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Bacteria are often isolated in stable chronic obstructive pulmonary disease (COPD). Whether fungi are also commonly present and associated with clinical and pathological features of disease is uncertain. We investigated the frequency of filamentous fungal culture and IgE sensitisation to Aspergillus fumigatus and the relationship to clinical outcomes in COPD subjects.COPD subjects were recruited to enter a 1-year observational study. Assessments of lung function, allergen testing and sputum analysis for inflammation, bacteria and fungus were undertaken in COPD subjects and healthy smoking and nonsmoking controls. Filamentous fungi were cultured at baseline in 49% (63 out of 128) of COPD subjects, of which 75% (47 out of 63) were A. fumigatus. Fungus was cultured in three out of 22 controls (two were A. fumigatus). The total sputum cell count and inhaled corticosteroid dosage were significantly increased in COPD patients with a positive filamentous fungal culture at baseline (p<0.05). Sensitisation to A. fumigatus was present in 13% of COPD subjects and was associated with worse lung function (forced expiratory volume in 1 s 39% predicted versus 51% predicted; p=0.01), but not related to filamentous fungal culture.A. fumigatus sensitisation is related to poor lung function. Positive filamentous fungal culture is a common feature of COPD. The clinical significance of this remains uncertain. Introduction Chronic obstructive pulmonary disease (COPD) is associated with significant morbidity and mortality [1]. It is characterised by irreversible airflow obstruction [2] with underlying emphysema and small airway obliteration, which co-exist commonly [3, 4]. Airways of patients with COPD are often “colonised” with potential pathogenic microorganisms [5] which give rise to increased airway inflammation [6]. Bacteria and viruses have been implicated as the major cause of COPD exacerbations, whereas the potential role of fungal colonisation and infection in the pathogenesis of COPD is poorly understood. The commonest fungal genus to cause pulmonary-associated fungal infections is Aspergillus [7], with a wide spectrum of syndromes, including saprophytic invasion, allergic disease and invasive aspergillosis [8], often due to Aspergillus fumigatus [7]. Additionally, allergic bronchopulmonary aspergillosis (ABPA) [9–12], found commonly in asthma and cystic fibrosis [13], is increasingly recognised in COPD [14]. Furthermore, sensitisation to A. fumigatus has been found to be associated with poor lung function in severe asthmatics [15]. Impairment in host defence systems in immunocompetent and immunocompromised patients, including those with COPD, is thus likely to promote susceptibility to fungal infections. How this impacts on important clinical outcomes in COPD such as disease severity, inflammation and exacerbations is currently unknown. We hypothesised that the presence of filamentous fungi in the airways of patients with COPD and sensitisation to A. fumigatus is associated with disease severity, airway inflammation and exacerbations. To study this, we carried out a longitudinal study to investigate fungal culture in patients with COPD during stable state and exacerbations. Subjects and measurements Subjects with COPD diagnosed according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines [2] were recruited as part of a larger biomarker-directed randomised controlled study [16]. Subjects with a diagnosis of asthma, active pulmonary tuberculosis or any other clinically relevant lung disease were excluded. All COPD subjects underwent lung function testing including reversibility testing [17]. Sputum was collected for analysis of routine microbiology for potential pathogenic microorganisms (defined as Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae, Staphylococcus aureus or Pseudomonas aeruginosa) [18] and mycology [15, 19]. In brief, ∼150 mg of undiluted sputum was inoculated onto potato dextrose agar plates containing chloramphenicol (16 μg•mL−1), gentamicin (4 μg•mL−1) and fluconazole (5 μg•mL−1). Plates were then incubated at 37°C and inspected daily for up to 7 days. A. fumigatus colonies were identified by colony formation and microscopy. PCR sequencing of the large subunit or the internal transcribed spacer region of the nuclear ribosomal operon was used for the detection of other filamentous fungi, as previously described [19]. Sputum was also processed to produce cytospins for assessment of sputum total and differential cell counts [20, 21]. Venous blood was collected for assessment of peripheral blood differential cell counts and serum C reactive protein. Health status and symptom scores were measured using the St George’s Respiratory Questionnaire (University of London, London, UK) [22], the Chronic Respiratory Disease Interviewer-Administered Questionnaire (CRQ) (McMaster University, Hamilton, Canada) [23] and the visual analogue scale (VAS) for the domains of cough, breathlessness, sputum production and sputum purulence [24]. Subjects were seen during stable-state and exacerbation visits; sample collection was performed prior to randomisation and institution of any therapy. Nonsmoking and nonobstructed smoking controls were invited to attend a single study visit, with lung function testing, sputum induction and demographic data collection. Skin-prick testing was used to assess atopy to Dermatophagoides pteronyssinus, dog, cat, grass pollen and to the fungi Alternaria alternata, A. fumigatus, Botrytis cinerea, Cladosporium herbarum and Penicillium chrysogenum (Alk-Abello, Hørsholm, Denmark). Total IgE levels, allergen-specific IgE antibody levels to cat, dog, timothy grass, D. pteronyssinus and A. fumigatus, and A. fumigatus-IgG levels were measured using the ImmunoCap 250 system (Phadia Ltd, Milton Keynes, UK). The study was approved by the Leicestershire, Northamptonshire and Rutland ethics committee and all subjects gave informed written consent. Statistical analysis Statistical analysis was performed using PRISM version 4 (GraphPad, San Diego, CA, USA) and SPSS version 16 (SPSS Inc., Chicago, IL, USA). Parametric and nonparametric data are presented as mean±SEM and median (interquartile range) unless otherwise stated. Log-transformed data are presented as geometric mean (95% CI). For comparison of unpaired or paired parametric or nonparametric groups, t-tests, Mann–Whitney U-test and Wilcoxon matched-pairs tests were used. For comparison of three groups or more for parametric and nonparametric variables the ANOVA or Kruskal–Wallis test was used, and the Chi-squared test was used for proportions. Repeatability of A. fumigatus culture was assessed in patients with two stable visits 3 months apart, using the Cohen κ statistic. Logistic regression analysis was used to assess the relationship of the variables of forced expiratory volume in 1 s (FEV1) % predicted, sputum eosinophil count (log-transformed), sputum neutrophil count (log-transformed) and exacerbation frequency with the presence of 1) A. fumigatus-only culture and 2) sensitisation to A. fumigatus using the block entry method. Standard multiple regression analysis was used to assess the relationship of airway inflammation, exacerbation frequency, fungal culture and fungal sensitisation with lung function. Logistic regression goodness-to-fit was performed using the Hosmer–Lemeshow Chi-squared and R-squared (true and pseudo-R2 for multiple and logistic regression, respectively) tests were used to estimate the variance explained by the model. A p-value of <0.05 was taken as the threshold of significance. Results Filamentous fungal culture and baseline demographic data were obtained in 128 patients with COPD (89 males and 39 females). The mean FEV1 in the COPD subjects was 48±3% pred. Control data were available in 22 (eight males and 14 females) subjects with a mean (range) age of 58 (41–79) years; mean (range) smoking history of 10 (0–30) pack-years and a mean FEV1 of 116±3% pred. The clinical characteristics of the COPD patients and controls are presented in table 1. A filamentous fungus was isolated in 49% (63 out of 128) of COPD patients and was predominantly Aspergillus or Penicillium species (online supplementary table E1). A. fumigatus was cultured in 37% (47 out of 128) of patients, while identification of any Aspergillus species was present in 42% (55 out of 128) of patients. Pathogenic bacteria (H. influenzae, M. catarrhalis, S. pneumoniae, S. aureus or P. aeruginosa) and A. fumigatus co-culture was found in 14% (18 out of 128) of patients at baseline stable state. The proportion of control subjects that had positive filamentous fungal cultures was significantly lower compared to COPD subjects (14% versus 49%; mean difference 36%, 95% CI 14–48%; p=0.002). View inlineView popup Patients with A. fumigatus were on a higher inhaled corticosteroid dose compared to those who were culture-negative (1628 versus 1389 μg beclomethasone dipropionate equivalent; mean difference 239, 95% CI 0–477; p=0.050). There were no differences in health status, exacerbation frequency or FEV1 % pred in COPD subjects who were A. fumigatus culture-positive compared to those who were A. fumigatus culture-negative (table 2). There was a significant difference in total and percentage sputum neutrophil count, but not in sputum eosinophils in patients with A. fumigatus compared to patients with no fungi or other filamentous fungi (fig. 1). The total sputum neutrophil count was the most independent predictor of A. fumigatus culture, with a sensitivity and specificity of 54% and 76%, respectively (table 3). Discussion Here we that in subjects with COPD, sensitisation to A. fumigatus was associated with poor lung function. Positive culture of filamentous fungi, especially that of A. fumigatus, was commonly found in the sputum of patients with COPD and was increased compared to controls. However, repeatability of A. fumigatus culture in stable state was poor and the prevalence did not change at exacerbations. The clinical significance of a positive filamentous fungal culture in COPD therefore remains uncertain. In our study, hypersensitivity to A. fumigatus was detected in 13% of the COPD subjects and was associated with reduced lung function independent of A. fumigatus sputum culture. This is consistent with previously observed findings in patients with severe asthma using the same fungal culture technique [15]. Whether sensitisation to A. fumigatus contributes to the cause of airflow obstruction or is a consequence of a damaged and remodelled airway and thus more likely in subjects with severe COPD is uncertain. In severe asthma the relationship between sensitisation and filamentous fungal culture suggests that persistent colonisation may promote sensitisation. Although we were unable to replicate the finding of colonisation in our study, it remains plausible that sensitisation reflects increased filamentous fungi exposure over time. 网站原创范文除特殊说明外一切图文作品权归所有；未经官方授权谢绝任何用途转载或刊发于媒体。如发生侵犯作品权现象，英语论文题目，保留一切法学追诉权。()，英语论文题目 |
