant tuberculosis in South Africa.

BACKGROUND: Updated World Health Organization (WHO) treatment guidelines prioritize all-oral drug-resistant tuberculosis (DR-TB) regimens. Several poorly tolerated drugs, such as amikacin and para-aminosalicylic acid (PAS), remain treatment options for DR-TB in WHO-recommended longer regimens as Group C drugs. Incomplete treatment with anti-TB drugs increases the risk of treatment failure, relapse, and death. We determined whether missed doses of individual anti-TB drugs, and reasons for their discontinuation, varied in closely monitored hospital settings prior to the 2020 WHO DR-TB treatment guideline updates. METHODS: We collected retrospective data on adult patients with microbiologically confirmed DR-TB between 2008 and 2015 who were selected for a study of acquired drug resistance in the Western Cape Province of South Africa. Medical records through mid-2017 were reviewed. Patients received directly observed treatment during hospitalization at specialized DR-TB hospitals. Incomplete treatment with individual anti-TB drugs, defined as the failure to take medication as prescribed, regardless of reason, was determined by comparing percent missed doses, stratified by HIV status and DR-TB regimen. We applied a generalized mixed effects model. RESULTS: Among 242 patients, 131 (54%) were male, 97 (40%) were living with HIV, 175 (72%) received second-line treatment prior to first hospitalization, and 191 (79%) died during the study period. At initial hospitalization, 134 (55%) patients had Mycobacterium tuberculosis with resistance to rifampicin and isoniazid (multidrug-resistant TB [MDR-TB]) without resistance to ofloxacin or amikacin, and 102 (42%) had resistance to ofloxacin and/or amikacin. Most patients (129 [53%]) had multiple hospitalizations and DST changes occurred in 146 (60%) by the end of their last hospital discharge. Incomplete treatment was significantly higher for amikacin (18%), capreomycin (18%), PAS (17%) and kanamycin (16%) than other DR-TB drugs (P<0.001), including ethionamide (8%), moxifloxacin (7%), terizidone (7%), ethambutol (7%), and pyrazinamide (6%). Among the most frequently prescribed drugs, second-line injectables had the highest rates of discontinuation for adverse events (range 0.56-1.02 events per year follow-up), while amikacin, PAS and ethionamide had the highest rates of discontinuation for patient refusal (range 0.51-0.68 events per year follow-up). Missed doses did not differ according to HIV status or anti-TB drug combinations. CONCLUSION: We found that incomplete treatment for second-line injectables and PAS during hospitalization was higher than for other anti-TB drugs. To maximize treatment success, interventions to improve person-centered care and mitigate adverse events may be necessary in cases when PAS or amikacin (2020 WHO recommended Group C drugs) are needed.

"Upcycling" known molecules and targets for drug-resistant TB.

Despite reinvigorated efforts in Tuberculosis (TB) drug discovery over the past 20 years, relatively few new drugs and candidates have emerged with clear utility against drug resistant TB. Over the same period, significant technological advances and learnings around target value have taken place. This has offered opportunities to re-assess the potential for optimization of previously discovered chemical matter against Mycobacterium tuberculosis (M.tb) and for reconsideration of clinically validated targets encumbered by drug resistance. A re-assessment of discarded compounds and programs from the "golden age of antibiotics" has yielded new scaffolds and targets against TB and uncovered classes, for example beta-lactams, with previously unappreciated utility for TB. Leveraging validated classes and targets has also met with success: booster technologies and efforts to thwart efflux have improved the potential of ethionamide and spectinomycin classes. Multiple programs to rescue high value targets while avoiding cross-resistance are making progress. These attempts to make the most of known classes, drugs and targets complement efforts to discover new chemical matter against novel targets, enhancing the chances of success of discovering effective novel regimens against drug-resistant TB.

Identification of potential biomarkers and pathogenesis in neutrophil-predominant severe asthma: A comprehensive bioinformatics analysis.

BACKGROUND: Airway neutrophilia has been associated with asthma severity and asthma exacerbations. This study attempted to identify biomarkers, pathogenesis, and therapeutic molecular targets for severe asthma in neutrophils using bioinformatics analysis. METHODS: Fifteen healthy controls and 3 patients with neutrophilic severe asthma were screened from the Gene Expression Omnibus (GEO) database. Based on the analysis of differentially expressed genes (DEGs), functional and pathway enrichment analyses, gene set enrichment analysis, protein-protein interaction network construction, and analysis were performed. Moreover, small-molecule drug candidates have also been identified. RESULTS: Three hundred and three upregulated and 59 downregulated genes were identified. Gene ontology function enrichment analyses were primarily related to inflammatory response, immune response, leukocyte migration, neutrophil chemotaxis, mitogen-activated protein kinase cascade, Jun N-terminal kinase cascade, I-kappaB kinase/nuclear factor-κB, and MyD88-dependent toll-like receptor signaling pathway. Pathway enrichment analyses and gene set enrichment analysis were mainly involved in cytokine-cytokine receptor interaction, the TNF signaling pathway, leukocyte transendothelial migration, and the NOD-like receptor signaling pathway. Furthermore, 1 important module and 10 hub genes (CXCL8, TLR2, CXCL1, ICAM1, CXCR4, FPR2, SELL, PTEN, TREM1, and LEP) were identified in the protein-protein interaction network. Moreover, indoprofen, mimosine, STOCK1N-35874, trapidil, iloprost, aminoglutethimide, ajmaline, levobunolol, ethionamide, cefaclor, dimenhydrinate, and bethanechol are potential drugs for the treatment of neutrophil-predominant severe asthma. CONCLUSION: This study identified potential biomarkers, pathogenesis, and therapeutic molecular targets for neutrophil-predominant severe asthma.

Effective uptake of folate-functionalized ethionamide-loaded hybrid system: targeting alveolar macrophages.

Aim: To assess the targeting ability of hybrid nanosystems functionalized with folate. It also aimed to reduce stomach intolerance by substituting the oral route for parenteral delivery. Method: The nanosystems, prepared by nanoprecipitation technique, utilized a one-step method to prepare nanoparticles followed by surface functionalization through adsorption. The prepared nanosystems underwent physical characterization, in vitro and in vivo evaluations. Result: The nanosystems were effective in targeting the alveolar macrophages. Ethionamide was released from the formulation over 5 days. Fourier-transform infrared results proved the structural characteristics, and the positive charge further improved the targeting efficacy on the functionalized system. Conclusion: The hybrid formulation improved the release characteristics. Reduction in dosing frequency due to prolonged release improves compliance with the dosage regimen.

Evaluation of the broth microdilution plate methodology for susceptibility testing of Mycobacterium tuberculosis in Peru.

BACKGROUND: Tuberculosis (TB) is a communicable, preventable and curable disease caused by the bacterium Mycobacterium tuberculosis (MTB). Peru is amongst the 30 countries with the highest burden of multidrug-resistant tuberculosis (MDR-TB) worldwide. In the fight against drug-resistant tuberculosis, the UKMYC6 microdilution plate was developed and validated by the CRyPTIC project. The objective of the study was to evaluate the use of the broth microdilution (BMD) plate methodology for susceptibility testing of drug-resistant MTB strains in Peru. METHODS: MTB strains isolated between 2015 and 2018 in Peru were used. 496 nationally-representative strains determined as drug-resistant by the routine 7H10 Agar Proportion Method (APM) were included in the present study. The Minimum Inhibitory Concentration (MIC) of 13 antituberculosis drugs were determined for each strain using the UKMYC6 microdilution plates. Diagnostic agreement between APM and BMD plate methodology was determined for rifampicin, isoniazid, ethambutol, ethionamide, kanamycin and levofloxacin. Phenotypes were set using binary (or ternary) classification based on Epidemiological cut-off values (ECOFF/ECV) proposed by the CRyPTIC project. Whole Genome Sequencing (WGS) was performed on strains with discrepant results between both methods. RESULTS: MIC distributions were determined for 13 first- and second-line anti-TB drugs, including new (bedaquiline, delamanid) and repurposed (clofazimine, linezolid) agents. MIC results were available for 80% (397/496) of the strains at 14 days and the remainder at 21 days. The comparative analysis determined a good agreement (0.64 ≤ k ≤ 0.79) for the drugs rifampicin, ethambutol, ethionamide and kanamycin, and the best agreement (k > 0.8) for isoniazid and levofloxacin. Overall, 12% of MIC values were above the UKMYC6 plate dilution ranges, most notably for the drugs rifampicin and rifabutin. No strain presented MICs higher than the ECOFF/ECV values for the new or repurposed drugs. Discrepant analysis using genotypic susceptibility testing by WGS supported half of the results obtained by APM (52%, 93/179) and half of those obtained by BMD plate methodology (48%, 86/179). CONCLUSIONS: The BMD methodology using the UKMYC6 plate allows the complete susceptibility characterization, through the determination of MICs, of drug-resistant MTB strains in Peru. This methodology shows good diagnostic performances for rifampicin, isoniazid, ethambutol, ethionamide, kanamycin and levofloxacin. It also allows for the characterization of MICs for other drugs used in previous years against tuberculosis, as well as for new and repurposed drugs recently introduced worldwide.

Pharmacokinetics of standard versus high-dose isoniazid for treatment of multidrug-resistant tuberculosis.

BACKGROUND: The WHO-endorsed shorter-course regimen for MDR-TB includes high-dose isoniazid. The pharmacokinetics of high-dose isoniazid within MDR-TB regimens has not been well described. OBJECTIVES: To characterize isoniazid pharmacokinetics at 5-15 mg/kg as monotherapy or as part of the MDR-TB treatment regimen. METHODS: We used non-linear mixed-effects modelling to evaluate the combined data from INHindsight, a 7 day early bactericidal activity study with isoniazid monotherapy, and PODRtb, an observational study of patients on MDR-TB treatment including terizidone, pyrazinamide, moxifloxacin, kanamycin, ethionamide and/or isoniazid. RESULTS: A total of 58 and 103 participants from the INHindsight and PODRtb studies, respectively, were included in the analysis. A two-compartment model with hepatic elimination best described the data. N-acetyltransferase 2 (NAT2) genotype caused multi-modal clearance, and saturable first-pass was observed beyond 10 mg/kg dosing. Saturable isoniazid kinetics predicted an increased exposure of approximately 50% beyond linearity at 20 mg/kg dosing. Participants treated with the MDR-TB regimen had a 65.6% lower AUC compared with participants on monotherapy. Ethionamide co-administration was associated with a 29% increase in isoniazid AUC. CONCLUSIONS: Markedly lower isoniazid exposures were observed in participants on combination MDR-TB treatment compared with monotherapy. Isoniazid displays saturable kinetics at doses >10 mg/kg. The safety implications of these phenomena remain unclear.

The small-molecule SMARt751 reverses Mycobacterium tuberculosis resistance to ethionamide in acute and chronic mouse models of tuberculosis.

The sensitivity of Mycobacterium tuberculosis, the pathogen that causes tuberculosis (TB), to antibiotic prodrugs is dependent on the efficacy of the activation process that transforms the prodrugs into their active antibacterial moieties. Various oxidases of M. tuberculosis have the potential to activate the prodrug ethionamide. Here, we used medicinal chemistry coupled with a phenotypic assay to select the N-acylated 4-phenylpiperidine compound series. The lead compound, SMARt751, interacted with the transcriptional regulator VirS of M. tuberculosis, which regulates the mymA operon encoding a monooxygenase that activates ethionamide. SMARt751 boosted the efficacy of ethionamide in vitro and in mouse models of acute and chronic TB. SMARt751 also restored full efficacy of ethionamide in mice infected with M. tuberculosis strains carrying mutations in the ethA gene, which cause ethionamide resistance in the clinic. SMARt751 was shown to be safe in tests conducted in vitro and in vivo. A model extrapolating animal pharmacokinetic and pharmacodynamic parameters to humans predicted that as little as 25 mg of SMARt751 daily would allow a fourfold reduction in the dose of ethionamide administered while retaining the same efficacy and reducing side effects.

Xpert MTB/XDR for detection of pulmonary tuberculosis and resistance to isoniazid, fluoroquinolones, ethionamide, and amikacin.

BACKGROUND: The World Health Organization (WHO) End TB Strategy stresses universal access to drug susceptibility testing (DST). DST determines whether Mycobacterium tuberculosis bacteria are susceptible or resistant to drugs. Xpert MTB/XDR is a rapid nucleic acid amplification test for detection of tuberculosis and drug resistance in one test suitable for use in peripheral and intermediate level laboratories. In specimens where tuberculosis is detected by Xpert MTB/XDR, Xpert MTB/XDR can also detect resistance to isoniazid, fluoroquinolones, ethionamide, and amikacin. OBJECTIVES: To assess the diagnostic accuracy of Xpert MTB/XDR for pulmonary tuberculosis in people with presumptive pulmonary tuberculosis (having signs and symptoms suggestive of tuberculosis, including cough, fever, weight loss, night sweats). To assess the diagnostic accuracy of Xpert MTB/XDR for resistance to isoniazid, fluoroquinolones, ethionamide, and amikacin in people with tuberculosis detected by Xpert MTB/XDR, irrespective of rifampicin resistance (whether or not rifampicin resistance status was known) and with known rifampicin resistance. SEARCH METHODS: We searched multiple databases to 23 September 2021. We limited searches to 2015 onwards as Xpert MTB/XDR was launched in 2020. SELECTION CRITERIA: Diagnostic accuracy studies using sputum in adults with presumptive or confirmed pulmonary tuberculosis. Reference standards were culture (pulmonary tuberculosis detection); phenotypic DST (pDST), genotypic DST (gDST),composite (pDST and gDST) (drug resistance detection). DATA COLLECTION AND ANALYSIS: Two review authors independently reviewed reports for eligibility and extracted data using a standardized form. For multicentre studies, we anticipated variability in the type and frequency of mutations associated with resistance to a given drug at the different centres and considered each centre as an independent study cohort for quality assessment and analysis. We assessed methodological quality with QUADAS-2, judging risk of bias separately for each target condition and reference standard. For pulmonary tuberculosis detection, owing to heterogeneity in participant characteristics and observed specificity estimates, we reported a range of sensitivity and specificity estimates and did not perform a meta-analysis. For drug resistance detection, we performed meta-analyses by reference standard using bivariate random-effects models. Using GRADE, we assessed certainty of evidence of Xpert MTB/XDR accuracy for detection of resistance to isoniazid and fluoroquinolones in people irrespective of rifampicin resistance and to ethionamide and amikacin in people with known rifampicin resistance, reflecting real-world situations. We used pDST, except for ethionamide resistance where we considered gDST a better reference standard. MAIN RESULTS: We included two multicentre studies from high multidrug-resistant/rifampicin-resistant tuberculosis burden countries, reporting on six independent study cohorts, involving 1228 participants for pulmonary tuberculosis detection and 1141 participants for drug resistance detection. The proportion of participants with rifampicin resistance in the two studies was 47.9% and 80.9%. For tuberculosis detection, we judged high risk of bias for patient selection owing to selective recruitment. For ethionamide resistance detection, we judged high risk of bias for the reference standard, both pDST and gDST, though we considered gDST a better reference standard. Pulmonary tuberculosis detection - Xpert MTB/XDR sensitivity range, 98.3% (96.1 to 99.5) to 98.9% (96.2 to 99.9) and specificity range, 22.5% (14.3 to 32.6) to 100.0% (86.3 to 100.0); median prevalence of pulmonary tuberculosis 91.3%, (interquartile range, 89.3% to 91.8%), (2 studies; 1 study reported on 2 cohorts, 1228 participants; very low-certainty evidence, sensitivity and specificity). Drug resistance detection People irrespective of rifampicin resistance - Isoniazid resistance: Xpert MTB/XDR summary sensitivity and specificity (95% confidence interval (CI)) were 94.2% (87.5 to 97.4) and 98.5% (92.6 to 99.7) against pDST, (6 cohorts, 1083 participants, moderate-certainty evidence, sensitivity and specificity). - Fluoroquinolone resistance: Xpert MTB/XDR summary sensitivity and specificity were 93.2% (88.1 to 96.2) and 98.0% (90.8 to 99.6) against pDST, (6 cohorts, 1021 participants; high-certainty evidence, sensitivity; moderate-certainty evidence, specificity). People with known rifampicin resistance - Ethionamide resistance: Xpert MTB/XDR summary sensitivity and specificity were 98.0% (74.2 to 99.9) and 99.7% (83.5 to 100.0) against gDST, (4 cohorts, 434 participants; very low-certainty evidence, sensitivity and specificity). - Amikacin resistance: Xpert MTB/XDR summary sensitivity and specificity were 86.1% (75.0 to 92.7) and 98.9% (93.0 to 99.8) against pDST, (4 cohorts, 490 participants; low-certainty evidence, sensitivity; high-certainty evidence, specificity). Of 1000 people with pulmonary tuberculosis, detected as tuberculosis by Xpert MTB/XDR: - where 50 have isoniazid resistance, 61 would have an Xpert MTB/XDR result indicating isoniazid resistance: of these, 14/61 (23%) would not have isoniazid resistance (FP); 939 (of 1000 people) would have a result indicating the absence of isoniazid resistance: of these, 3/939 (0%) would have isoniazid resistance (FN). - where 50 have fluoroquinolone resistance, 66 would have an Xpert MTB/XDR result indicating fluoroquinolone resistance: of these, 19/66 (29%) would not have fluoroquinolone resistance (FP); 934 would have a result indicating the absence of fluoroquinolone resistance: of these, 3/934 (0%) would have fluoroquinolone resistance (FN). - where 300 have ethionamide resistance, 296 would have an Xpert MTB/XDR result indicating ethionamide resistance: of these, 2/296 (1%) would not have ethionamide resistance (FP); 704 would have a result indicating the absence of ethionamide resistance: of these, 6/704 (1%) would have ethionamide resistance (FN). - where 135 have amikacin resistance, 126 would have an Xpert MTB/XDR result indicating amikacin resistance: of these, 10/126 (8%) would not have amikacin resistance (FP); 874 would have a result indicating the absence of amikacin resistance: of these, 19/874 (2%) would have amikacin resistance (FN). AUTHORS' CONCLUSIONS: Review findings suggest that, in people determined by Xpert MTB/XDR to be tuberculosis-positive, Xpert MTB/XDR provides accurate results for detection of isoniazid and fluoroquinolone resistance and can assist with selection of an optimised treatment regimen. Given that Xpert MTB/XDR targets a limited number of resistance variants in specific genes, the test may perform differently in different settings. Findings in this review should be interpreted with caution. Sensitivity for detection of ethionamide resistance was based only on Xpert MTB/XDR detection of mutations in the inhA promoter region, a known limitation. High risk of bias limits our confidence in Xpert MTB/XDR accuracy for pulmonary tuberculosis. Xpert MTB/XDR's impact will depend on its ability to detect tuberculosis (required for DST), prevalence of resistance to a given drug, health care infrastructure, and access to other tests.

Mutation in Eth A protein of Mycobacterium tuberculosis conferred drug tolerance against enthinoamide in Mycobacterium smegmatis mc2155.

EthA is an NADPH-specific flavin adenine dinucleotide (FAD) containing monooxygenase that activates the -second-line drug ethionamide (ETH). ETH gets converted to an active form after interaction with the EthA (monooxygenase) protein. Upon activation, ETH interacts with NAD+ to form an ETH-NAD adduct, which hampers the activity of InhA (Enoyl-[(acyl-carrier-protein) reductase (NADH)]. This, in turn, inhibits the cell wall synthesis, thus killing the Mycobacterium tuberculosis (Mtb). Mutations in the EthA gene can modulate ETH activation. The mutation at 202 position (Val202-Leu) of EthA protein has been reported frequently in ETH resistance. In this study, the effect of this mutation on the function of the EthA protein was examined through structural and functional analysis. Molecular docking of wild type and mutated EthA protein with ETH were compared to inspect the effect of mutation on molecular mechanism of drug resistant. Docking results corroborated that the lower docking score of the mutant protein, larger binding cavity, and lower affinity towards ETH resulted in a less compact and energetically less stable structure than the wild type protein. The computational outcome was authenticated by in-vitro experiments. The wild type and mutated genes were cloned and expressed in M. smegmatis, a surrogate host. Antibiotic susceptibility testing demonstrated that the mutant showed high growth and survival in the presence of the ETH drug. Overall, the results indicated that a mutation in the intergenic region of EthA protein could result in the altered conversion of ETH to the active form, resulting in differential ETH sensitivity for M. smegmatis carrying the wild type and mutant gene.

Phosphatidylcholine (18:0/20:4), a potential biomarker to predict ethionamide-induced hepatic steatosis in rats.

Ethionamide (ETH), a second-line drug for multidrug-resistant tuberculosis, is known to cause hepatic steatosis in rats and humans. To investigate predictive biomarkers for ETH-induced steatosis, we performed lipidomics analysis using plasma and liver samples collected from rats treated orally with ETH at 30 and 100 mg/kg for 14 days. The ETH-treated rats developed hepatic steatosis with Oil Red O staining-positive vacuolation in the centrilobular hepatocytes accompanied by increased hepatic contents of triglycerides (TG) and decreased plasma TG and total cholesterol levels. A multivariate analysis for lipid profiles revealed differences in each of the 35 lipid species in the plasma and liver between the control and the ETH-treated rats. Of those lipids, phosphatidylcholine (PC) (18:0/20:4) decreased dose-dependently in both the plasma and liver. Moreover, serum TG-rich very low-density lipoprotein (VLDL) levels, especially the large particle fraction of VLDL composed of PC containing arachidonic acid (20:4) involved in hepatic secretion of TG, were decreased dose-dependently. In conclusion, the decreased PC (18:0/20:4) in the liver, possibly leading to suppression of hepatic TG secretion, was considered to be involved in the pathogenesis of the ETH-induced hepatic steatosis. Therefore, plasma PC (18:0/20:4) levels are proposed as mechanism-related biomarkers for ETH-induced hepatic steatosis.

Use of Whole-Genome Sequencing to Predict Mycobacterium tuberculosis Complex Drug Resistance from Early Positive Liquid Cultures.

Our objective was to evaluate the performance of whole-genome sequencing (WGS) from early positive liquid cultures for predicting Mycobacterium tuberculosis complex (MTBC) drug resistance. Clinical isolates were obtained from tuberculosis patients at Shanghai Pulmonary Hospital (SPH). Antimicrobial susceptibility testing (AST) was performed, and WGS from early Bactec mycobacterial growth indicator tube (MGIT) 960-positive liquid cultures was performed to predict the drug resistance using the TB-Profiler informatics platform. A total of 182 clinical isolates were enrolled in this study. Using phenotypic AST as the gold standard, the overall sensitivity and specificity for WGS were, respectively, 97.1% (89.8 to 99.6%) and 90.4% (83.4 to 95.1%) for rifampin, 91.0% (82.4 to 96.3%) and 95.2% (89.1 to 98.4%) for isoniazid, 100.0% (89.4 to 100.0%) and 87.3% (80.8 to 92.1%) for ethambutol, 96.6% (88.3 to 99.6%) and 61.8% (52.6 to 70.4%) for streptomycin, 86.8% (71.9 to 95.6%) and 95.8% (91.2 to 98.5%) for moxifloxacin, 86.5% (71.2 to 91.5%) and 95.2% (90.3 to 98.0%) for ofloxacin, 100.0% (54.1 to 100.0%) and 67.6% (60.2 to 74.5%) for amikacin, 100.0% (63.1 to 100.0%) and 67.2% (59.7 to 74.2%) for kanamycin, 62.5% (24.5 to 91.5%) and 88.5% (82.8 to 92.8%) for ethionamide, 33.3% (4.3 to 77.7%) and 98.3% (95.1 to 99.7%) for para-aminosalicylic acid, and 0.0% (0.0 to 12.3%) and 100.0% (97.6 to 100.0%) for cycloserine. The concordances of WGS-based AST and phenotypic AST were as follows: rifampin (92.9%), isoniazid (93.4%), ethambutol (89.6%), streptomycin (73.1%), moxifloxacin (94.0%), ofloxacin (93.4%), amikacin (68.7%), kanamycin (68.7%), ethionamide (87.4%), para-aminosalicylic acid (96.2%) and cycloserine (84.6%). We conclude that WGS could be a promising approach to predict MTBC resistance from early positive liquid cultures. IMPORTANCE In this study, we used whole-genome sequencing (WGS) from early positive liquid (MGIT) cultures instead of solid cultures to predict drug resistance of 182 Mycobacterium tuberculosis complex (MTBC) clinical isolates to predict drug resistance using the TB-Profiler informatics platform. Our study indicates that WGS may be a promising method for predicting MTBC resistance using early positive liquid cultures.

Design, synthesis and molecular docking studies of imidazole and benzimidazole linked ethionamide derivatives as inhibitors of InhA and antituberculosis agents.

To explore effective antituberculosis agents, a new class of imidazoles and benzimidazoles linked ethionamide analogs were designed and synthesized. The elemental analysis, 1H NMR, 13C NMR and mass spectral data were used to characterize all of the novel analogs. In vitro activity against Mycobacterium tuberculosis (Mtb) H37Rv was assessed for all of the target compounds. The hydroxy and nitrile moieties on the imidazole ring, as well as the hydroxy and methoxy groups on the benzimidazole ring connected to the ethionamide side chain, were shown to be advantageous. In our cell viability experiment against the Vero cell line, all of the compounds were non-cytotoxic even at 100 µM. To confirm the powerful analogs target identification, we investigated their in vitro inhibitory action on an M. tuberculosis InhA over-expressing (Mtb InhA-OE) strain, which yielded MICs nearly twice those of the Mtb H37Rv strain. Furthermore, the results of molecular docking confirmed the experimental findings. Additionally, the molecules were evaluated in silico for ADMET and drug similarity features. The experimental observation enables the newly generated ethionamide derivatives to be attractive candidates for the creation of newer and better anti-TB agents.