Culture-free proof of Mycobacterium tuberculosis - a new assay for viable bacteria

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Linezolid resistance of Mycobacterium tuberculosis – associated mutations and risk factors: cross-sectional retrospective analytical study

Nicolae Testemitanu State University of Medicine and Pharmacy, Chisinau, Republic of Moldova, Chiril Draganiuc Phthisiopneumology Institute, Chisinau, Republic of Moldova, German Centre for Infection Research (DZIF), Partner site Hamburg-Lübeck-Borstel- Riems, Clinical Infectious Diseases, Research Center Borstel, Borstel Germany, National and Supranational Reference Center for Mycobacteria, Research Center Borstel, Borstel Germany, Molecular and Experimental Mycobacteriology, Research Center Borstel, Borstel, Germany, Respiratory Medicine & International Health, University of Lübeck, Lübeck, Germany, Department of Medicine, Umeå University, Umeå, Sweden, Global TB Program, Baylor College of Medicine and Texas Children´s Hospital, Houston, TX, USAIntroducere. Linezolidul (LNZ) este unul dintre principalele medicamente utilizate în tratamentul tuberculozei multidrog- rezistente (TB-MDR). La moment determinantele genetice asociate cu rezistenţa la LNZ nu sunt pe deplin caracterizate. Scopul studiului a fost de a descrie mutaţiile asociate cu rezistenţa la LNZ prin aplicarea secvenţierii întregului genom (WGS) și evaluarea factorilor de risc asociaţi cu rezistenţa la LNZ pe un set de tulpini de Mycobacterium tuberculosis (MTB) izolate de la bolnavii de TB-MDR din Republica Moldova. Material și metode. A fost realizat un studiu retrospectiv transversal pe un set de tulpini de MTB preluate din biobanca Laboratorului Naţional de Referinţă în Microbiologia Tuberculozei (LNRM). Au fost identificate toate tulpinile MTB stocate în perioada 2017-2018, obţinute din sputa pacienţilor cu TB-MDR, care au administrat LNZ în schema de tratament pe parcursul ultimilor doi ani, indiferent de durata administrării LNZ. Au fost considerate eligibile pentru studiu doar tulpinile MTB izolate de la pacienţii cu o expunere cumulativă la LNZ mai mare de 30 de zile. Datele WGS ale tulpinilor de MTB incluse în studiu au fost comparate cu rezultatele testelor fenotipice de sensibilitate pe medii lichide pentru 3 concentraţii de LNZ (0,5 mg/L; 1,0 mg/L; 2,0 mg/L). Caracteristicile clinice ale pacienţilor din studiu au fost preluate din Registrul naţional electronic al bolnavilor cu TB (SIME-TB). Regresia logistică multiplă a fost efectuată pentru a determina factorii de risc pentru rezistenţa la LNZ. Rezultate. 52/74 (70,3%) tulpini MTB au fost incluse în studiu, dintre acestea 15 (28,8%) au fost fenotipic rezistente la LNZ, dintre care în cazul a 12/52 (23,1%) tulpini a fost găsită o corelarea genotipică cu rezistenţa la LNZ. În 8 cazuri au fost găsite mutaţii în gena rplC (460T>C). În alte 4 cazuri, au fost identificate mutaţii în gena rrl (2746G>A; 2814G>T; 2810A>C; 2270G>T). S-a determinat o asociere semnificativă între rezistenţa la LNZ și numărul de medicamente active din regimul de tratament TB-MDR (OR 0,23; 95%CI -0,03 - 0,70; p = 0,04). O asociere mai slabă s-a stabilit cu numărul de doze de LNZ administrate anterior (OR 1,01; 95%CI 1,004-1,03; p = 0,03). Concluzii. La majoritatea tulpinilor de MTB studiate, rezistenţa la LNZ a fost asociată cu mutaţii în genele rrl și rplC. Numărul insuficient de medicamente active în regimul de tratament al TB-MDR crește șansele de apariţie a rezistenţei la LNZ.Introduction. Linezolid (LNZ) is one of the main drugs used for multidrug-resistant tuberculosis (MDR-TB) treatment. Genetic targets associated with resistance to LNZ are not fully characterized. The aim of the study was to describe mutations associated with LNZ resistance by applying whole genome sequencing (WGS) and to assess the risk factors for LNZ resistance in a set of M. tuberculosis clinical isolates from the Republic of Moldova. Material and methods. We conducted a retrospective cross-sectional study on a set of MTB isolates retrieved from the biobank of National Reference Laboratory for Tuberculosis Microbiology (NRLM). We identified all sputum culture isolates stored during 2017-2018, obtained from adult MDR-TB patients who had LNZ as part of their MDR-TB treatment at any time during two years before the collection corresponding sputum sample. Only isolates from patients with a cumulative exposure to LNZ longer than 30 days were considered eligible for the study. We performed WGS of the MTB strains and compared these results with liquid culture-based susceptibility tests on 3 concentrations of LNZ (0.5 mg/L; 1.0 mg/L; 2.0 mg/L). The clinical characteristics of the study patients were retrieved form the National TB Data Base (SIME-TB). The multiple regression analysis was performed to assess risk factors for LNZ resistance. Results. 52/74 (70,3%) isolates were included into the study of them 15 (28,8%) were phenotypically LNZ resistant. However only in 12/52 (23,1%) isolates genotypic correlates of LNZ resistant was found. In 8 cases mutations were detected in the rplC gene (460T>C). In other 4 cases the identified mutation implied rrl gene (2746G>A; 2814G>T; 2810A>C; 2270G>T). In a multivariate logistic regression model, significant association between LNZ resistance and the number of the active drugs in the MDR-TB treatment regimen (OR 0,23; 95%CI 0,03 – 0,70; p = 0,04) was found. A weaker association was found with the number of the previously taken LNZ doses (OR 1,01; 95%CI 1,004 – 1,03; p=0,03). Conclusions. In most of the studied MTB isolates LNZ resistance was associated with mutations in the rrl and rplC genes. A reduce number of the active drugs in the LNZ containing treatment regimen increase the chance of LNZ resistance emergence

Pulmonary vasculitis due to infection with Mycobacterium goodii : A case report

A 57-year-old Caucasian woman suffered from dyspnea on exertion. One year following a supposed pulmonary embolism event, a chronic thromboembolic vasculopathy was diagnosed and a pulmonary thromboendarterectomy was performed. However, a granulomatous pulmonary arterial vasculitis was identified upon examination. DNA of Mycobacterium goodii was detected as the most likely causative agent. Anti-inflammatory and anti-mycobacterial therapy was initiated for more than 12 months. Regular PET-CT scans revealed improvement under therapy. The last PET-CT did not show any tracer uptake following 10 months of therapy

Rapid diagnosis of pulmonary tuberculosis by combined molecular and immunological methods.

Diagnosing pulmonary tuberculosis (TB) may be delayed until culture results become available.We ascertained the accuracy of a stepwise diagnostic algorithm for the rapid diagnosis of pulmonary TB by GeneXpert from sputum and/or bronchoalveolar lavage (BAL) followed by a Mycobacterium tuberculosis-specific BAL ELISPOT assay in patients with a suspected diagnosis of pulmonary TB at a clinical referral centre in Germany.Among 166 patients with a presumptive diagnosis of pulmonary TB, 81 cases were confirmed by M. tuberculosis culture from sputum and/or BAL. In 66 out of 81 (81.5%) cases, patients initially had M. tuberculosis detected by GeneXpert from sputum; in addition, six out of 81 (7.4%) cases were diagnosed by GeneXpert on BAL fluid (together 72 out of 81 (88.9%) patients). Out of the remaining nine patients with negative GeneXpert results from sputum and BAL, BAL ELISPOT identified eight patients with culture-confirmed TB correctly (median time to culture positivity 26 days). At a cut-off of >4000 early secretory antigenic target-6- or culture filtrate protein-10-specific interferon-γ-producing lymphocytes per 1 000 0000 lymphocytes, the specificity of the BAL ELISPOT for active TB was 97%.In low TB incidence countries, nearly all patients with active pulmonary TB can be identified within the first few days of clinical presentation using a stepwise strategy with GeneXpert and BAL ELISPOT

Brief Research Report: Serum clara cell 16 kDa protein levels are increased in patients hospitalized for severe SARS-CoV-2 or sepsis infection

Background Clara cell 16 kDa protein (CC16) is a secretory protein primarily expressed in epithelial cells in the lungs. Previous studies show that CC16 exerts anti-inflammatory and immune-modulatory properties in both acute and chronic pulmonary diseases. However, despite the evidence of CC16's high biomarker potential, evaluation of its role in infectious diseases is yet very limited. Methods Serum CC16 concentrations were measured by ELISA and assessed in two different types of severe infections. Using a case-control study design, patients treated for either severe SARS-CoV-2 or severe non-pulmonary sepsis infection were compared to age- and sex-matched healthy human subjects. Results Serum CC16 was significantly increased in both types of infection (SARS-CoV-2: 96.22 ± 129.01 ng/ml vs. healthy controls: 14.05 ± 7.48 ng/ml, p = 0.022; sepsis: 35.37 ± 28.10 ng/ml vs. healthy controls: 15.25 ± 7.51 ng/ml, p = 0.032) but there were no distinct differences between infections with and without pulmonary focus (p = 0.089). Furthermore, CC16 serum levels were positively correlated to disease duration and inversely to the platelet count in severe SARS-CoV-2 infection. Conclusions Increased CC16 serum levels in both SARS-CoV-2 and sepsis reinforce the high potential as a biomarker for epithelial cell damage and bronchoalveolar-blood barrier leakage in pulmonary as well as non-pulmonary infectious diseases

Developing biomarkers assays to accelerate tuberculosis drug development : defining target product profiles

Funding: This study is supported by the UNITE4TB consortium (www.unite4tb.org). UNITE4TB has received funding from the Innovative Medicines Initiative 2 Joint Undertaking (JU) under grant agreement No 101007873. The JU receives support from the European Union’s Horizon 2020 research and innovation programme and EFPIA, Deutsches Zentrum für Infektionsforschung e. V. (DZIF), and Ludwig-Maximilians-Universität München (LMU; Munich, Germany). EFPIA/AP contribute to 50% of funding, whereas the contribution of DZIF and the LMU Hospital Munich has been granted by the German Federal Ministry of Education and Research. CL is supported by DZIF under grant TTU-TB 02.709.Drug development for tuberculosis is hindered by the methodological limitations in the definitions of patient outcomes, particularly the slow organism growth and difficulty in obtaining suitable and representative samples throughout the treatment. We developed target product profiles for biomarker assays suitable for early-phase and late-phase clinical drug trials by consulting subject-matter experts on the desirable performance and operational characteristics of such assays for monitoring of tuberculosis treatment in drug trials. Minimal and optimal criteria were defined for scope, intended use, pricing, performance, and operational characteristics of the biomarkers. Early-stage trial assays should accurately quantify the number of viable bacilli, whereas late-stage trial assays should match the number, predict relapse-free cure, and replace culture conversion endpoints. The operational criteria reflect the infrastructure and resources available for drug trials. The effective tools should define the sterilising activity of the drug and lower the probability of treatment failure or relapse in people with tuberculosis. The target product profiles outlined in this Review should guide and de-risk the development of biomarker-based assays suitable for phase 2 and 3 clinical drug trials.Peer reviewe

Tuberculostearic Acid-Containing Phosphatidylinositols as Markers of Bacterial Burden in Tuberculosis

One-fourth of the global human population is estimated to be infected with strains of the Mycobacterium tuberculosis complex (MTBC), the causative agent of tuberculosis (TB). Using lipidomic approaches, we show that tuberculostearic acid (TSA)-containing phosphatidylinositols (PIs) are molecular markers for infection with clinically relevant MTBC strains and signify bacterial burden. For the most abundant lipid marker, detection limits of ∼10$^{2}$ colony forming units (CFUs) and ∼10$^{3}$ CFUs for bacterial and cell culture systems were determined, respectively. We developed a targeted lipid assay, which can be performed within a day including sample preparation─roughly 30-fold faster than in conventional methods based on bacterial culture. This indirect and culture-free detection approach allowed us to determine pathogen loads in infected murine macrophages, human neutrophils, and murine lung tissue. These marker lipids inferred from mycobacterial PIs were found in higher levels in peripheral blood mononuclear cells of TB patients compared to healthy individuals. Moreover, in a small cohort of drug-susceptible TB patients, elevated levels of these molecular markers were detected at the start of therapy and declined upon successful anti-TB treatment. Thus, the concentration of TSA-containing PIs can be used as a correlate for the mycobacterial burden in experimental models and in vitro systems and may prospectively also provide a clinically relevant tool to monitor TB severity

Perspectives for systems biology in the management of tuberculosis

Standardised management of tuberculosis may soon be replaced by individualised, precision medicine-guided therapies informed with knowledge provided by the field of systems biology. Systems biology is a rapidly expanding field of computational and mathematical analysis and modelling of complex biological systems that can provide insights into mechanisms underlying tuberculosis, identify novel biomarkers, and help to optimise prevention, diagnosis and treatment of disease. These advances are critically important in the context of the evolving epidemic of drug-resistant tuberculosis. Here, we review the available evidence on the role of systems biology approaches - human and mycobacterial genomics and transcriptomics, proteomics, lipidomics/metabolomics, immunophenotyping, systems pharmacology and gut microbiomes - in the management of tuberculosis including prediction of risk for disease progression, severity of mycobacterial virulence and drug resistance, adverse events, comorbidities, response to therapy and treatment outcomes. Application of the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) approach demonstrated that at present most of the studies provide "very low" certainty of evidence for answering clinically relevant questions. Further studies in large prospective cohorts of patients, including randomised clinical trials, are necessary to assess the applicability of the findings in tuberculosis prevention and more efficient clinical management of patients.Publisher PDFPeer reviewe