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 3e84) 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 firstline investigation in patients with tuberculous intrathoracic lymphadenopathy.

BackgroundPCR inhibition by nucleic acid extracts is a well known yet poorly described phenomenon. Inhibition assessment generally depends on the assumption that inhibitors affect all PCR reactions to the same extent; i.e. that the reaction of interest and the control reaction are equally susceptible to inhibition. To test this assumption we performed inhibition assessment on DNA extracts from human urine samples, fresh urine and EDTA using different PCR reactions.ResultsWhen copurified inhibitors were assessed using two different PCR reactions one reaction appeared to be inhibited whilst the other was not. Further experiments using various concentrations of unextracted urine to inhibit six different PCR reactions revealed that susceptibility to inhibition was highly variable between reactions. Similar results were obtained using EDTA as the PCR inhibitor. We could find no obvious explanation why one reaction should be more susceptible to inhibition than another, although a possible association with amplicon GC content was noted.ConclusionThese findings have serious implications for all PCR-based gene expression studies, including the relatively new PCR array method, and for both qualitative and quantitative PCR-based molecular diagnostic assays, suggesting that careful consideration should be given to inhibition compatibility when conducting PCR analyses. We have demonstrated unequivocally that it is not safe to assume that different PCR reactions are equally susceptible to inhibition by substances co-purified in nucleic acid extracts.

SummaryBackgroundStaphylococcus aureus bacteraemia is a common cause of severe community-acquired and hospital-acquired infection worldwide. We tested the hypothesis that adjunctive rifampicin would reduce bacteriologically confirmed treatment failure or disease recurrence, or death, by enhancing early S aureus killing, sterilising infected foci and blood faster, and reducing risks of dissemination and metastatic infection.MethodsIn this multicentre, randomised, double-blind, placebo-controlled trial, adults (≥18 years) with S aureus bacteraemia who had received ≤96 h of active antibiotic therapy were recruited from 29 UK hospitals. Patients were randomly assigned (1:1) via a computer-generated sequential randomisation list to receive 2 weeks of adjunctive rifampicin (600 mg or 900 mg per day according to weight, oral or intravenous) versus identical placebo, together with standard antibiotic therapy. Randomisation was stratified by centre. Patients, investigators, and those caring for the patients were masked to group allocation. The primary outcome was time to bacteriologically confirmed treatment failure or disease recurrence, or death (all-cause), from randomisation to 12 weeks, adjudicated by an independent review committee masked to the treatment. Analysis was intention to treat. This trial was registered, number ISRCTN37666216, and is closed to new participants.FindingsBetween Dec 10, 2012, and Oct 25, 2016, 758 eligible participants were randomly assigned: 370 to rifampicin and 388 to placebo. 485 (64%) participants had community-acquired S aureus infections, and 132 (17%) had nosocomial S aureus infections. 47 (6%) had meticillin-resistant infections. 301 (40%) participants had an initial deep infection focus. Standard antibiotics were given for 29 (IQR 18–45) days; 619 (82%) participants received flucloxacillin. By week 12, 62 (17%) of participants who received rifampicin versus 71 (18%) who received placebo experienced treatment failure or disease recurrence, or died (absolute risk difference −1·4%, 95% CI −7·0 to 4·3; hazard ratio 0·96, 0·68–1·35, p=0·81). From randomisation to 12 weeks, no evidence of differences in serious (p=0·17) or grade 3–4 (p=0·36) adverse events were observed; however, 63 (17%) participants in the rifampicin group versus 39 (10%) in the placebo group had antibiotic or trial drug-modifying adverse events (p=0·004), and 24 (6%) versus six (2%) had drug interactions (p=0·0005).InterpretationAdjunctive rifampicin provided no overall benefit over standard antibiotic therapy in adults with S aureus bacteraemia.FundingUK National Institute for Health Research Health Technology Assessment.

Background: Pneumocystis pneumonia (PCP) is conventionally diagnosed by identifying Pneumocystis jirovecii in lower respiratory tract samples using cytochemical stains. Molecular diagnosis of PCP is potentially more sensitive. Methods: A study was undertaken to use an extensively optimised real-time polymerase chain reaction (PCR) using primers designed to hybridise with the P jirovecii heat shock protein 70 (HSP70) gene to quantify P jirovecii DNA in bronchoalveolar lavage (BAL) fluid from HIV-infected patients with and without PCP, and to compare this assay with conventional PCR targeting the P jirovecii mitochondrial large subunit rRNA gene sequence (mt LSU rRNA). Results: Sixty-one patients had 62 episodes of PCP (defined by detection of P jirovecii in BAL fluid by cytochemical stains and typical clinical presentation). Quantifiable HSP70 DNA was detected in 61/62 (range ,13-18 608 copies/reaction; median ,332) and was detectable but below the limit of quantification (,5 copies/reaction) in 1/62. Seventy-one other patients had 74 episodes with alternative diagnoses. Quantifiable HSP70 DNA was detectable in 6/74 (8%) episodes (range ,6-590 copies/reaction; median ,14) and detectable but below the limit of quantification in 34/74 (46%). Receiver-operator curve analysis (cut-off .10 copies/ reaction) showed a clinical sensitivity of 98% (95% 91% to 100%) and specificity of 96% (95% CI 87% to 99%) for diagnosis of PCP. By contrast, clinical sensitivity of mt LSU rRNA PCR was 97% (95% CI 89% to 99%) and specificity was 68% (95% CI 56% to 78%). Conclusion: The HSP70 real-time PCR assay detects P jirovecii DNA in BAL fluid and may have a diagnostic application. Quantification of P jirovecii DNA by real-time PCR may also discriminate between colonisation with P jirovecii and infection.

BACKGROUND. Novel rapid diagnostics for active tuberculosis (TB) are required to overcome the time delays and inadequate sensitivity of current microbiological tests that are critically dependent on sampling the site of disease. Multiparametric blood transcriptomic signatures of TB have been described as potential diagnostic tests. We sought to identify the best transcript candidates as host biomarkers for active TB, extend the evaluation of their specificity by comparison with other infectious diseases, and to test their performance in both pulmonary and extrapulmonary TB.METHODS. Support vector machine learning, combined with feature selection, was applied to new and previously published blood transcriptional profiles in order to identify the minimal TB‑specific transcriptional signature shared by multiple patient cohorts including pulmonary and extrapulmonary TB, and individuals with and without HIV-1 coinfection.RESULTS. We identified and validated elevated blood basic leucine zipper transcription factor 2 (BATF2) transcript levels as a single sensitive biomarker that discriminated active pulmonary and extrapulmonary TB from healthy individuals, with receiver operating characteristic (ROC) area under the curve (AUC) scores of 0.93 to 0.99 in multiple cohorts of HIV-1–negative individuals, and 0.85 in HIV-1–infected individuals. In addition, we identified and validated a potentially novel 4-gene signature comprising CD177, haptoglobin, immunoglobin J chain, and galectin 10 that discriminated active pulmonary and extrapulmonary TB from other febrile infections, giving ROC AUCs of 0.94 to 1.CONCLUSIONS. Elevated blood BATF2 transcript levels provide a sensitive biomarker that discriminates active TB from healthy individuals, and a potentially novel 4-gene transcriptional signature differentiates between active TB and other infectious diseases in individuals presenting with fever.FUNDING. MRC, Wellcome Trust, Rosetrees Trust, British Lung Foundation, NIHR.