An evaluation of methods for the isolation of nontuberculous mycobacteria from patients with cystic fibrosis, bronchiectasis and patients assessed for lung transplantation.

BACKGROUND: RGM medium is an agar-based, selective culture medium designed for the isolation of nontuberculous mycobacteria (NTM) from the sputum of patients with cystic fibrosis (CF). We evaluated RGM medium for the detection of NTM in patients with CF (405 samples), bronchiectasis (323 samples) and other lung diseases necessitating lung transplantation (274 samples). METHODS: In total, 1002 respiratory samples from 676 patients were included in the study. Direct culture on RGM medium, with incubation at two temperatures (30 °C and 37 °C), was compared with conventional culture of decontaminated samples for acid-fast bacilli (AFB) using both a solid medium (Löwenstein-Jensen medium) and a liquid medium (the Mycobacterial Growth Indicator Tube; MGIT). RESULTS: For all three patient groups, significantly more isolates of NTM were recovered using RGM medium incubated at 30 °C than by any other method (sensitivity: 94.6% vs. 22.4% for conventional AFB culture; P < 0.0001). Significantly more isolates of Mycobacterium abscessus complex were isolated on RGM at 30 °C than by AFB culture (sensitivity: 96.1% vs. 58.8%; P < 0.0001). The recovery of Mycobacterium avium complex was also greater using RGM medium at 30 °C compared to AFB culture (sensitivity: 83% vs. 70.2%), although this difference was not statistically significant and a combination of methods was necessary for optimal recovery (P = 0.21). CONCLUSIONS: In the largest study of RGM medium to date, we reaffirm its utility for isolation of NTM from patients with CF. Furthermore; we show that it also provides an effective tool for culture of respiratory samples from patients with bronchiectasis and other lung diseases.

Multidimensional severity assessment in bronchiectasis: an analysis of seven European cohorts.

INTRODUCTION: Bronchiectasis is a multidimensional disease associated with substantial morbidity and mortality. Two disease-specific clinical prediction tools have been developed, the Bronchiectasis Severity Index (BSI) and the FACED score, both of which stratify patients into severity risk categories to predict the probability of mortality. METHODS: We aimed to compare the predictive utility of BSI and FACED in assessing clinically relevant disease outcomes across seven European cohorts independent of their original validation studies. RESULTS: The combined cohorts totalled 1612. Pooled analysis showed that both scores had a good discriminatory predictive value for mortality (pooled area under the curve (AUC) 0.76, 95% CI 0.74 to 0.78 for both scores) with the BSI demonstrating a higher sensitivity (65% vs 28%) but lower specificity (70% vs 93%) compared with the FACED score. Calibration analysis suggested that the BSI performed consistently well across all cohorts, while FACED consistently overestimated mortality in 'severe' patients (pooled OR 0.33 (0.23 to 0.48), p<0.0001). The BSI accurately predicted hospitalisations (pooled AUC 0.82, 95% CI 0.78 to 0.84), exacerbations, quality of life (QoL) and respiratory symptoms across all risk categories. FACED had poor discrimination for hospital admissions (pooled AUC 0.65, 95% CI 0.63 to 0.67) with low sensitivity at 16% and did not consistently predict future risk of exacerbations, QoL or respiratory symptoms. No association was observed with FACED and 6 min walk distance (6MWD) or lung function decline. CONCLUSION: The BSI accurately predicts mortality, hospital admissions, exacerbations, QoL, respiratory symptoms, 6MWD and lung function decline in bronchiectasis, providing a clinically relevant evaluation of disease severity.

TNFα from classically activated macrophages accentuates epithelial to mesenchymal transition in obliterative bronchiolitis.

Bronchiolitis obliterans syndrome is characterized by fibrotic obliteration of small airways which severely impairs graft function and survival after lung transplantation. Bronchial epithelial cells from the transplanted lung can undergo epithelial to mesenchymal transition and this can be accentuated by activated macrophages. Macrophages demonstrate significant plasticity and change phenotype in response to their microenvironment. In this study we aimed to identify secretory products from macrophages that might be therapeutic targets for limiting the inflammatory accentuation of epithelial to mesenchymal transition in bronchiolitis obliterans syndrome. TNFα, IL-1ß and IL-8 are elevated in bronchoalveolar lavage from lung transplant patients prior to diagnosis of bronchiolitis obliterans syndrome. Classically activated macrophages secrete more TNFα and IL-1ß than alternatively activated macrophages and dramatically accentuate TGF-ß1-driven epithelial to mesenchymal transition in bronchial epithelial cells isolated from lung transplant patients. Blocking TNFα, but not IL-1ß, inhibits the accentuation of epithelial to mesenchymal transition. In a pilot unblinded therapeutic intervention in five patients with progressive bronchiolitis obliterans syndrome, anti-TNFα treatment improved forced expiratory volume in 1 second and 6-min walk distances in four patients. Our data identify TNFα as a potential new therapeutic target in bronchiolitis obliterans syndrome deserving of a randomized placebo controlled clinical trial.

Pseudomonas aeruginosa accentuates epithelial-to-mesenchymal transition in the airway.

Epithelial-to-mesenchymal transition (EMT) has been implicated in the dysregulated epithelial wound repair that contributes to obliterative bronchiolitis (OB) after lung transplantation. Acquisition of Pseudomonas aeruginosa in the transplanted airway has been shown to be a risk factor for the development of OB. We investigated the potential of P. aeruginosa to drive EMT in primary bronchial epithelial cells (PBECs) isolated from lung transplant recipients. Changes in the expression of epithelial and mesenchymal markers was assessed in cells challenged with clinical isolates of P. aeruginosa or co-cultured with P. aeruginosa-activated monocytic cells (THP-1) in the presence or absence of transforming growth factor (TGF)-ß1. P. aeruginosa did not drive or accentuate TGF-ß1-driven EMT directly. Co-culturing P. aeruginosa-activated THP-1 cells with PBECs did not drive EMT. However, co-culturing P. aeruginosa-activated THP-1 cells with PBECs significantly accentuated TGF-ß1-driven EMT. P. aeruginosa, via the activation of monocytic cells, can accentuate TGF-ß1-driven EMT. These in vitro observations may help explain the in vivo clinical observation of a link between acquisition of P. aeruginosa and an increased risk of developing OB.

Volatile biomarkers of Pseudomonas aeruginosa in cystic fibrosis and noncystic fibrosis bronchiectasis.

AIMS: The purpose of this study was to determine whether volatile organic compounds specific to Pseudomonas aeruginosa could be detected in clinical sputum specimens. METHODS AND RESULTS: Patients were recruited from specialist bronchiectasis and cystic fibrosis clinics. The gold standard for diagnosing Ps. aeruginosa infection was a positive sputum culture. About 72 sputum headspace samples taken from patients at risk of or known to have prior Ps. aeruginosa infection were analysed by solid phase micro-extraction mass spectrometry. 2-nonanone was a marker in Ps. aeruginosa in sputum headspace gas with sensitivity of 72% and specificity of 88%. A combination of volatile compounds, a sputum library of 17 compounds with 2-nonanone, increased sensitivity in the detection of Ps. aeruginosa to 91% with specificity of 88%. CONCLUSIONS: In contrast to the 48-hour turnaround for classical microbiological culture, these results were available within 1-2 h. These data demonstrate the potential for rapid and accurate diagnosis of Ps. aeruginosa infection from sputum samples. SIGNIFICANCE AND IMPACT OF THE STUDY: 2-Nonanone is a compound requiring further study in the exhaled breath as it may improve diagnostic of Ps. aeruginosa infection when combined with other reported volatile markers.

Assessment of sample handling practices on microbial activity in sputum samples from patients with cystic fibrosis.

AIM: The aim of this study was to quantitatively and qualitatively assess the effect of sample storage on the metabolically active microbial community found in sputum samples from patients with cystic fibrosis (CF). METHODS: Sputum samples were collected and split in two equal aliquots one of which was immersed in RNAlater and refrigerated immediately, the second stored at room temperature for 24 h and RNAlater was subsequently added. mRNA was extracted, and RT-PCR-DGGE and qPCR analysis of the bacterial and fungal communities was carried out. RESULTS: Significant differences in the bacterial communities between the two protocols were observed but there were no significant difference seen in the fungal community analyses. Analysis by qPCR demonstrated that room temperature storage gave statistically significant increases in eubacteria and Pseudomonas spp. and a statistically significant decrease in those of Haemophilus influenzae. CONCLUSIONS: The analysis of metabolically active microbial communities from CF sputum using molecular techniques indicated that samples should be stored at 4 degrees C upon addition of RNAlater to obtain an accurate depiction of the CF lung microbiota. Also, storing respiratory samples at room temperature may cause an over representation of Pseudomonas aeruginosa and mask the presence of other clinically significant organisms.

Outcomes of lung transplantation for cystic fibrosis in a large UK cohort.

BACKGROUND: Lung transplantation is an important option to treat patients with advanced cystic fibrosis (CF) lung disease. The outcomes of a large UK cohort of CF lung transplantation recipients is reported. METHODS: Retrospective review of case notes and transplantation databases. RESULTS: 176 patients with CF underwent lung transplantation at our centre. The majority (168) had bilateral sequential lung transplantation. Median age at transplantation was 26 years. Diabetes was common pretransplantation (40%). Polymicrobial infection was common in individual recipients. A diverse range of pathogens were encountered, including the Burkholderia cepacia complex (BCC). The bronchial anastomotic complication rate was 2%. Pulmonary function (forced expiratory volume in 1 s % predicted) improved from a pretransplantation median of 0.8 l (21% predicted) to 2.95 l (78% predicted) at 1 year following transplantation. We noted an acute rejection rate of 41% within the first month. Our survival values were 82% survival at 1 year, 70% at 3 years, 62% at 5 years and 51% at 10 years. Patients with BCC infection had poorer outcomes and represented the majority of those who had a septic death. Data are presented on those free from these infections. Bronchiolitis obliterans syndrome (BOS) and sepsis were common causes of death. Freedom from BOS was 74% at 5 years and 38% at 10 years. Biochemical evidence of renal dysfunction was common although renal replacement was infrequently required (<5%). CONCLUSION: Lung transplantation is an important therapeutic option in patients with CF even in those with more complex microbiology. Good functional outcomes are noted although transplantation associated morbidities accrue with time.

Occupational asthma caused by Arabidopsis thaliana: a case of laboratory plant allergy.

A 36-yr-old male never-smoker with an 8-yr history of hay fever but no past history of asthma undertook a 3-yr research project involving the plant Arabidopsis thaliana. The subject was based in a small laboratory with an attached growing room. After 30 months of research, he began to develop breathlessness within 5-10 min of entering the laboratory. Initial investigations confirmed asthma with airflow obstruction (forced expiratory volume in one second (FEV(1))/forced vital capacity was 3.01/4.75 L; predicted values were 3.67/4.43 L) and increased airway responsiveness. Serial peak expiratory flow measurements showed a work-related pattern. A supervised workplace challenge test led to a fall in FEV(1) from the baseline value of 3.10 L to 1.95 L within 20 min of entering the growing room. Skin-prick solutions were prepared from Arabidopsis leaves and flower heads; positive 4-mm responses were obtained to the flower heads (i.e. to the pollen). Arabidopsis is a member of the Brassicaceae family. It is used extensively in plant biology research as its genome is small, has been fully sequenced and is easily manipulated. The present article represents the first reported case of occupational asthma due to Arabidopsis thaliana.

A qualitative synthesis of gastro-oesophageal reflux in bronchiectasis: Current understanding and future risk.

Gastro-oesophageal reflux disease (GORD) is a common comorbidity in bronchiectasis, and is often associated with poorer outcomes. The cause and effect relationship between GORD and bronchiectasis has not yet been fully elucidated and a greater understanding of the pathophysiology of the interaction and potential therapies is required. This review explores the underlying pathophysiology of GORD, its clinical presentation, risk factors, commonly applied diagnostic tools, and a detailed synthesis of original articles evaluating the prevalence of GORD, its influence on disease severity and current management strategies within the context of bronchiectasis. The prevalence of GORD in bronchiectasis ranges from 26% to 75%. Patients with co-existing bronchiectasis and GORD were found to have an increased mortality and increased bronchiectasis severity, manifest by increased symptoms, exacerbations, hospitalisations, radiological extent and chronic infection, with reduced pulmonary function and quality of life. The pathogenic role of Helicobacter pylori infection in bronchiectasis, perhaps via aspiration of gastric contents, also warrants further investigation. Our index of suspicion for GORD should remain high across the spectrum of disease severity in bronchiectasis. Identifying GORD in bronchiectasis patients may have important therapeutic and prognostic implications, although clinical trial evidence that treatment targeted at GORD can improve outcomes in bronchiectasis is currently lacking.

Intragraft proliferating T lymphocytes are associated with moderate acute pulmonary rejection.

Acute allograft rejection is characterized by infiltration of the donor organ by host lymphoid cells, predominantly T lymphocytes. However, the site of proliferation and clonal expansion of alloreactive T lymphocytes is not well defined in man. A group of normal transbronchial biopsies (TBB, n=9) from clinically well lung transplant recipients was compared to TBB showing acute rejection (at least grade A2, n=9), using CD3- and Ki67-specific antibodies to double-label proliferating T lymphocytes. Few double-labeled lymphocytes were present in the normal biopsies (range, 0-3 cells). However, five of the rejection biopsies contained significant numbers of proliferating T lymphocytes (range, 19-47; Fisher's exact test; P=0.029). Furthermore, this positive group contained all three cases of grade A3 rejection in the study, as well as a case with persistent grade A2 rejection on follow-up biopsy. These data demonstrate that T lymphocytes do proliferate in transplanted human lungs; such proliferation is associated with more severe rejection.

Donor-acquired small cell lung cancer following pulmonary transplantation.

We describe a case of donor-acquired small cell lung cancer after pulmonary transplantation for cystic fibrosis. The recipient was an ex-smoker with minimal smoking history and had been abstinent for 20 years. At the time of death, the donor chest radiographic finding was normal. The recipient had multiple posttransplant bronchoscopies and a normal CT scan result at 4 months after transplantation. The recipient presented 13 months after transplantation with metastatic disease. He did not respond to chemotherapy and died shortly thereafter. Molecular genetic techniques revealed that the primary tumor and metastases were different to recipient tissues, confirming the donor origin.

Effects of plant size and water relations on gas exchange and growth of the desert shrub Larrea tridentata.

Larrea tridentata is a xerophytic evergreen shrub, dominant in the arid regions of the southwestern United States. We examined relationships between gasexchange characteristics, plant and soil water relations, and growth responses of large versus small shrubs of L. tridentata over the course of a summer growing season in the Chihuahuan Desert of southern New Mexico, USA. The soil wetting front did not reach 0.6 m, and soils at depths of 0.6 and 0.9 m remained dry throughout the summer, suggesting that L. tridentata extracts water largely from soil near the surface. Surface soil layers (<0.3 m) were drier under large plants, but predawn xylem water potentials were similar for both plant sizes suggesting some access to deeper soil moisture reserves by large plants. Stem elongation rates were about 40% less in large, reproductively active shrubs than in small, reproductively inactive shrubs. Maximal net photosynthetic rates (Pmax) occurred in early summer (21.3 µ mol m-2 s-1), when pre-dawn xylem water potential (XWP) reached ca. -1 MPa. Although both shrub sizes exhibited similar responses to environmental factors, small shrubs recovered faster from short-term drought, when pre-dawn XWP reached about -4.5 MPa and Pmax decreased to only ca. 20% of unstressed levels. Gas exchange measurements yielded a strong relationship between stomatal conductance and photosynthesis, and the relationship between leaf-to-air vapor pressure deficit and stomatal conductance was found to be influenced by pre-dawn XWP. Our results indicate that stomatal responses to water stress and vapor pressure deficit are important in determining rates of carbon gain and water loss in L. tridentata.

Lack of association between KIR and HLA-C type and susceptibility to idiopathic bronchiectasis.

INTRODUCTION: Idiopathic bronchiectasis is a poorly defined disease characterised by persistent inflammation, infection and progressive lung damage. Natural killer (NK) cells provide a major defense against infection, through the interaction of their surface receptors, including the activating and inhibitory killer immunoglobulin-like receptors (KIR), and human leukocyte antigens (HLA) class I molecules. Homozygosity for HLA-C has been shown in a single study to confer increased genetic susceptibility to idiopathic bronchiectasis. We aimed to assess whether the KIR and HLA repertoire, alone or in combination, may influence the risk of developing idiopathic bronchiectasis, in an independent replication study. METHODS: In this prospective, observational, case-control association study, 79 idiopathic bronchiectasis patients diagnosed following extensive aetiological investigation were compared with 98 anonymous, healthy, age, sex and ethnically-matched controls attending blood donor sessions in the same geographical location. DNA extraction was performed according to standardised techniques. Determination of presence or absence of KIR genes was performed by a sequence specific oligonucleotide probe method. Allele frequencies for the proposed KIR, HLA-B and HLA-C risk alleles both individually and in combinations were compared. RESULTS: We found no significant differences in allele frequency between the idiopathic bronchiectasis and control samples, whether considering HLA-C group homozygosity alone or in combination with the KIR type. DISCUSSION: Our results do not show an association between HLA-C and KIR and therefore do not confirm previous positive findings. This may be explained by the lower frequency of HLA-C1 group homozygosity in the control population of the previous study (27.2%), compared to 42.3% in our study, which is consistent with the genetic profiling of control groups across the UK. The previous positive association study may therefore have been driven by an anomalous control group. Further larger prospective multicentre replication studies are needed to determine if an association exists.

Fatigue in bronchiectasis.

BACKGROUND: Fatigue is a complex, disabling symptom in non-CF bronchiectasis (nCF-Br). Fatigue can be formally measured using the validated fatigue impact scale (FIS). The relationship between fatigue and clinically important factors such as airflow obstruction, breathlessness or Pseudomonas aeruginosa infection in nCF-Br is unclear. AIM: To measure the correlation between FIS scores and markers of disease severity in nCF-Br. DESIGN: A prospective cohort study. METHODS: Patients attending a specialist service were studied. Lung function (FEV(1)% predicted), Medical Research Council dyspnoea score (MRCD), sputum culture results and FIS were recorded. Patients were categorized according to sputum culture into three subgroups: Pseudomonas 'colonization', 'isolation' and neither. RESULTS: One hundred and seventeen consecutive patients were included. Average FEV(1)% predicted was 64% (SD ±28%). Twelve (10%) patients had Pseudomonas aeruginosa isolation; 47 (40%) patients had P. aeruginosa colonization. Fatigue levels were similar in patients with and without colonization (median 38 versus 32, P = 0.155). Significant fatigue (FIS > 40) was similar in all three Pseudomonas subgroups (P = 0.31, chi-square). Fatigue correlated with MRCD score (r = 0.57, P < 0.001) and FEV(1)% predicted (r = -0.30, P = 0.001). FEV(1)% predicted was lower in patients who had ever isolated or been colonized with P. aeruginosa (P ≤ 0.001). CONCLUSION: There are significant correlations between FIS score and MRCD score and FEV(1)% predicted in bronchiectasis. Pseudomonas aeruginosa infection appears to be associated with poorer lung function, and higher MRCD scores, yet there is no significant association between P. aeruginosa status and fatigue.

Inhaled corticosteroids and the treatment of lymphocytic bronchiolitis following lung transplantation.

Airway rejection after lung transplantation is recognized histologically as lymphocytic bronchiolitis (LB). We hypothesized that inhaled steroids could control LB and that changes in exhaled nitric oxide (eNO) would correlate with the development of LB and also have a role in monitoring response to treatment. A cohort of 120 lung transplant (LT) recipients attending for review and biopsy had eNO measurements, FEV1, lavage microbiology, and biopsy histology performed prospectively. Wilcoxon signed-rank test was used to assess the significance of changes in eNO and FEV1. The coefficient of reproducibility of eNO measurement in stable recipients was 2.36 ppb. Fourteen developed graft dysfunction owing to isolated LB and were treated with inhaled budesonide 800 microg twice daily. They showed significant increases in eNO at diagnosis, median (range) 10.9 ppb (4.6 to 48) ppb compared with baseline, 4.33 (1.0 to 10.76), p = 0.008, and a decrease in FEV1. After inhaled treatment, both eNO and FEV1 returned to baseline values. Seven developed acute vascular rejection (with or without LB) and were treated with oral corticosteroids; no changes in eNO occurred at diagnosis or after treatment. Serial eNO measurements provide a useful noninvasive method of identifying airway inflammation in LT recipients. Inhaled budesonide may be a useful addition to systemic immunosuppressants in controlling airway inflammation posttransplant.

Reticular basement membrane thickening in airways of lung transplant recipients is not affected by inhaled corticosteroids.

INTRODUCTION: Chronic rejection is a major problem for all lung transplant programmes, which is functionally manifested by fixed airflow limitation, Bronchiolitis Obliterans Syndrome (BOS). The inclusion of a Pre-BOS category, BOS(0 approximately p), in newly revised guidelines, recognizes the potential importance of early changes. We have previously demonstrated reticular basement membrane (Rbm) thickening in clinically stable lung transplant recipients free from BOS. The present study extends this, testing the hypothesis that inhaled corticosteroid (ICS) therapy will lead to a decrease in Rbm thickness in lung transplant recipients. METHODS: A parallel group, bronchoscopic intervention study of clinically stable lung allograft recipients, free from BOS, but with evidence of airway inflammation. Following baseline assessment of Rbm thickening, subjects were randomized to 3 months of either chlorofluorocarbon-driven beclomethasone diproprionate (BDP) 400 microg b.i.d., or a formulation designed to yield at least an equivalent dose, hydrofluoroalkane-driven BDP, 200 microg b.i.d. RESULTS: Three months treatment with a moderate dose of ICS, including a formulation designed for preferential small airway deposition, had no effect on Rbm thickening (13+/-3 vs. 14+/-5 microm post-ICS). CONCLUSION: Our data would suggest that airway remodelling can occur early in lung allografts and is not affected by moderate dose ICS therapy. Longitudinal studies are required to describe the pathophysiological processes involved in BOS, and specifically to elucidate potential relationships between airway remodelling, airflow obstruction and allograft failure.

Preliminary findings of quorum signal molecules in clinically stable lung allograft recipients.

BACKGROUND: Infection with bacteria such as Pseudomonas is common in lung allograft recipients, particularly during chronic rejection. Analysis of sputum samples from patients with cystic fibrosis infected with Pseudomonas aeruginosa or Burkholderia cepacia has indicated the presence of bacterial N-acylhomoserine lactones (AHLs) quorum sensing signalling molecules. AHLs not only control the expression of bacterial virulence genes but are also involved in stimulating the maturation of antibiotic resistant biofilms and host chemokine release. It was hypothesised that AHLs may be detected even in clinically stable lung transplant recipients free of clinical infection or rejection. METHODS: Three 60 ml samples of bronchoalveolar lavage (BAL) fluid were taken from nine stable lung transplant recipients 3-12 months after transplantation. Detection of AHLs was carried out on dichloromethane extracted supernatants using the bioluminescence based AHL reporter plasmid pSB1075. This responds to the presence of AHLs with long acyl chains (C10-C14), generating light. Synthetic AHLs were included as positive controls. RESULTS: Five of the nine BAL fluid supernatants exhibited AHL activity, suggesting the presence of AHLs with long N-acyl chains. There was no correlation between the levels of AHLs detected or their absence and BAL fluid microbiology or diagnosis before transplantation. CONCLUSIONS: This is the first evidence for the presence of AHL quorum sensing signals in human lung allograft recipients, even in subjects with no rejection or apparent infection. Further longitudinal follow up of these preliminary findings is required to elucidate potential links with infection, rejection, and allograft deterioration.

Prevalence and clonality of Burkholderia cepacia complex genomovars in UK patients with cystic fibrosis referred for lung transplantation.

BACKGROUND: It has previously been reported that patients infected with Burkholderia cenocepacia (genomovar III) before lung transplantation have a poorer outcome than those with other B. cepacia complex infections. METHODS: An extensive study was conducted to determine the prevalence and clonality of B. cepacia complex genomovars isolated from patients referred for transplant assessment between 1989 to the present and, where appropriate, whether strain type was related to transplant outcome. RESULTS: Isolates from 29 patients were identified as B. cepacia complex organisms by molecular analysis. Thirteen patients (45%) were infected with the highly transmissible ET-12 strain of B. cenocepacia recA lineage III-A, while all remaining patients were infected with genetically unique B. cenocepacia, B. multivorans, and B. vietnamiensis strains. All previously reported deaths following transplantation were associated with ET-12 infection. CONCLUSIONS: The ET-12 strain is the predominant cause of B. cenocepacia infections in patients with cystic fibrosis referred to our pulmonary transplant centre and is associated with poor transplant outcomes using standard treatment regimens.