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T cells play a prominent role in orchestrating the immune response to viral diseases, but their role in the clinical presentation and subsequent immunity to SARS-CoV-2 infection remains poorly understood. As part of a population-based survey of the municipality of Vo', Italy, conducted after the initial SARS-CoV-2 outbreak, we sampled the T cell receptor (TCR) repertoires of the population 2 months after the initial PCR survey and followed up positive cases 9 and 15 months later. At 2 months, we found that 97.0% (98 of 101) of cases had elevated levels of TCRs associated with SARS-CoV-2. T cell frequency (depth) was increased in individuals with more severe disease. Both depth and diversity (breadth) of the TCR repertoire were positively associated with neutralizing antibody titers, driven mostly by CD4+ T cells directed against spike protein. At the later time points, detection of these TCRs remained high, with 90.7% (78 of 96) and 86.2% (25 of 29) of individuals having detectable signal at 9 and 15 months, respectively. Forty-three individuals were vaccinated by month 15 and showed a significant increase in TCRs directed against spike protein. Taken together, these results demonstrate the central role of T cells in mounting an immune defense against SARS-CoV-2 that persists out to 15 months.

Infectious and inflammatory stimuli elicit the generation of chitinase-3-like protein-1 (CHI3L1), involved in tissue damage, repair and remodeling. We evaluated whether plasma CHI3L1 at disease onset predicts clinical outcome of patients with Coronavirus 2019 (COVID-19) disease. Blood from 191 prospectively followed COVID-19 patients were collected at hospital admission between March 18th and May 5th, 2020. Plasma from 80 survivors was collected one month post-discharge. Forty age- and sex-matched healthy volunteers served as controls. Primary outcome was transfer to intensive care unit (ICU) or death. CHI3L1 was higher in COVID-19 patients than controls (p < 0.0001). Patients with unfavorable outcome (41 patients admitted to ICU, 47 died) had significantly higher CHI3L1 levels than non-ICU survivors (p < 0.0001). CHI3L1 levels abated in survivors one month post-discharge, regardless of initial disease severity (p < 0.0001), although remaining higher than controls (p < 0.05). Cox regression analysis revealed that CHI3L1 levels predict primary outcome independently of age, sex, comorbidities, degree of respiratory insufficiency and systemic inflammation or time from symptom onset to sampling (p < 0.0001). Kaplan-Meier curve analysis confirmed that patients with CHI3L1 levels above the median (361 ng/mL) had a poorer prognosis (log rank test, p < 0.0001). Plasma CHI3L1 is increased in COVID-19 patients and predicts adverse outcome., (© 2022. The Author(s).)

Background: Chromogranin A (CgA) and its fragment vasostatin I (VS-I) are secreted in the blood by endocrine/neuroendocrine cells and regulate stress responses. Their involvement in Coronavirus 2019 disease (COVID-19) has not been investigated., Methods: CgA and VS-I plasma concentrations were measured at hospital admission from March to May 2020 in 190 patients. 40 age- and sex-matched healthy volunteers served as controls. CgA and VS-I levels relationship with demographics, comorbidities and disease severity was assessed through Mann Whitney U test or Spearman correlation test. Cox regression analysis and Kaplan Meier survival curves were performed to investigate the impact of the CgA and VS-I levels on in-hospital mortality., Results: Median CgA and VS-I levels were higher in patients than in healthy controls (CgA: 0.558 nM [interquartile range, IQR 0.358-1.046] vs 0.368 nM [IQR 0.288-0.490] respectively, p = 0.0017; VS-I: 0.357 nM [IQR 0.196-0.465] vs 0.144 nM [0.144-0.156] respectively, p<0.0001). Concentration of CgA, but not of VS-I, significantly increased in patients who died (n = 47) than in survivors (n = 143) (median 0.948 nM [IQR 0.514-1.754] vs 0.507 nM [IQR 0.343-0.785], p = 0.00026). Levels of CgA were independent predictors of in-hospital mortality (hazard ratio 1.28 [95% confidence interval 1.077-1.522], p = 0.005) when adjusted for age, number of comorbidities, respiratory insufficiency degree, C-reactive protein levels and time from symptom onset to sampling. Kaplan Meier curves revealed a significantly increased mortality rate in patients with CgA levels above 0.558 nM (median value, log rank test, p = 0.001)., Conclusion: Plasma CgA levels increase in COVID-19 patients and represent an early independent predictor of mortality., Competing Interests: NO authors have competing interests.

Mycobacterium abscessus is the etiological agent of severe pulmonary infections in vulnerable patients, such as those with cystic fibrosis (CF), where it represents a relevant cause of morbidity and mortality. Treatment of pulmonary infections caused by M. abscessus remains extremely difficult, as this species is resistant to most classes of antibiotics, including macrolides, aminoglycosides, rifamycins, tetracyclines, and β-lactams. Here, we show that apoptotic body like liposomes loaded with phosphatidylinositol 5-phosphate (ABL/PI5P) enhance the antimycobacterial response, both in macrophages from healthy donors exposed to pharmacological inhibition of cystic fibrosis transmembrane conductance regulator (CFTR) and in macrophages from CF patients, by enhancing phagosome acidification and reactive oxygen species (ROS) production. The treatment with liposomes of wild-type as well as CF mice, intratracheally infected with M. abscessus, resulted in about a 2-log reduction of pulmonary mycobacterial burden and a significant reduction of macrophages and neutrophils in bronchoalveolar lavage fluid (BALF). Finally, the combination treatment with ABL/PI5P and amikacin, to specifically target intracellular and extracellular bacilli, resulted in a further significant reduction of both pulmonary mycobacterial burden and inflammatory response in comparison with the single treatments. These results offer the conceptual basis for a novel therapeutic regimen based on antibiotic and bioactive liposomes, used as a combined host- and pathogen-directed therapeutic strategy, aimed at the control of M. abscessus infection, and of related immunopathogenic responses, for which therapeutic options are still limited. IMPORTANCE Mycobacterium abscessus is an opportunistic pathogen intrinsically resistant to many antibiotics, frequently linked to chronic pulmonary infections, and representing a relevant cause of morbidity and mortality, especially in immunocompromised patients, such as those affected by cystic fibrosis. M. abscessus-caused pulmonary infection treatment is extremely difficult due to its high toxicity and long-lasting regimen with life-impairing side effects and the scarce availability of new antibiotics approved for human use. In this context, there is an urgent need for the development of an alternative therapeutic strategy that aims at improving the current management of patients affected by chronic M. abscessus infections. Our data support the therapeutic value of a combined host- and pathogen-directed therapy as a promising approach, as an alternative to single treatments, to simultaneously target intracellular and extracellular pathogens and improve the clinical management of patients infected with multidrug-resistant pathogens such as M. abscessus.

A laboratory validation study was conducted to assess the equivalence of Xpert MTB/RIF Ultra testing on the GeneXpert System and the GeneXpert Omni System ('Omni') for tuberculosis and rifampicin resistance. High concordance of the two devices was demonstrated for well-characterized clinical samples as well as control materials, with controls tested on Omni at normal and challenging environmental conditions (i.e. 35°C, 90% relative humidity). Equivalence of the Cts for all probes was also shown. Equivalence was demonstrated for the Omni and GeneXpert devices for tuberculosis and rifampicin resistance detection for a diverse range of clinical specimens and environmental conditions., Competing Interests: The authors have declared that no competing interests exist.

Background and Aims: Patients infected with SARS-CoV-2 range from asymptomatic, to mild, moderate or severe disease evolution including fatal outcome. Thus, early predictors of clinical outcome are highly needed. We investigated markers of neural tissue damage as a possible early sign of multisystem involvement to assess their clinical prognostic value on survival or transfer to intensive care unit (ICU)., Methods: We collected blood from 104 patients infected with SARS-CoV-2 the day of admission to the emergency room and measured blood neurofilament light chair (NfL), glial fibrillary acidic protein (GFAP), ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), and total tau protein levels., Results: We found that NfL, GFAP, and tau were significantly increased in patients with fatal outcome, while NfL and UCH-L1 in those needing ICU transfer. ROC and Kaplan-Meier curves indicated that total tau levels at admission accurately predict mortality., Conclusions: Blood neural markers may provide additional prognostic value to conventional biomarkers used to predict COVID-19 outcome., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Host inflammation contributes to determine whether SARS-CoV-2 infection causes mild or life-threatening disease. Tools are needed for early risk assessment., Methods: We studied in 111 COVID-19 patients prospectively followed at a single reference Hospital fifty-three potential biomarkers including alarmins, cytokines, adipocytokines and growth factors, humoral innate immune and neuroendocrine molecules and regulators of iron metabolism. Biomarkers at hospital admission together with age, degree of hypoxia, neutrophil to lymphocyte ratio (NLR), lactate dehydrogenase (LDH), C-reactive protein (CRP) and creatinine were analysed within a data-driven approach to classify patients with respect to survival and ICU outcomes. Classification and regression tree (CART) models were used to identify prognostic biomarkers., Results: Among the fifty-three potential biomarkers, the classification tree analysis selected CXCL10 at hospital admission, in combination with NLR and time from onset, as the best predictor of ICU transfer (AUC [95% CI] = 0.8374 [0.6233-0.8435]), while it was selected alone to predict death (AUC [95% CI] = 0.7334 [0.7547-0.9201]). CXCL10 concentration abated in COVID-19 survivors after healing and discharge from the hospital., Conclusions: CXCL10 results from a data-driven analysis, that accounts for presence of confounding factors, as the most robust predictive biomarker of patient outcome in COVID-19., (© 2021. The Author(s).)

Background: Tuberculosis (TB) unevenly affects individuals across the globe, especially in rural areas of low-income countries. Aim of the study was to assess the impact of social protection to increase TB awareness on treatment outcomes among TB patients in a rural area of Senegal. Materials & methods: The study, conducted in Fimela district (Senegal) from 1 January 2010 to 31 December 2019 and the intervention started from 31 January 2013, includes activities to increase awareness, active case finding, active follow-up and social protection. Results: Overall, 435 subjects - mainly male and young - were included in the analysis. Among TB cases, 94% had pulmonary involvement, 87% had no previous TB history, and 6% resulted positive HIV. Improved outcome was observed once intervention began (from 71 to 91%, p < 0.001); whereas mortality decreased (from 15 to 5%; p < 0.001), especially for those HIV co-infected for whom TB mortality rate dropped from 70 to 29%. Conclusion: After beginning the cooperation program, TB treatment success increased as a result of the decline of mortality, especially in people living with HIV.

Nontypeable Haemophilus influenzae (NTHi) is commonly isolated from airways of patients suffering from chronic respiratory diseases, such as COPD or cystic fibrosis (CF). However, to what extent NTHi long-term infection contributes to the lung inflammatory burden during chronic airway disease is still controversial. Here, we exploited human respiratory samples from a small cohort of CF patients and found that patients chronically infected with NTHi had significantly higher levels of interleukin (IL)-8 and CXCL1 than those who were not infected. To better define the impact of chronic NTHi infection in fuelling inflammatory response in chronic lung diseases, we developed a new mouse model using both laboratory and clinical strains. Chronic NTHi infection was associated with chronic inflammation of the lung, characterised by recruitment of neutrophils and cytokine release keratinocyte-derived chemokine (KC), macrophage inflammatory protein 2 (MIP-2), granulocyte colony-stimulating factor (G-CFS), IL-6, IL-17A and IL-17F) at 2 and 14 days post-infection. An increased burden of T-cell-mediated response (CD4 + and γδ cells) and higher levels of pro-matrix metalloproteinase 9 (pro-MMP9), known to be associated with tissue remodelling, were observed at 14 days post-infection. Of note we found that both CD4 + IL-17 + cells and levels of IL-17 cytokines were enriched in mice at advanced stages of NTHi chronic infection. Moreover, by immunohistochemistry we found CD3 + , B220 + and CXCL-13 + cells localised in bronchus-associated lymphoid tissue-like structures at day 14. Our results demonstrate that chronic NTHi infection exerts a pro-inflammatory activity in the human and murine lung and could therefore contribute to the exaggerated burden of lung inflammation in patients at risk., Competing Interests: Conflict of interest: F. Saliu has nothing to disclose. Conflict of interest: G. Rizzo has nothing to disclose. Conflict of interest: A. Bragonzi has nothing to disclose. Conflict of interest: L. Cariani has nothing to disclose. Conflict of interest: D.M. Cirillo has nothing to disclose. Conflict of interest: C. Colombo has nothing to disclose. Conflict of interest: V. Daccò has nothing to disclose. Conflict of interest: D. Girelli has nothing to disclose. Conflict of interest: S. Rizzetto has nothing to disclose. Conflict of interest: B. Sipione has nothing to disclose. Conflict of interest: C. Cigana has nothing to disclose. Conflict of interest: N.I. Lorè has nothing to disclose., (Copyright ©ERS 2021.)

We surveyed availability of phenotypic drug susceptibility testing for drug-resistant Mycobacterium tuberculosis in Europe. Of 27 laboratories, 17 tested for linezolid, 11 for clofazimine, 9 for bedaquiline, and 6 for delamanid during 2019. Our findings indicate that testing capacity for newer and repurposed tuberculosis drugs exists, but its availability is limited.

We describe the design, development, analytical performance, and a limited clinical evaluation of the 10-color Xpert MTB/XDR assay (CE-IVD only, not for sale in the United States). This assay is intended as a reflex test to detect resistance to isoniazid (INH), fluoroquinolones (FLQ), ethionamide (ETH), and second-line injectable drugs (SLIDs) in unprocessed sputum samples and concentrated sputum sediments which are positive for Mycobacterium tuberculosis The Xpert MTB/XDR assay simultaneously amplifies eight genes and promoter regions in M. tuberculosis and analyzes melting temperatures ( T m s) using sloppy molecular beacon (SMB) probes to identify mutations associated with INH, FLQ, ETH, and SLID resistance. Results can be obtained in under 90 min using 10-color GeneXpert modules. The assay can differentiate low- versus high-level resistance to INH and FLQ as well as cross-resistance versus individual resistance to SLIDs by identifying mutation-specific T m s or T m patterns generated by the SMB probes. The assay has a limit of detection comparable to that of the Xpert MTB/RIF assay and successfully detected 16 clinically significant mutations in a challenge set of clinical isolate DNA. In a clinical study performed at two sites with 100 sputum and 214 clinical isolates, the assay showed a sensitivity of 94% to 100% and a specificity of 100% for all drugs except for ETH compared to that of sequencing. The sensitivity and specificity were in the same ranges as those of phenotypic drug-susceptibility testing. Used in combination with a primary tuberculosis diagnostic test, this assay should expand the capacity for detection of drug-resistant tuberculosis near the point of care., (Copyright © 2021 American Society for Microbiology.)

Major epidemics, including some that qualify as pandemics, such as severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), HIV, influenza A (H1N1)pdm/09 and most recently COVID-19, affect the lung. Tuberculosis (TB) remains the top infectious disease killer, but apart from syndemic TB/HIV little is known regarding the interaction of viral epidemics and pandemics with TB. The aim of this consensus-based document is to describe the effects of viral infections resulting in epidemics and pandemics that affect the lung (MERS, SARS, HIV, influenza A (H1N1)pdm/09 and COVID-19) and their interactions with TB. A search of the scientific literature was performed. A writing committee of international experts including the European Centre for Disease Prevention and Control Public Health Emergency (ECDC PHE) team, the World Association for Infectious Diseases and Immunological Disorders (WAidid), the Global Tuberculosis Network (GTN), and members of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Study Group for Mycobacterial Infections (ESGMYC) was established. Consensus was achieved after multiple rounds of revisions between the writing committee and a larger expert group. A Delphi process involving the core group of authors (excluding the ECDC PHE team) identified the areas requiring review/consensus, followed by a second round to refine the definitive consensus elements. The epidemiology and immunology of these viral infections and their interactions with TB are discussed with implications for diagnosis, treatment and prevention of airborne infections (infection control, viral containment and workplace safety). This consensus document represents a rapid and comprehensive summary on what is known on the topic., Competing Interests: Conflict of interest: G.B. Migliori has nothing to disclose. Conflict of interest: M. Raviglione has nothing to disclose. Conflict of interest: G. MacGregor-Skinner has nothing to disclose. Conflict of interest: G. Sotgiu has nothing to disclose. Conflict of interest: J-W. Alffenaar has nothing to disclose. Conflict of interest: S. Tiberi has nothing to disclose. Conflict of interest: C. Adlhoch has nothing to disclose. Conflict of interest: T. Alonzi has nothing to disclose. Conflict of interest: S. Archuleta has nothing to disclose. Conflict of interest: S. Brusin has nothing to disclose. Conflict of interest: E. Cambau has nothing to disclose. Conflict of interest: M.R. Capobianchi has nothing to disclose. Conflict of interest: C. Castilletti has nothing to disclose. Conflict of interest: R. Centis has nothing to disclose. Conflict of interest: D.M. Cirillo has nothing to disclose. Conflict of interest: L. D'Ambrosio has nothing to disclose. Conflict of interest: G. Delogu has nothing to disclose. Conflict of interest: S.M.R. Esposito has nothing to disclose. Conflict of interest: J. Figueroa has nothing to disclose. Conflict of interest: J.S. Friedland has nothing to disclose. Conflict of interest: B.C.H. Ho has nothing to disclose. Conflict of interest: G. Ippolito has nothing to disclose. Conflict of interest: M. Jankovic has nothing to disclose. Conflict of interest: H.Y. Kim has nothing to disclose. Conflict of interest: S. Rosales Klintz has nothing to disclose. Conflict of interest: C. Ködmön has nothing to disclose. Conflict of interest: E. Lalle has nothing to disclose. Conflict of interest: Y.S. Leo has nothing to disclose. Conflict of interest: C-C. Leung has nothing to disclose. Conflict of interest: A-G. Märtson has nothing to disclose. Conflict of interest: M.G. Melazzini has nothing to disclose. Conflict of interest: S. Najafi Fard has nothing to disclose. Conflict of interest: P. Penttinen has nothing to disclose. Conflict of interest: L. Petrone has nothing to disclose. Conflict of interest: E. Petruccioli has nothing to disclose. Conflict of interest: E. Pontali has nothing to disclose. Conflict of interest: L. Saderi has nothing to disclose. Conflict of interest: M. Santin has nothing to disclose. Conflict of interest: A. Spanevello has nothing to disclose. Conflict of interest: R. van Crevel has nothing to disclose. Conflict of interest: M.J. van der Werf has nothing to disclose. Conflict of interest: D. Visca has nothing to disclose. Conflict of interest: M. Viveiros has nothing to disclose. Conflict of interest: J-P. Zellweger has nothing to disclose. Conflict of interest: A. Zumla has nothing to disclose. Conflict of interest: D. Goletti has nothing to disclose. Conflict of interest: C.W.M. Ong has nothing to disclose., (Copyright ©ERS 2020.)

Targeted next-generation sequencing (tNGS) has emerged as a comprehensive alternative to existing methods for drug susceptibility testing (DST) of Mycobacterium tuberculosis from patient sputum samples for clinical diagnosis of drug-resistant tuberculosis (DR-TB). However, the complexity of sequencing platforms has limited their uptake in low-resource settings. The goal of this study was to evaluate the use of the tNGS-based DST solution Genoscreen Deeplex Myc-TB, for use on the compact, low-cost Oxford Nanopore Technologies MinION sequencer. One hundred four DNA samples extracted from smear-positive sputum sediments, previously sequenced using the Deeplex assay on an Illumina MiniSeq, were resequenced on MinION after applying a custom library preparation. MinION read quality, mapping statistics, and variant calling were computed using an in-house pipeline and compared to the reference MiniSeq data. The average percentage of MinION reads mapped to an H37RV reference genome was 90.8%, versus 99.5% on MiniSeq. The mean depths of coverage were 4,151× and 4,177× on MinION and MiniSeq, respectively, with heterogeneous distribution across targeted genes. Composite reference coverage breadth was >99% for both platforms. We observed full concordance between technologies in reporting the clinically relevant drug-resistant markers, including full gene deletions. In conclusion, we demonstrated that the workflow and sequencing data obtained from Deeplex on MinION are comparable to those for the MiniSeq, despite the higher raw error rates on MinION, with the added advantage of MinION's portability, versatility, and low capital costs. Targeted NGS on MinION is a promising DST solution for rapidly providing clinically relevant data to manage complex DR-TB cases., (Copyright © 2020 Cabibbe et al.)

False-susceptible phenotypic drug-susceptibility testing (DST) results for pyrazinamide due to mutations with MICs close to the critical concentration (CC) confound the classification of pncA resistance mutations, leading to an underestimate of the specificity of genotypic DST. This could be minimized by basing treatment decisions on well-understood mutations and by adopting an area of technical uncertainty for phenotypic DST rather than only testing the CC, as is current practice for the Mycobacterium tuberculosis complex., (Copyright © 2020 Köser et al.)

On December 31, 2019, the Chinese government officially announced the identification of a new type of coronavirus (SARS-CoV-2) as the etiological cause of a severe acute respiratory syndrome in Wuhan city, Hubei Province. Over the next weeks, SARS-CoV-2 caused a global pandemic as officially declared by the WHO on March 11, 2020, with confirmed cases and deaths in more than 166 countries. We are experiencing a worldwide phenomenon of unprecedented social and economic consequences. Since the beginning of the COVID-19 outbreak, there have been fears that the epidemic could strongly impact weaker healthcare systems in poor-resource settings, especially in Sub-Saharan Africa (SSA). The 2 million Chinese nationals that live and work in Africa could potentially contribute to the spread of COVID-19 on the continent., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

It is relevant to evaluate MDR-tuberculosis in prisons and its impact on the global epidemiology of this disease. However, systematic molecular epidemiology programs in prisons are lacking. A health-screening program performed on arrival for inmates transferred from Peruvian prisons to Spain led to the diagnosis of five MDR-TB cases from one of the biggest prisons in Latin America. They grouped into two MIRU-VNTR-clusters (Callao-1 and Callao-2), suggesting a reservoir of two prevalent MDR strains. A high-rate of overexposure was deduced because one of the five cases was coinfected by a pansusceptible strain. Callao-1 strain was also identified in 2018 in a community case in Spain who had been in the same Peruvian prison in 2002-5. A strain-specific-PCR tailored from WGS data was implemented in Peru, allowing the confirmation that these strains were currently responsible for the majority of the MDR cases in that prison, including a new mixed infection.

Background: Treatment outcomes of the shorter regimen for rifampicin-resistant tuberculosis are not completely established. We report on these outcomes two years after treatment completion among patients enrolled in an observational cohort study in nine African countries., Methods: 1,006 patients treated with the nine-month regimen were followed every six months with sputum cultures up to 24 months after treatment completion. The risk of any unfavourable outcome, of failure and relapse, and of death during and after treatment was analysed according to patient's characteristics and initial drug susceptibility by Cox proportional hazard models., Findings: Respectively 67.8% and 57.2% patients had >=1 culture result six months and 12 months after treatment completion. Fourteen relapses were diagnosed. The probability of relapse-free success was 79.3% (95% confidence interval [CI] 76.6-82.0%) overall, 80.9% (95% CI 78.0-84.0%) among HIV-negative and 72.5% (95% CI 66.5-78.9%) among HIV-infected patients. Initial fluoroquinolone (adjusted hazard ratio [aHR] 6.7 [95% CI 3.4-13.1]) and isoniazid resistance (aHR 9.4 [95% CI 1.3-68.0]) were significantly associated with increased risk of failure/relapse and of any unfavourable outcome., Interpretation: The close to 80% relapse-free success indicates the good outcome of the regimen in low-and middle-income settings. Results confirm the lesser effectiveness of the regimen in patients with initial resistance to fluoroquinolones and support the use of high-dose isoniazid, but do not support exclusion of patients for resistance to drugs other than fluoroquinolones., Funding: Expertise-France and Agence Française de Développement., Competing Interests: The authors have no conflict of interests to disclose., (© 2020 Published by Elsevier Ltd.)

Isolation of metallo-β-lactamase-producing, carbapenem-resistant, Pseudomonas aeruginosa strains is increasingly being documented worldwide; their presence constitutes a public health threat. Here, we report draft genome sequences of two New Delhi metallo-β-lactamase-1-producing, multidrug-resistant, P. aeruginosa strains of sequence type 235 that were isolated from the surgical wound of two patients hospitalized in the same ward., (Copyright © 2020 Tafaj et al.)

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., Competing Interests: No competing interests were disclosed., (Copyright: © 2019 Hunt M et al.)

Tuberculosis is the primary infectious disease killer worldwide, with a growing threat from multidrug-resistant cases. Unfortunately, classic growth-based phenotypic drug susceptibility testing (DST) remains difficult, costly, and time consuming, while current rapid molecular testing options are limited by the diversity of antimicrobial-resistant genotypes that can be detected at once. Next-generation sequencing (NGS) offers the opportunity for rapid, comprehensive DST without the time or cost burden of phenotypic tests and can provide useful information for global surveillance. As access to NGS expands, it will be important to ensure that results are communicated clearly, consistent, comparable between laboratories, and associated with clear guidance on clinical interpretation of results. In this viewpoint article, we summarize 2 expert workshops regarding a standardized report format, focusing on relevant variables, terminology, and required minimal elements for clinical and laboratory reports with a proposed standardized template for clinical reporting NGS results for Mycobacterium tuberculosis., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America.)

Whole genome sequencing (WGS) of Mycobacterium tuberculosis has rapidly progressed from a research tool to a clinical application for the diagnosis and management of tuberculosis and in public health surveillance. This development has been facilitated by drastic drops in cost, advances in technology and concerted efforts to translate sequencing data into actionable information. There is, however, a risk that, in the absence of a consensus and international standards, the widespread use of WGS technology may result in data and processes that lack harmonization, comparability and validation. In this Review, we outline the current landscape of WGS pipelines and applications, and set out best practices for M. tuberculosis WGS, including standards for bioinformatics pipelines, curated repositories of resistance-causing variants, phylogenetic analyses, quality control and standardized reporting.

In a 12-month nationwide study on the prevalence of drug-resistant tuberculosis (TB) in Lebanon, we identified 3 multidrug-resistant cases and 3 extensively drug-resistant TB cases in refugees, migrants, and 1 Lebanon resident. Enhanced diagnostics, particularly in major destinations for refugees, asylum seekers, and migrant workers, can inform treatment decisions and may help prevent the spread of drug-resistant TB.

Drug-resistant tuberculosis poses a persistent public health threat. The ReSeqTB platform is a collaborative, curated knowledgebase, designed to standardize and aggregate global Mycobacterium tuberculosis complex (MTBC) variant data from whole genome sequencing (WGS) with phenotypic drug susceptibility testing (DST) and clinical data. We developed a unified analysis variant pipeline (UVP) ( https://github.com/CPTR-ReSeqTB/UVP ) to identify variants and assign lineage from MTBC sequence data. Stringent thresholds and quality control measures were incorporated in this open source tool. The pipeline was validated using a well-characterized dataset of 90 diverse MTBC isolates with conventional DST and DNA Sanger sequencing data. The UVP exhibited 98.9% agreement with the variants identified using Sanger sequencing and was 100% concordant with conventional methods of assigning lineage. We analyzed 4636 publicly available MTBC isolates in the ReSeqTB platform representing all seven major MTBC lineages. The variants detected have an above 94% accuracy of predicting drug based on the accompanying DST results in the platform. The aggregation of variants over time in the platform will establish confidence-graded mutations statistically associated with phenotypic drug resistance. These tools serve as critical reference standards for future molecular diagnostic assay developers, researchers, public health agencies and clinicians working towards the control of drug-resistant tuberculosis.

Background: The World Health Organization recommends drug-susceptibility testing of Mycobacterium tuberculosis complex for all patients with tuberculosis to guide treatment decisions and improve outcomes. Whether DNA sequencing can be used to accurately predict profiles of susceptibility to first-line antituberculosis drugs has not been clear., Methods: We obtained whole-genome sequences and associated phenotypes of resistance or susceptibility to the first-line antituberculosis drugs isoniazid, rifampin, ethambutol, and pyrazinamide for isolates from 16 countries across six continents. For each isolate, mutations associated with drug resistance and drug susceptibility were identified across nine genes, and individual phenotypes were predicted unless mutations of unknown association were also present. To identify how whole-genome sequencing might direct first-line drug therapy, complete susceptibility profiles were predicted. These profiles were predicted to be susceptible to all four drugs (i.e., pansusceptible) if they were predicted to be susceptible to isoniazid and to the other drugs or if they contained mutations of unknown association in genes that affect susceptibility to the other drugs. We simulated the way in which the negative predictive value changed with the prevalence of drug resistance., Results: A total of 10,209 isolates were analyzed. The largest proportion of phenotypes was predicted for rifampin (9660 [95.4%] of 10,130) and the smallest was predicted for ethambutol (8794 [89.8%] of 9794). Resistance to isoniazid, rifampin, ethambutol, and pyrazinamide was correctly predicted with 97.1%, 97.5%, 94.6%, and 91.3% sensitivity, respectively, and susceptibility to these drugs was correctly predicted with 99.0%, 98.8%, 93.6%, and 96.8% specificity. Of the 7516 isolates with complete phenotypic drug-susceptibility profiles, 5865 (78.0%) had complete genotypic predictions, among which 5250 profiles (89.5%) were correctly predicted. Among the 4037 phenotypic profiles that were predicted to be pansusceptible, 3952 (97.9%) were correctly predicted., Conclusions: Genotypic predictions of the susceptibility of M. tuberculosis to first-line drugs were found to be correlated with phenotypic susceptibility to these drugs. (Funded by the Bill and Melinda Gates Foundation and others.).

The UKMYC5 plate is a 96-well microtiter plate designed by the CRyPTIC Consortium (Comprehensive Resistance Prediction for Tuberculosis: an International Consortium) to enable the measurement of MICs of 14 different antituberculosis (anti-TB) compounds for >30,000 clinical Mycobacterium tuberculosis isolates. Unlike the MYCOTB plate, on which the UKMYC5 plate is based, the UKMYC5 plate includes two new (bedaquiline and delamanid) and two repurposed (clofazimine and linezolid) compounds. UKMYC5 plates were tested by seven laboratories on four continents by use of a panel of 19 external quality assessment (EQA) strains, including H37Rv. To assess the optimal combination of reading method and incubation time, MICs were measured from each plate by two readers, using three methods (mirrored box, microscope, and Vizion digital viewing system), after 7, 10, 14, and 21 days of incubation. In addition, all EQA strains were subjected to whole-genome sequencing and phenotypically characterized by the 7H10/7H11 agar proportion method (APM) and by use of MGIT960 mycobacterial growth indicator tubes. We concluded that the UKMYC5 plate is optimally read using the Vizion system after 14 days of incubation, achieving an interreader agreement of 97.9% and intra- and interlaboratory reproducibility rates of 95.6% and 93.1%, respectively. The mirrored box had a similar reproducibility. Strains classified as resistant by APM, MGIT960, or the presence of mutations known to confer resistance consistently showed elevated MICs compared to those for strains classified as susceptible. Finally, the UKMYC5 plate records intermediate MICs for one strain for which the APM measured MICs close to the applied critical concentration, providing early evidence that the UKMYC5 plate can quantitatively measure the magnitude of resistance to anti-TB compounds that is due to specific genetic variation., (Copyright © 2018 Rancoita et al.)

Background: Staphylococcus aureus and Pseudomonas aeruginosa are key bacterial pathogens of the respiratory tract in patients with cystic fibrosis (CF). Although P. aeruginosa chronic bronchial infection is associated with a poorer prognosis, the consequences of S. aureus colonization on CF outcomes are controversial., Methods: In this paper, murine models of infection resembling traits of the CF human airways disease have been revisited using an infection schedule that mimics the sequence of events of pulmonary disease in CF patients. First, mice were infected with S. aureus, embedded in agar beads; this was followed by P. aeruginosa infection and analysis of bacterial load, leukocyte infiltration, and lung tissue damage., Results: We reveal that (1) S. aureus promotes severe lesions including abscess formation, (2) S. aureus increases the risk of subsequent chronic P. aeruginosa respiratory infection, and (3) once the chronic infection has been established, P. aeruginosa influences most of the inflammatory responses independent of S. aureus., Conclusions: Our findings established the significance of S. aureus colonization per se and the impact on the subsequent P. aeruginosa infection. This would point towards a thorough assessment for the need of treatment against S. aureus., (© The Author(s) 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: The Xpert MTB/RIF assay is an automated molecular test that has improved the detection of tuberculosis and rifampicin resistance, but its sensitivity is inadequate in patients with paucibacillary disease or HIV. Xpert MTB/RIF Ultra (Xpert Ultra) was developed to overcome this limitation. We compared the diagnostic performance of Xpert Ultra with that of Xpert for detection of tuberculosis and rifampicin resistance., Methods: In this prospective, multicentre, diagnostic accuracy study, we recruited adults with pulmonary tuberculosis symptoms presenting at primary health-care centres and hospitals in eight countries (South Africa, Uganda, Kenya, India, China, Georgia, Belarus, and Brazil). Participants were allocated to the case detection group if no drugs had been taken for tuberculosis in the past 6 months or to the multidrug-resistance risk group if drugs for tuberculosis had been taken in the past 6 months, but drug resistance was suspected. Demographic information, medical history, chest imaging results, and HIV test results were recorded at enrolment, and each participant gave at least three sputum specimen on 2 separate days. Xpert and Xpert Ultra diagnostic performance in the same sputum specimen was compared with culture tests and drug susceptibility testing as reference standards. The primary objectives were to estimate and compare the sensitivity of Xpert Ultra test with that of Xpert for detection of smear-negative tuberculosis and rifampicin resistance and to estimate and compare Xpert Ultra and Xpert specificities for detection of rifampicin resistance. Study participants in the case detection group were included in all analyses, whereas participants in the multidrug-resistance risk group were only included in analyses of rifampicin-resistance detection., Findings: Between Feb 18, and Dec 24, 2016, we enrolled 2368 participants for sputum sampling. 248 participants were excluded from the analysis, and 1753 participants were distributed to the case detection group (n=1439) and the multidrug-resistance risk group (n=314). Sensitivities of Xpert Ultra and Xpert were 63% and 46%, respectively, for the 137 participants with smear-negative and culture-positive sputum (difference of 17%, 95% CI 10 to 24); 90% and 77%, respectively, for the 115 HIV-positive participants with culture-positive sputum (13%, 6·4 to 21); and 88% and 83%, respectively, across all 462 participants with culture-positive sputum (5·4%, 3·3 to 8·0). Specificities of Xpert Ultra and Xpert for case detection were 96% and 98% (-2·7%, -3·9 to -1·7) overall, and 93% and 98% for patients with a history of tuberculosis. Xpert Ultra and Xpert performed similarly in detecting rifampicin resistance., Interpretation: For tuberculosis case detection, sensitivity of Xpert Ultra was superior to that of Xpert in patients with paucibacillary disease and in patients with HIV. However, this increase in sensitivity came at the expense of a decrease in specificity., Funding: Government of Netherlands, Government of Australia, Bill & Melinda Gates Foundation, Government of the UK, and the National Institute of Allergy and Infectious Diseases., (Copyright © 2018 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

A clear understanding of the genetic basis of antibiotic resistance in Mycobacterium tuberculosis is required to accelerate the development of rapid drug susceptibility testing methods based on genetic sequence.Raw genotype-phenotype correlation data were extracted as part of a comprehensive systematic review to develop a standardised analytical approach for interpreting resistance associated mutations for rifampicin, isoniazid, ofloxacin/levofloxacin, moxifloxacin, amikacin, kanamycin, capreomycin, streptomycin, ethionamide/prothionamide and pyrazinamide. Mutation frequencies in resistant and susceptible isolates were calculated, together with novel statistical measures to classify mutations as high, moderate, minimal or indeterminate confidence for predicting resistance.We identified 286 confidence-graded mutations associated with resistance. Compared to phenotypic methods, sensitivity (95% CI) for rifampicin was 90.3% (89.6-90.9%), while for isoniazid it was 78.2% (77.4-79.0%) and their specificities were 96.3% (95.7-96.8%) and 94.4% (93.1-95.5%), respectively. For second-line drugs, sensitivity varied from 67.4% (64.1-70.6%) for capreomycin to 88.2% (85.1-90.9%) for moxifloxacin, with specificity ranging from 90.0% (87.1-92.5%) for moxifloxacin to 99.5% (99.0-99.8%) for amikacin.This study provides a standardised and comprehensive approach for the interpretation of mutations as predictors of M. tuberculosis drug-resistant phenotypes. These data have implications for the clinical interpretation of molecular diagnostics and next-generation sequencing as well as efficient individualised therapy for patients with drug-resistant tuberculosis., Competing Interests: Conflict of interest: Disclosures can be found alongside this article at erj.ersjournals.com, (Copyright ©ERS 2017.)

Background: Phylogenetic studies of bacteria have been based so far either on a single gene (usually the 16S rRNA) or on concatenated housekeeping genes. For what concerns the genus Mycobacterium these approaches support the separation of rapidly and slowly growing species and the clustering of most species in well-defined phylogenetic groups. The advent of high-throughput shotgun sequencing leads us to revise conventional taxonomy of mycobacteria on the light of genomic data. For this purpose we investigated 88 newly sequenced species in addition to 60 retrieved from GenBank and used the Average Nucleotide Identity pairwise scores to reconstruct phylogenetic relationships within this genus., Results: Our analysis confirmed the separation of slow and rapid growers and the intermediate position occupied by the M. terrae complex. Among the rapid growers, the species of the M. chelonae-abscessus complex belonged to the most ancestral cluster. Other major clades of rapid growers included the species related to M. fortuitum and M. smegmatis and a large grouping containing mostly environmental species rarely isolated from humans. The members of the M. terrae complex appeared as the most ancestral slow growers. Among slow growers two deep branches led to the clusters of species related to M. celatum and M. xenopi and to a large group harboring most of the species more frequently responsible of disease in humans, including the major pathogenic mycobacteria (M. tuberculosis, M. leprae, M. ulcerans). The species previously grouped in the M. simiae complex were allocated in a number of sub-clades; of them, only the one including the species M. simiae identified the real members of this complex. The other clades included also species previously not considered related to M. simiae. The ANI analysis, in most cases supported by Genome to Genome Distance and by Genomic Signature-Delta Difference, showed that a number of species with standing in literature were indeed synonymous., Conclusions: Genomic data revealed to be much more informative in comparison with phenotype. We believe that the genomic revolution enabled by high-throughput shotgun sequencing should now be considered in order to revise the conservative approaches still informing taxonomic sciences., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Drug Resistant Tuberculosis (DRTB) is an emerging problem world-wide. In order to control the disease and decrease the number of cases overtime a prompt diagnosis followed by an appropriate treatment should be provided to patients. Phenotypic DST based on liquid automated culture has greatly reduced the time needed to generate reliable data but has the drawback to be expensive and prone to contamination in the absence of appropriate infrastructures. In the past 10 years molecular biology tools have been developed. Those tools target the main mutations responsible for DRTB and are now globally accessible in term of cost and infrastructures needed for the implementation. The dissemination of the Xpert MTB/rif has radically increased the capacity to perform the detection of rifampicin resistant TB cases. One of the main challenges for the large scale implementation of molecular based tests is the emergence of conflicting results between phenotypic and genotypic tests. This mines the confidence of clinicians in the molecular tests and delays the initiation of an appropriate treatment. A new technique is revolutionizing the genotypic approach to DST: the WGS by Next-Generation Sequencing technologies. This methodology promises to become the solution for a rapid access to universal DST, able indeed to overcome the limitations of the current phenotypic and genotypic assays. Today the use of the generated information is still challenging in decentralized facilities due to the lack of automation for sample processing and standardization in the analysis.The growing knowledge of the molecular mechanisms at the basis of drug resistance and the introduction of high-performing user-friendly tools at peripheral level should allow the very much needed accurate diagnosis of DRTB in the near future.

The taxonomic position of members of the Mycobacterium abscessus complex has been the subject of intensive investigation and, in some aspects confusion, in recent years as a result of varying approaches to genetic data interpretation. Currently, the former species Mycobacterium massiliense and Mycobacterium bolletii are grouped together as Mycobacterium abscessus subsp. bolletii. They differ greatly, however, as the former M. bolletii has a functional erm(41) gene that confers inducible resistance to macrolides, the primary therapeutic antimicrobials for M. abscessus, while in the former M. massiliense the erm(41) gene is non-functional. Furthermore, previous whole genome studies of the M. abscessus group support the separation of M. bolletii and M. massiliense. To shed further light on the population structure of Mycobacterium abscessus, 43 strains and three genomes retrieved from GenBank were subjected to pairwise comparisons using three computational approaches: verage ucleotide dentity, enome to enome istance and single nucleotide polymorphism analysis. The three methods produced overlapping results, each demonstrating three clusters of strains corresponding to the same number of taxonomic entities. The distances were insufficient to warrant distinction at the species level, but met the criteria for differentiation at the subspecies level. Based on prior erm(41)-related phenotypic data and current genomic data, we conclude that the species M. abscessus encompasses, in adjunct to the presently recognized subspecies M. abscessus subsp. abscessus and M. abscessus subsp. bolletii, a third subspecies for which we suggest the name M. abscessus subsp. massiliense comb. nov. (type strain CCUG 48898T=CIP 108297T=DSM 45103T=KCTC 19086T).

Aim of this study was to investigate a pseudo-outbreak of Mycobacterium gordonae analyzing isolates detected from clinical and environmental samples. Mycobacterium gordonae was detected in 7 out of 497 broncho-alveolar lavage (BAL) samples after bronchoscopy procedure in patients admitted to a teaching hospital between January and April 2013. During this pseudo-outbreak clinical, epidemiological, environmental and molecular investigations were performed. None of the patients met the criteria for non-tuberculous mycobacterial (NTM) lung disease and were treated for M. gordonae lung disease. Environmental investigation revealed M. gordonae in 3 samples: in tap water and in the water supply channel of the washer disinfector. All the isolates were subjected to genotyping by pulsed-field gel electrophoresis (PFGE). The PFGE revealed that only patients' isolates presented the same band pattern but no correlation with the environmental strain was detected. Surveillance of the outbreak and the strict adherence to the reprocessing procedure and its supplies resulted afterwards in no detection of M. gordonae in clinical respiratory samples. Clinical surveillance of patients was crucial to establish the start of NTM treatment. Regular screening of tap water and endoscopic equipment should be adopted to compare the clinical strains with the environmental ones when an outbreak occurs.

The name 'Mycobacterium angelicum' dates back to 2003 when it was suggested for a slowly growing mycobacterium isolated from freshwater angelfish. This name is revived here and the novel species is proposed on the basis of the polyphasic characterization of four strains including the original one. The four strains presented 100 % 16S rRNA gene sequence similarity with Mycobacterium szulgai but clearly differed from M. szulgai for the milky white aspect of the colonies. The sequence similarity with the type strain of M. szulgai ranged, in eight additionally investigated genetic targets, from 78.9 to 94.3 %, an evident contrast with the close relatedness that emerged at the level of 16S rRNA gene. The average nucleotide identity between the genomes of M. szulgai DSM 44166T and strain 126/5/03T (type strain of the novel species) was 92.92 %, and supported the status of independent species. The confirmation of the name Mycobacterium angelicum sp. nov. is proposed, with strain 126/5/03T ( = CIP 109313T = DSM 45057T) as the type strain.

Continued progress in addressing challenges associated with detection and management of tuberculosis requires new diagnostic tools. These tools must be able to provide rapid and accurate information for detecting resistance to guide selection of the treatment regimen for each patient. To achieve this goal, globally representative genotypic, phenotypic, and clinical data are needed in a standardized and curated data platform. A global partnership of academic institutions, public health agencies, and nongovernmental organizations has been established to develop a tuberculosis relational sequencing data platform (ReSeqTB) that seeks to increase understanding of the genetic basis of resistance by correlating molecular data with results from drug susceptibility testing and, optimally, associated patient outcomes. These data will inform development of new diagnostics, facilitate clinical decision making, and improve surveillance for drug resistance. ReSeqTB offers an opportunity for collaboration to achieve improved patient outcomes and to advance efforts to prevent and control this devastating disease., (Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Resistance to fluoroquinolones (FLQ) and second-line injectable drugs (SLID) is steadily increasing, especially in eastern European countries, posing a serious threat to effective tuberculosis (TB) infection control and adequate patient management. The availability of rapid molecular tests for the detection of extensively drug-resistant TB (XDR-TB) is critical in areas with high rates of multidrug-resistant TB (MDR-TB) and XDR-TB and limited conventional drug susceptibility testing (DST) capacity. We conducted a multicenter study to evaluate the performance of the new version (v2.0) of the Genotype MTBDRsl assay compared to phenotypic DST and sequencing on a panel of 228 Mycobacterium tuberculosis isolates and 231 smear-positive clinical specimens. The inclusion of probes for the detection of mutations in the eis promoter region in the MTBDRsl v2.0 test resulted in a higher sensitivity for detection of kanamycin resistance for both direct and indirect testing (96% and 95.4%, respectively) than that seen with the original version of the assay, whereas the test sensitivities for detection of FLQ resistance remained unchanged (93% and 83.6% for direct and indirect testing, respectively). Moreover, MTBDRsl v2.0 showed better performance characteristics than v1.0 for the detection of XDR-TB, with high specificity and sensitivities of 81.8% and 80.4% for direct and indirect testing, respectively. MTBDRsl v2.0 thus represents a reliable test for the rapid detection of resistance to second-line drugs and a useful screening tool to guide the initiation of appropriate MDR-TB treatment., (Copyright © 2015 Tagliani et al.)

Physicians are more and more often challenged by difficult-to-treat cases of TB. They include patients infected by strains of Mycobacterium tuberculosis that are resistant to at least isoniazid and rifampicin (multidrug-resistant TB) or to at least one fluoroquinolone (FQ) and one injectable, second-line anti-TB drug in addition to isoniazid and rifampicin (extensively drug-resistant TB). The drug treatment of these cases is very long, toxic, and expensive, and, unfortunately, the proportion of unsatisfactory outcomes is still considerably high. Although FQs and pyrazinamide (PZA) are backbone drugs in the available anti-TB regimens, several uncertainties remain about their mechanisms of action and even more remain about the mechanisms leading to drug resistance. From a clinical point of view, a better understanding of the genetic basis of drug resistance will aid (1) clinicians to provide quality clinical management to both drug-susceptible and drug-resistant TB cases (while preventing emergence of further resistance), and (2) developers of new molecular-based diagnostic assays to better direct their research efforts toward a new generation of sensitive, specific, cheap, and easy-to-use point-of-care diagnostics. In this review we provide an update on the molecular mechanisms leading to FQ- and PZA-resistance in M tuberculosis.

Tuberculosis remains a major global public health challenge. Although incidence is decreasing, the proportion of drug-resistant cases is increasing. Technical and operational complexities prevent Mycobacterium tuberculosis drug susceptibility phenotyping in the vast majority of new and retreatment cases. The advent of molecular technologies provides an opportunity to obtain results rapidly as compared to phenotypic culture. However, correlations between genetic mutations and resistance to multiple drugs have not been systematically evaluated. Molecular testing of M. tuberculosis sampled from a typical patient continues to provide a partial picture of drug resistance. A database of phenotypic and genotypic testing results, especially where prospectively collected, could document statistically significant associations and may reveal new, predictive molecular patterns. We examine the feasibility of integrating existing molecular and phenotypic drug susceptibility data to identify associations observed across multiple studies and demonstrate potential for well-integrated M. tuberculosis mutation data to reveal actionable findings., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Rationale: In the absence of active tuberculosis, a positive tuberculin skin test (TST) or interferon-γ release assay (IGRA) result defines latent infection with Mycobacterium tuberculosis, although test results may vary depending on immunodeficiency., Objectives: This study compared the performance of TST and IGRAs in five different groups of immunocompromised patients, and evaluated their ability to identify those at risk for development of tuberculosis., Methods: Immunocompromised patients with HIV infection, chronic renal failure, rheumatoid arthritis, solid-organ or stem-cell transplantation, and healthy control subjects were evaluated head-to-head by the TST, QuantiFERON-TB-Gold in-tube test (ELISA), and T-SPOT.TB test (enzyme-linked immunospot) at 17 centers in 11 European countries. Development of tuberculosis was assessed during follow-up., Measurements and Main Results: Frequencies of positive test results varied from 8.7 to 15.9% in HIV infection (n = 768), 25.3 to 30.6% in chronic renal failure (n = 270), 25.0% to 37.2% in rheumatoid arthritis (n = 199), 9.0 to 20.0% in solid-organ transplant recipients (n = 197), 0% to 5.8% in stem-cell transplant recipients (n = 103), and 11.2 to 15.2% in immunocompetent control subjects (n = 211). Eleven patients (10 with HIV infection and one solid-organ transplant recipient) developed tuberculosis during a median follow-up of 1.8 (interquartile range, 0.2-3.0) years. Six of the 11 patients had a negative or indeterminate test result in all three tests at the time of screening. Tuberculosis incidence was generally low, but higher in HIV-infected individuals with a positive TST (3.25 cases per 100 person-years) than with a positive ELISA (1.31 cases per 100 person-years) or enzyme-linked immunospot result (1.78 cases per 100 person-years). No cases of tuberculosis occurred in patients who received preventive chemotherapy., Conclusions: Among immunocompromised patients evaluated in this study, progression toward tuberculosis was highest in HIV-infected individuals and was poorly predicted by TST or IGRAs. Clinical trial registered with www.clinicaltrials.gov (NCT 00707317).

Extrapulmonary tuberculosis (EPTB) accounts for a significant proportion of tuberculosis cases worldwide. Nevertheless, the diagnosis is often delayed or even missed due to insidious clinical presentation and poor performance of diagnostic tests. Culture, the classical gold standard for tuberculosis, suffers from increased technical and logistical constraints in EPTB cases. In this review the authors outline current diagnostic options for the main forms of EPTB. The authors also discuss the opportunities and challenges linked in particular to microbiological diagnostics and to the attempts to find a new gold standard test for EPTB. Finally, new biomarkers and tests currently under evaluation are hopefully on the way to introduce significant improvements in EPTB diagnosis, for which clinical suspicion will nevertheless be essential.

The central proteins for protection against tuberculosis are attributed to interferon-γ, tumor necrosis factor-α, interleukin (IL)-6 and IL-1β, while IL-10 primarily suppresses anti-mycobacterial responses. Several studies found alteration of expression profile of genes involved in anti-mycobacterial responses in macrophages and natural killer (NK) cells from active and latent tuberculosis and from tuberculosis and healthy controls. This alteration of cellular composition might be regulated by microRNAs (miRNAs). Albeit only 1% of the genomic transcripts in mammalian cells encode miRNA, they are predicted to control the activity of more than 60% of all protein-coding genes and they have a huge influence in pathogenesis theory, diagnosis and treatment approach to some diseases. Several miRNAs have been found to regulate T cell differentiation and function and have critical role in regulating the innate function of macrophages, dendritic cells and NK cells. Here, we have reviewed the role of miRNAs implicated in tuberculosis infection, especially related to their new roles in the molecular pathology of tuberculosis immunology and as new targets for future tuberculosis diagnostics., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Mycobacterium tuberculosis (Mtb) has evolved multiple mechanisms to manipulate its cellular niche for its own advantage. Many efforts have been made to understand basal mechanisms of mycobacterial infections. However, the underlying molecular regulation is not fully understood. Recently, a new class of non-coding, small RNAs, called microRNAs (miRNAs), has emerged as important regulators in biological processes, and their involvement in mycobacterial infection has been identified, thus opening a new field of research., Methods: This study aimed to determine by TaqMan Low Density Array the host genome-wide miRNA expression profile of primary human monocyte-derived macrophages (MDM) infected with two members of the Mtb complex: virulent Mtb H37Rv and the non-virulent vaccine strain Mycobacterium bovis Bacillus Calmette-Guerin (BCG) in comparison with chemically-inactivated Mtb bacilli., Results: The findings of this study showed that infection of MDM with H37Rv or BCG results in a signature of miRNA expression mostly overlapping between the two mycobacteria. A substantially different signature emerged from infection with killed virulent bacilli, suggesting an active influence of live intracellular bacteria on cellular miRNA metabolism. Specifically, Mtb induced miRNA signature is composed of miRNAs well established in immune regulation, miR-155 and miR-146a, as well as a set of miRNAs newly associated with Mtb infection: miR-145, miR-222(∗), miR-27a and miR-27b. All of these miRNAs are predicted to target important immune-related genes., Conclusions: This study signifies the miRNA host response upon intracellular mycobacterial infection in macrophages, providing new aspects of regulation in host-pathogen interactions, at post-transcriptional levels., (Copyright © 2013. Published by Elsevier Ltd.)

If tuberculosis (TB) is to be eliminated as a global health problem in the foreseeable future, improved detection of patients, earlier diagnosis and timely identification of rifampicin resistance will be critical. New diagnostics released in recent years have improved this perspective but they require investments in laboratory infrastructure, biosafety and staff specialisation beyond the means of many resource-constrained settings where most patients live. Xpert MTB/RIF, a new assay employing automated nucleic acid amplification to detect Mycobacterium tuberculosis, as well as mutations that confer rifampicin resistance, holds the promise to largely overcome these operational challenges. In this article we position Xpert MTB/RIF in today's TB diagnostic landscape and describe its additional potential as an adjunct to surveillance and surveys, taking into account considerations of pricing and ethics. In what could serve as a model for the future formulation of new policy on diagnostics, we trace the unique process by which the World Health Organization consulted international expertise and systematically assessed published evidence and freshly emerging experience from the field ahead of its endorsement of the Xpert MTB/RIF technology in 2010, summarise subsequent research findings and guidance on who to test and how, and provide perspectives on scaling up the new technology.

Despite the improvements in the global fight against tuberculosis (TB), critical points still remain and fuel the epidemic. Even today, only 30% of the estimate number of people suffering from TB worldwide are correctly diagnosed, and lower proportions of cases are diagnosed in high-TB-burden, low-resource settings. Current TB diagnostics are still suboptimal in their performance for childhood TB, smear-negative TB, extrapulmonary TB, HIV-TB and drug-resistant TB. Furthermore, there is no gold standard test for the identification of latent TB infection status. Improving diagnosis is therefore a strategic goal in TB research, and the pipeline of diagnostic tools is rapidly growing: new ways of performing "old" tests (e.g. sputum smear microscopy) and completely innovative tools (e.g. new technologies for molecular diagnosis) are under investigation or have already been endorsed by WHO. Some of the structural limits of current TB diagnostics are likely to be overcome by such new tools, but research is still needed. Finally, the roll-out of new technologies and the development of newer ones will necessarily have to take into account the diagnostic needs of each context they are directed to (point-of-need testing approach), together with the logistic, economic and technical constraints present in the majority of high-TB-burden settings.

We evaluate the performance of the GenoType® MTBDRsl (Hain Lifescience Nehren, Germany) for the detection of second-line resistant tuberculosis and we correlate the frequency of mutations to different Mycobacterium tuberculosis genotypes. We tested 175 strains and 59 clinical specimens interpreting the results according to the Standards for Reporting of Diagnostic Accuracy recommendations. All the strains were also investigated by spoligotyping and Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats typing. The performances of the MTBDRsl in detecting resistance to fluoroquinolones (FQ), second-line injectable drugs (SLID), and ethambutol (EMB) on clinical isolates were similar (specificity ∼ 99%, sensitivity ∼ 70%, and positive predictive value (PPV) ∼ 99%). Of the 59 respiratory specimens, three samples were classified as "indeterminate". The specificity in detecting resistances was similar for FQs and EMB 100% (95% CI 92.7-100%) and 100% (95% CI 83.9-100%), respectively with a PPV of 100% (95% CI 64.6-100%) and 100% (95% CI 87.9-100%), respectively. Detection of SLID showed a specificity of 89.1% (95% CI 77.0-95.3%) and a PPV of 58.3% (95% CI 32.0-80.7%). Sensitivity for FQ-resistance detection was 100% (95% CI 64.6-100%), whereas for SLID and EMB it was 89.1% (95% CI 77.0-95.3%) and 86.1% (95% CI 71.3-93.9%), respectively. We detected a significant association between mutations in the rrs gene and Beijing lineage. The MTBDRsl can be used to "rule in" extensively drug-resistant strains of tuberculosis in a high risk group; the low sensitivity and negative predicted value (NPV) make confirmation by conventional drug susceptibility testing mandatory when mutations are not identified. NPV for SLID is higher in Beijing strains, showing that the predictive values of the molecular tests are related to the genetic background.