Melissa Wickremasinghe

Transcriptional blood signatures distinguish pulmonary tuberculosis, pulmonary sarcoidosis, pneumonias and lung cancers.

Rationale New approaches to define factors underlying the immunopathogenesis of pulmonary diseases including sarcoidosis and tuberculosis are needed to develop new treatments and biomarkers. Comparing the blood transcriptional response of tuberculosis to other similar pulmonary diseases will advance knowledge of disease pathways and help distinguish diseases with similar clinical presentations. Objectives To determine the factors underlying the immunopathogenesis of the granulomatous diseases, sarcoidosis and tuberculosis, by comparing the blood transcriptional responses in these and other pulmonary diseases. Methods We compared whole blood genome-wide transcriptional profiles in pulmonary sarcoidosis, pulmonary tuberculosis, to community acquired pneumonia and primary lung cancer and healthy controls, before and after treatment, and in purified leucocyte populations. Measurements and Main Results An Interferon-inducible neutrophil-driven blood transcriptional signature was present in both sarcoidosis and tuberculosis, with a higher abundance and expression in tuberculosis. Heterogeneity of the sarcoidosis signature correlated significantly with disease activity. Transcriptional profiles in pneumonia and lung cancer revealed an over-abundance of inflammatory transcripts. After successful treatment the transcriptional activity in tuberculosis and pneumonia patients was significantly reduced. However the glucocorticoid-responsive sarcoidosis patients showed a significant increase in transcriptional activity. 144-blood transcripts were able to distinguish tuberculosis from other lung diseases and controls. Conclusions Tuberculosis and sarcoidosis revealed similar blood transcriptional profiles, dominated by interferon-inducible transcripts, while pneumonia and lung cancer showed distinct signatures, dominated by inflammatory genes. There were also significant differences between tuberculosis and sarcoidosis in the degree of their transcriptional activity, the heterogeneity of their profiles and their transcriptional response to treatment.

Utility of endobronchial ultrasound-guided transbronchial needle aspiration in patients with tuberculous intrathoracic lymphadenopathy: a multicentre study

Background Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) has emerged as an important tool for the diagnosis and staging of lung cancer but its role in the diagnosis of tuberculous intrathoracic lymphadenopathy has not been established. The aim of this study was to describe the diagnostic utility of EBUS-TBNA in patients with intrathoracic lymphadenopathy due to tuberculosis (TB). Methods 156 consecutive patients with isolated intrathoracic TB lymphadenitis were studied across four centres over a 2-year period. Only patients with a confirmed diagnosis or unequivocal clinical and radiological response to antituberculous treatment during follow-up for a minimum of 6 months were included. All patients underwent routine clinical assessment and a CT scan prior to EBUS-TBNA. Demographic data, HIV status, pathological findings and microbiological results were recorded. Results EBUS-TBNA was diagnostic of TB in 146 patients (94%; 95% CI 88% to 97%). Pathological findings were consistent with TB in 134 patients (86%). Microbiological investigations yielded a positive culture of TB in 74 patients (47%) with a median time to positive culture of 16 days (range 3–84) and identified eight drug-resistant cases (5%). Ten patients (6%) did not have a specific diagnosis following EBUS; four underwent mediastinoscopy which confirmed the diagnosis of TB while six responded to empirical antituberculous therapy. There was one complication requiring an inpatient admission. Conclusions EBUS-TBNA is a safe and effective first-line investigation in patients with tuberculous intrathoracic lymphadenopathy.

Rv3615c is a highly immunodominant RD1 (Region of Difference 1)-dependent secreted antigen specific for Mycobacterium tuberculosis infection.

The 6-kDa early secretory antigenic target of Mycobacterium tuberculosis (ESAT-6) and the 10-kDa culture filtrate antigen (CFP-10), encoded in region of difference 1 (RD1) and secreted by the ESAT-6 system 1 (Esx-1) secretion system, are the most immunodominant and highly M. tuberculosis (MTB)-specific antigens. These attributes are responsible for their primary importance in tuberculosis (TB) immunodiagnosis and vaccine development. Rv3615c [Esx-1 substrate protein C (EspC)], encoded outside RD1, is similar in size and sequence homology to CFP-10 and ESAT-6, suggesting it might be a target of cellular immunity in TB. Using ex vivo enzyme-linked immunospot- and flow cytometry-based cytokine-secretion assay, we comprehensively assessed cellular immune responses to EspC in patients with active TB, latently infected persons, and uninfected bacillus Calmette–Guérin (BCG)-vaccinated controls. EspC was at least as immunodominant as ESAT-6 and CFP-10 in both active and latent TB infection. EspC contained broadly recognized CD4+ and CD8+ epitopes, inducing a predominantly CD4+ T-cell response that comprised functional T-cell subsets secreting both IFN-γ and IL-2 as well as functional T-cell subsets secreting only IFN-γ. Surprisingly, T-cell responses to EspC were as highly specific (93%) for MTB infection as responses to ESAT-6 and CFP-10, with only 2 of 27 BCG-vaccinated controls responding to each antigen. Using quantitative proteomics and metabolically labeled mutant and genetically complemented MTB strains, we identified the mechanism of the specificity of anti-EspC immunity as the Esx-1 dependence of EspC secretion. The high immunodominance of EspC, equivalent to that of ESAT-6 and CFP-10, makes it a TB vaccine candidate, and its high specificity confers strong potential for T-cell–based immunodiagnosis.

Rituximab in severe, treatment‐refractory interstitial lung disease

In patients with severe interstitial lung disease (ILD) progressing despite conventional immunosuppression, rituximab, a B‐lymphocyte depleting monoclonal antibody, may offer an effective rescue therapy.

Enumeration of functional T-cell subsets by fluorescence-immunospot defines signatures of pathogen burden in tuberculosis.

Background IFN-γ and IL-2 cytokine-profiles define three functional T-cell subsets which may correlate with pathogen load in chronic intracellular infections. We therefore investigated the feasibility of the immunospot platform to rapidly enumerate T-cell subsets by single-cell IFN-γ/IL-2 cytokine-profiling and establish whether immunospot-based T-cell signatures distinguish different clinical stages of human tuberculosis infection. Methods We used fluorophore-labelled anti-IFN-γ and anti-IL-2 antibodies with digital overlay of spatially-mapped colour-filtered images to enumerate dual and single cytokine-secreting M. tuberculosis antigen-specific T-cells in tuberculosis patients and in latent tuberculosis infection (LTBI). We validated results against established measures of cytokine-secreting T-cells. Results Fluorescence-immunospot correlated closely with single-cytokine enzyme-linked-immunospot for IFN-γ-secreting T-cells and IL-2-secreting T-cells and flow-cytometry-based detection of dual IFN-γ/IL-2-secreting T-cells. The untreated tuberculosis signature was dominated by IFN-γ-only-secreting T-cells which shifted consistently in longitudinally-followed patients during treatment to a signature dominated by dual IFN-γ/IL-2-secreting T-cells in treated patients. The LTBI signature differed from active tuberculosis, with higher proportions of IL-2-only and IFN-γ/IL-2-secreting T-cells and lower proportions of IFN-γ-only-secreting T-cells. Conclusions Fluorescence-immunospot is a quantitative, accurate measure of functional T-cell subsets; identification of cytokine-signatures of pathogen burden, distinct clinical stages of M. tuberculosis infection and long-term immune containment suggests application for treatment monitoring and vaccine evaluation.

Evaluation of screening methods for identification of patients with chronic rheumatological disease requiring tuberculosis chemoprophylaxis prior to commencement of TNF-α antagonist therapy

Background Patients undergoing tumour necrosis factor (TNF)-α antagonist therapy are at increased risk of latent tuberculosis infection (LTBI) reactivation. The aim of this study was to determine the optimum available screening strategy for identifying patients for tuberculosis (TB) chemoprophylaxis. Methods We conducted a prospective observational study of consecutive adults with chronic rheumatological disease referred for LTBI screening prior to commencement of TNF-α antagonist therapy. All patients included had calculation of TB risk according to age, ethnicity and year of UK entry, as described in the 2005 British Thoracic Society (BTS) guidelines and measurement of tuberculin skin test (TST) and T.Spot.TB. Results There were 187 patients included in the study, with 157 patients (84%) taking immunosuppressants. 137 patients would require further risk stratification according to the BTS algorithm, with 110 (80.3%) classified as being at low risk of having LTBI. There were 39 patients (35.5%) who were categorised as low risk but were either TST and/or T.Spot positive and would not have received chemoprophylaxis according to the BTS algorithm. Combination of all three methods (risk stratification and/or positive T.Spot and/or positive TST) identified 66 patients out of 137 who would potentially be offered chemoprophylaxis, which was greater than any single test or two-test combination. Conclusion Performing both a TST and T.Spot in patients on immunosuppressants prior to commencement of TNF-α antagonist therapy gives an additional yield of potential LTBI compared with use of risk stratification tables alone. Our results suggest that use of all three screening modalities gives the highest yield of patients potentially requiring chemoprophylaxis.

Performance of Xpert MTB/RIF in the Diagnosis of Tuberculous Mediastinal Lymphadenopathy by Endobronchial Ultrasound

RATIONALE The Xpert (GeneXpert) MTB/RIF, an integrated polymerase chain reaction assay, has not been systematically studied in extrapulmonary and in particular mediastinal tuberculosis (TB). OBJECTIVES To investigate the performance of Xpert MTB/RIF in the diagnosis of intrathoracic nodal TB in a large tertiary urban medical center in the UK. METHODS We collected clinical, cytological, and microbiological data from two cohorts: 116 consecutive patients referred with mediastinal lymphadenopathy with detailed diagnostic information obtained, and an immediately subsequent second cohort of 52 consecutive patients with microbiologically confirmed mediastinal TB lymphadenopathy. All data were derived between January 2010 and October 2012. All patients underwent endobronchial ultrasound and transbronchial needle aspiration (TBNA). The performance of a single Xpert MTB/RIF assay alongside standard investigations, cytology, and microscopy/culture was evaluated against culture-confirmed TB. MEASUREMENTS AND MAIN RESULTS Microbiologically confirmed TB mediastinal lymphadenopathy was diagnosed in a total of 88 patients from both cohorts. Three culture-negative cases with associated caseating granulomatous inflammation on TBNA were given a probable diagnosis. A single Xpert MTB/RIF assay demonstrated overall sensitivity for culture-positive TB of 72.6% (62.3-81.0%). Xpert specificity from cohort 1 was 96.3% (89.1-99.1%). The positive predictive value was 88.9% (69.7-97.1%), negative predictive value was 86.5% (76.9-92.1%), and odds ratio was 51.3 (24.0-98.0) for correctly identifying culture-positive disease. Xpert captured all microscopy-positive cases (14 of 14) and the majority of microscopy-negative cases (48 of 71, 67.6%). Among the cases that were culture positive by TBNA, Xpert identified two-thirds of the multiple drug-resistant TB cases, leading to immediate regimen change up to 5 weeks ahead of positive cultures. The use of Xpert combined with cytology increased the sensitivity to 96.6%. CONCLUSIONS Xpert MTB/RIF provides a rapid, useful, and accurate test to diagnose mediastinal nodal TB in intermediate-incidence settings. The additional use of TBNA cytology further enhances the sensitivity of Xpert. This combination can facilitate rapid risk assessment and prompt TB treatment.

Sarcoidosis and tuberculosis cytokine profiles: indistinguishable in bronchoalveolar lavage but different in blood.

Background The clinical, radiological and pathological similarities between sarcoidosis and tuberculosis can make disease differentiation challenging. A complicating factor is that some cases of sarcoidosis may be initiated by mycobacteria. We hypothesised that immunological profiling might provide insight into a possible relationship between the diseases or allow us to distinguish between them. Methods We analysed bronchoalveolar lavage (BAL) fluid in sarcoidosis (n = 18), tuberculosis (n = 12) and healthy volunteers (n = 16). We further investigated serum samples in the same groups; sarcoidosis (n = 40), tuberculosis (n = 15) and healthy volunteers (n = 40). A cross-sectional analysis of multiple cytokine profiles was performed and data used to discriminate between samples. Results We found that BAL profiles were indistinguishable between both diseases and significantly different from healthy volunteers. In sera, tuberculosis patients had significantly lower levels of the Th2 cytokine interleukin-4 (IL-4) than those with sarcoidosis (p = 0.004). Additional serum differences allowed us to create a linear regression model for disease differentiation (within-sample accuracy 91%, cross-validation accuracy 73%). Conclusions These data warrant replication in independent cohorts to further develop and validate a serum cytokine signature that may be able to distinguish sarcoidosis from tuberculosis. Systemic Th2 cytokine differences between sarcoidosis and tuberculosis may also underly different disease outcomes to similar respiratory stimuli.

Post-bronchoscopy sputum: Improving the diagnostic yield in smear negative pulmonary TB

INTRODUCTION Patients with suspected active Pulmonary Tuberculosis (PTB) who are Acid-Fast Bacilli (AFB) smear negative or non-productive of sputum may undergo bronchoalveolar lavage. However, post-bronchoscopy sputum (PBS) sampling is not routine. The aim of this study was to establish the potential diagnostic value of PBS sampling. METHODS A retrospective study of patients attending a London University hospital with microbiologically confirmed PTB between January 2004 and December 2010. Patients who were AFB smear negative or non-productive of sputum were eligible if sputum sampling was performed within 7 days of bronchoscopy. RESULTS Over the study period, 236 patients had microbiologically confirmed smear negative PTB of which 57 patients were eligible for the study. 15 patients (26.3%) were infected with HIV. 19 patients (33.3%) converted to AFB sputum smear positivity post-bronchoscopy and 5 patients (8.8%) were exclusively AFB sputum smear positive on PBS microscopy. Mycobacterium tuberculosis was cultured from the PBS of 43 patients (75.4%) and of these, 4 (7.0%) were exclusively PBS culture positive. CONCLUSION PBS analysis can provide a simple method of rapidly diagnosing pulmonary tuberculosis. In this cohort, M. tuberculosis culture yield was increased by 7% through PBS sampling. This study has important infection control implications with nearly one third of patients becoming more infectious after bronchoscopy.

Proteomic Analysis of Kveim Reagent Identifies Targets of Cellular Immunity in Sarcoidosis

Background Kveim-reagent (Kv) skin testing was a historical method of diagnosing sarcoidosis. Intradermal injection of treated sarcoidosis spleen tissue resulted in a granuloma response at injection site by 4–6 weeks. Previous work indicates proteins as the possible trigger of this reaction. We aimed to identify Kv-specific proteins and characterise the ex vivo response of Peripheral Blood Mononuclear Cells (PBMCs) from sarcoidosis, tuberculosis and healthy control patients when stimulated with both Kv and selected Kv-specific proteins. Methods Kv extracts were separated by 1D-SDS-PAGE and 2D-DIGE and then underwent mass spectrometric analysis for protein identification. Sarcoidosis and control PBMCs were first stimulated with Kv and then with three selected recombinant protein candidates which were identified from the proteomic analysis. PBMC secreted cytokines were subsequently measured by Multiplex Cytokine Assay. Results We observed significantly increased IFN-γ and TNF-α secretion from Kv-stimulated PBMCs of sarcoidosis patients vs. PBMCs from healthy volunteers (IFN-γ: 207.2 pg/mL vs. 3.86 pg/mL, p = 0.0018; TNF-α: 2375 pg/mL vs. 42.82 pg/mL, p = 0.0003). Through proteomic approaches we then identified 74 sarcoidosis tissue-specific proteins. Of these, 3 proteins (vimentin, tubulin and alpha-actinin-4) were identified using both 1D-SDS-PAGE and 2D-DIGE. Data are available via ProteomeXchange with identifier PXD005150. Increased cytokine secretion was subsequently observed with vimentin stimulation of sarcoidosis PBMCs vs. tuberculosis PBMCs (IFN-γ: 396.6 pg/mL vs 0.1 pg/mL, p = 0.0009; TNF-α: 1139 pg/mL vs 0.1 pg/mL, p<0.0001). This finding was also observed in vimentin stimulation of sarcoidosis PBMCs compared to PBMCs from healthy controls (IFN-γ: 396.6 pg/mL vs. 0.1 pg/mL, p = 0.014; TNF-α: 1139 pg/mL vs 42.29 pg/mL, p = 0.027). No difference was found in cytokine secretion between sarcoidosis and control PBMCs when stimulated with either tubulin or alpha-actinin-4. Conclusions Stimulation with both Kveim reagent and vimentin induces a specific pro-inflammatory cytokine secretion from sarcoidosis PBMCs. Further investigation of cellular immune responses to Kveim-specific proteins may identify novel biomarkers to assist the diagnosis of sarcoidosis.