Annette Jepson

Rhinovirus Infection Induces Degradation of Antimicrobial Peptides and Secondary Bacterial Infection in Chronic Obstructive Pulmonary Disease

RATIONALE Chronic obstructive pulmonary disease (COPD) exacerbations are associated with virus (mostly rhinovirus) and bacterial infections, but it is not known whether rhinovirus infections precipitate secondary bacterial infections. OBJECTIVES To investigate relationships between rhinovirus infection and bacterial infection and the role of antimicrobial peptides in COPD exacerbations. METHODS We infected subjects with moderate COPD and smokers and nonsmokers with normal lung function with rhinovirus. Induced sputum was collected before and repeatedly after rhinovirus infection and virus and bacterial loads measured with quantitative polymerase chain reaction and culture. The antimicrobial peptides secretory leukoprotease inhibitor (SLPI), elafin, pentraxin, LL-37, α-defensins and β-defensin-2, and the protease neutrophil elastase were measured in sputum supernatants. MEASUREMENTS AND MAIN RESULTS After rhinovirus infection, secondary bacterial infection was detected in 60% of subjects with COPD, 9.5% of smokers, and 10% of nonsmokers (P < 0.001). Sputum virus load peaked on Days 5-9 and bacterial load on Day 15. Sputum neutrophil elastase was significantly increased and SLPI and elafin significantly reduced after rhinovirus infection exclusively in subjects with COPD with secondary bacterial infections, and SLPI and elafin levels correlated inversely with bacterial load. CONCLUSIONS Rhinovirus infections are frequently followed by secondary bacterial infections in COPD and cleavage of the antimicrobial peptides SLPI and elafin by virus-induced neutrophil elastase may precipitate these secondary bacterial infections. Therapy targeting neutrophil elastase or enhancing innate immunity may be useful novel therapies for prevention of secondary bacterial infections in virus-induced COPD exacerbations.

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.

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.

Undetected Multidrug-Resistant Tuberculosis Amplified by First-line Therapy in Mixed Infection

Infections with >1 Mycobacterium tuberculosis strain(s) are underrecognized. We show, in vitro and in vivo, how first-line treatment conferred a competitive growth advantage to amplify a multidrug-resistant M. tuberculosis strain in a patient with mixed infection. Diagnostic techniques that identify mixed tubercle bacilli populations are needed to curb the spread of multidrug resistance.

Waterborne Elizabethkingia meningoseptica in Adult Critical Care.

This outbreak might reflect improved diagnostic testing, indicating that E. meningoseptica is a pseudo-emerging pathogen.

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.

P19 GeneXpert MTB.Rif assay improves the diagnostic yield of EBUS-TBNA in smear-negative intra-thoracic tuberculous lymphadenopathy

Introduction and Objectives Tuberculosis notifications in the UK continue to rise and the diagnosis of both disease and drug resistance can be challenging. Endobronchial ultrasound (EBUS) and EBUS-guided transbronchial nodal aspirates (TBNA) have been shown recently to be a safe and effective tool in the diagnosis of intra-thoracic TB lymphadenopathy. New molecular techniques, notably the GeneXpert MTB.Rif system (Cepheid) have shown great promise in the diagnosis of pulmonary disease but have not been evaluated in intra-thoracic nodal disease. Methods As part of an ongoing study, consecutive patients with intra-thoracic lymphadenopathy were prospectively studied within our tertiary EBUS service between January 2010 and March 2011. In addition to standard cytological and microbiological investigations, a single GeneXpert MTB.Rif assay was performed on EBUS-TBNA samples. Using established methods, a final diagnosis was given of definite/highly probable TB, possible TB or not TB/alternative diagnosis. Performance of the GeneXpert MTB.Rif assay was then evaluated in the context of these final diagnoses. Results 74 patients (3 HIV-positive) underwent EBUS-TBNA sampling. Nineteen have been diagnosed with definite/highly probable TB to date. A single GeneXpert assay had a sensitivity of 67% (8/12) from culture-positive TBNA. 11/15 (73%) of patients with a positive culture from any tissue and 13/19 (68%) patients classed as definite/highly probable TB had positive GeneXpert results. One case of confirmed MDR-TB was correctly identified and treatment started promptly. Fifteen patients had positive GeneXpert MTB.Rif results from EBUS-TBNA: 13/15 were given immediate TB treatment. One of the remaining two cases without strong microbiological or cytological findings was subsequently diagnosed with active tuberculosis supported by evidence of PET-positive mediastinal lymph nodes. The other case appears not to have active disease and remains under follow-up. Conclusions A single GeneXpert MTB.Rif assay has good sensitivity in the context of culture-positive intra-thoracic tuberculous lymphadenopathy and can provide an immediate diagnosis of likely MDR-TB. Positive PCR results were seen in two patients where conventional techniques were inconclusive and in one provided the main support for the diagnosis. These results suggest the addition of the GeneXpert MTB.Rif assay to the investigation of intra-thoracic nodal disease improves diagnostic yield.

S15 Detection of bacteria in sputum following experimental rhinovirus infection is more common in COPD than controls subjects

Introduction and Objectives There is increasing evidence that the majority of acute exacerbations of COPD (AECOPD) are caused by virus infection, and rhinoviruses are the most frequently identified species. Bacteria are also responsible for AECOPD but the relationship between these two is poorly understood. To investigate this further bacterial culture was performed in sputum samples collected from GOLD stage II COPD subjects (n=9) and non-obstructed smoking (n=10) and non-smoking (n=11) controls enrolled in a rhinovirus challenge study. Methods Rhinovirus infection was confirmed with quantitative PCR performed on nasal lavage and sputum samples collected at baseline and days 3, 5, 9, 12, 15, 21 and 42 post virus inoculation. Semi-quantitative bacterial detection was performed in sputum samples by a CPA-accredited microbiological laboratory. Any subject that had bacteria detected on or after day 9 was defined as “bacteria positive” and those with none detected were defined “bacteria negative”. Species frequently associated with respiratory illness were defined as pathogenic (S pneumoniae, H influenzae, M catarrhalis and S aureus) and any others as non-pathogenic. Results No bacteria were detected in baseline sputum samples. Peak virus load occurred on day 9 with maximum bacterial colonies identified on day 15. There were significantly more bacteria positive subjects in the COPD group (67%) with the majority of control subjects (81%) being classified as bacteria negative, (Abstract S15 figure 1, χ2 p=0.04). COPD subjects with bacteria detected at any time point in the study had significantly more pathogenic species in their sputum samples (n=8/8) compared to controls (n=1/9), (χ2 p=0.0001). No non-pathogenic bacteria were detected in COPD subjects.Abstract S15 Figure 1 Number of subjects in each study group with bacteria negative or bacteria positive sputum samples on or following day 9, (χ2 p=0.04). Conclusions Detection of bacteria is common after rhinovirus infection, with the peak occurring 6 days after maximum virus load. COPD subjects are more likely to have pathogenic bacteria detected than controls following virus infection. Mechanisms responsible for this phenomenon merit further investigation.

P55 Post-bronchoscopy sputum: increasing the diagnostic yield in smear negative pulmonary tuberculosis

Background The prevalence of smear negative pulmonary tuberculosis (PTB) is increasing. At many centers, active PTB suspects who are Acid-Fast Bacilli (AFB) smear negative or non-productive of sputum undergo fiber optic bronchoscopy for bronchoalveolar lavage but post bronchoscopy sputum (PBS) sampling is not routine. The aim of the study was to establish the clinical utility of PBS sampling in this subgroup of patients with active PTB. Methods A retrospective study of all patients attending a central London University hospital with microbiologically confirmed PTB between January 2004 and December 2009. Patients who were AFB smear negative or non-productive of sputum were eligible for the study if a sputum sample was obtained within 7 days of bronchoscopy. Results The cohort (n=50) was heterogeneous—29 were male (58%), 12 were infected with HIV (24%), 19 were of African origin (38%), 17 were white Caucasian (34%) and four were from the Indian subcontinent (8%). 15 patients (30%) converted to AFB sputum smear positivity post bronchoscopy and five patients (10%) were exclusively AFB sputum smear positive on PBS microscopy. M tuberculosis was cultured from the PBS of 40 patients (80%) and four of these (8%) were exclusively PBS culture positive (Abstract P55 Figure 1). Two of these four patients were infected with HIV.Abstract P55 Figure 1 M tuberculosis culture positive results for pre-bronchoscopy, BAL and PBS samples. Conclusion Sampling sputum post bronchoscopy can provide a previously underutilized method of making a rapid diagnosis of PTB and reduce the number of patients who are treated on an empiric basis, particularly in the context of sputum smear negative or non-productive disease. Importantly it can increase culture yield by up to 8% hence allowing for a greater proportion of appropriate treatment of drug resistant strains. PBS sampling is also a key infection control measure that should be considered following bronchoscopy. Further studies are now required to establish the duration of smear positivity post bronchoscopy in patients who were previously considered non-infectious but in the light of this data, we consider it best practice to only de-isolate such patients when their infective status can be ascertained with at least one post-bronchoscopy sputum sample.