Joshua Agbetile

IgE Sensitization to Aspergillus fumigatus Is Associated with Reduced Lung Function in Asthma

RATIONALE The importance of Aspergillus fumigatus sensitization and colonization of the airways in patients with asthma is unclear. OBJECTIVES To define the relationship between the clinical and laboratory features of A. fumigatus-associated asthma. METHODS We studied 79 patients with asthma (89% classed as GINA 4 or 5) classified into 3 groups according to A. fumigatus sensitization: (1) IgE-sensitized (immediate cutaneous reactivity > 3 mm and/or IgE > 0.35 kU/L); (2) IgG-only-sensitized (IgG > 40 mg/L); and (3) nonsensitized. These were compared with 14 healthy control subjects. Sputum culture was focused toward detection of A. fumigatus and compared with clinical assessment data. MEASUREMENTS AND MAIN RESULTS A. fumigatus was cultured from 63% of IgE-sensitized patients with asthma (n = 40), 39% of IgG-only-sensitized patients with asthma (n = 13), 31% of nonsensitized patients with asthma (n = 26) and 7% of healthy control subjects (n = 14). Patients sensitized to A. fumigatus compared with nonsensitized patients with asthma had lower lung function (postbronchodilator FEV₁ % predicted, mean [SEM]: 68 [±5]% versus 88 [±5]%; P < 0.05), more bronchiectasis (68% versus 35%; P < 0.05), and more sputum neutrophils (median [interquartile range]: 80.9 [50.1-94.1]% versus 49.5 [21.2-71.4]%; P < 0.01). In a multilinear regression model, A. fumigatus-IgE sensitization and sputum neutrophil differential cell count were important predictors of lung function (P = 0.016), supported by culture of A. fumigatus (P = 0.046) and eosinophil differential cell count (P = 0.024). CONCLUSIONS A. fumigatus detection in sputum is associated with A. fumigatus-IgE sensitization, neutrophilic airway inflammation, and reduced lung function. This supports the concept that development of fixed airflow obstruction in asthma is consequent upon the damaging effects of airway colonization with A. fumigatus.

Aspergillus fumigatus during stable state and exacerbations of COPD

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. A. fumigatus sensitisation links to poor COPD lung function; clinical significance of positive fungal culture is unclear http://ow.ly/qfr5q

Routine processing procedures for isolating filamentous fungi from respiratory sputum samples may underestimate fungal prevalence.

Colonization of the airways by filamentous fungi can occur in asthma, chronic obstructive pulmonary disease (COPD) and cystic fibrosis. A recent study found IgE sensitization to Aspergillus fumigatus to be associated with reduced lung function. Significantly higher rates of A. fumigatus were detected in sputum from asthmatics sensitized to this fungus compared to non-sensitized asthmatics. The rate of positive cultures was far higher than equivalent historical samples analysed by the local clinical laboratory following protocols recommended by the UK Health Protection Agency (HPA). This study compares the HPA procedure with our sputum processing method, whereby sputum plugs are separated from saliva and aliquots of approximately 150 mg are inoculated directly onto potato dextrose agar. A total of 55 sputum samples from 41 patients with COPD were analyzed, comparing fungal recovery of five dilutions of sputa on two media. Isolation of A. fumigatus in culture was significantly higher using the research approach compared to the HPA standard method for mycological investigations (P < 0.001). There was also a significant difference in the recovery rate of A. fumigatus (P < 0.05) between media. This highlights the need for a standardized approach to fungal detection which is more sensitive than the method recommended by the HPA

Effectiveness of voriconazole in the treatment of Aspergillus fumigatus–associated asthma (EVITA3 study)

BACKGROUND IgE sensitization to Aspergillus fumigatus and a positive sputum fungal culture result are common in patients with refractory asthma. It is not clear whether these patients would benefit from antifungal treatment. OBJECTIVES We sought to determine whether a 3-month course of voriconazole improved asthma-related outcomes in patients with asthma who are IgE sensitized to A fumigatus. METHODS Asthmatic patients who were IgE sensitized to A fumigatus with a history of at least 2 severe exacerbations in the previous 12 months were treated for 3 months with 200 mg of voriconazole twice daily, followed by observation for 9 months, in a double-blind, placebo-controlled, randomized design. Primary outcomes were improvement in quality of life at the end of the treatment period and a reduction in the number of severe exacerbations over the 12 months of the study. RESULTS Sixty-five patients were randomized. Fifty-nine patients started treatment (32 receiving voriconazole and 27 receiving placebo) and were included in an intention-to-treat analysis. Fifty-six patients took the full 3 months of medication. Between the voriconazole and placebo groups, there were no significant differences in the number of severe exacerbations (1.16 vs 1.41 per patient per year, respectively; mean difference, 0.25; 95% CI, 0.19-0.31), quality of life (change in Asthma Quality of Life Questionnaire score, 0.68 vs 0.88; mean difference between groups, 0.2; 95% CI, -0.05 to -0.11), or any of our secondary outcome measures. CONCLUSION We were unable to show a beneficial effect of 3 months of treatment with voriconazole in patients with moderate-to-severe asthma who were IgE sensitized to A fumigatus on either the rate of severe exacerbations, quality of life, or other markers of asthma control.

Isolation of Aspergillus fumigatus from sputum is associated with elevated airborne levels in homes of patients with asthma

Indoor bioaerosols, such as mold spores, have been associated with respiratory symptoms in patients with asthma; however, dose-response relationships and guidelines on acceptable levels are lacking. Furthermore, a causal link between mold exposure and respiratory infections or asthma remains to be established. The aim of this study was to determine indoor concentrations of Aspergillus fumigatus and a subset of clinically relevant fungi in homes of people with asthma, in relation to markers of airways colonization and sensitization. Air and dust samples were collected from the living room of 58 properties. Fungal concentrations were quantified using mold-specific quantitative PCR and compared with traditional microscopic analysis of air samples. Isolation of A. fumigatus from sputum was associated with higher airborne concentrations of the fungus in patient homes (P = 0.04), and a similar trend was shown with Aspergillus/Penicillium-type concentrations analyzed by microscopy (P = 0.058). No association was found between airborne levels of A. fumigatus and sensitization to this fungus, or dustborne levels of A. fumigatus and either isolation from sputum or sensitization. The results of this study suggest that the home environment should be considered as a potential source of fungal exposure, and elevated home levels may predispose people with asthma to airways colonization.

S133 Eosinophilic airway inflammation is associated with FEV1 decline in severe asthma

Background Severe asthma is a multidimensional disease, with recent evidence supporting the notion that eosinophilic airway inflammation (EAI) is an important driver for exacerbations. In addition EAI has been shown to be associated with airflow limitation in cross sectional studies. However, it remains to be established whether EAI may drive FEV1 decline. Methods The severe asthma registry at Glenfield hospital, Leicester, was screened for patients with a physician diagnosis of asthma and at least 5 years of longitudinal data recording sputum eosinophils, pre- and post-bronchodilator spirometry, inhaled corticosteroid usage as well as standard demographic indices during stable scheduled follow-up visits. Linear mixed effects models were used to investigate the effect of log sputum eosinophils as a time varying covariate on decline of post bronchodilator FEV1. Models were iteratively compared and refined using standard information criteria. Other fixed effects in the final model were, time and the interaction terms for time * log sputum eosinophils and time *daily dose of inhaled corticosteroids and pack years smoked. Individual variations in the slopes and intercepts of time and time*log sputum eosinophils were considered by adding them iteratively as random effects. A first-order autoregressive correlation structure was used to model covariance of random effects. Results 92 patients, 46% male with severe asthma were identified from a registry cohort of 686 between 2000 and 2009. The mean (sem) age was 54(12.9) years and age of onset 23 (2.1) years. The mean (range) duration of follow-up and number of visits were 6 years (4.6–10.5), 2.7/year. We found a significant interaction between sputum eosinophils, time and post bronchodilator FEV1. Indicating a net decline (95% CI) of −16.8 mls(25.8–7.8 mls) /annum/log unit increase in sputum eosinophils (F(1, 43.4); p<0.0001). In contrast there was a net decline 95%CI of −0.015 mls (0.029 to 0.0014 mls)/annum/mcg of inhaled beclamethasonediproprionate daily (F(1,726); p=0.031). Conclusion Eosinophilic airway inflammation is associated with a significant decline in FEV1 in severe asthma.

P131 Outdoor fungal spore levels, lung function and symptoms in patients with asthma and aspergillus sensitisation

Background IgE sensitisation to Aspergillus fumigatus is seen in a significant proportion of patients with refractory asthma. The EVITA3 study recently showed that three months’ treatment with voriconazole did not improve asthma-related outcomes in this patient group. It is not known whether daily variations in outdoor fungal spore levels are associated with concomitant fluctuations in symptoms and lung function in patients with Aspergillus-associated asthma. Methods Participants in the EVITA3 study kept daily diaries of peak expiratory flow (PEF) and asthma symptoms during their follow-up period. These diary records were retrospectively analysed together with contemporaneous fungal spore levels measured locally, in those patients (n = 36) who consented to the secondary use of their clinical and research data. Participants also underwent skin-prick tests for Aspergillus, Alternaria, Cladosporium, Penicillium and Botrytis. For each participant, cross-correlation was used to investigate the relationship between PEF and local spore counts of Aspergillus/Penicillium, Alternaria, Cladosporium, Botrytis, Sporobolomyces, Tilletiopsis, and Didymella. Group-level relationships were investigated using linear mixed models for PEF and generalised estimating equations for daily symptom scores, with participants stratified by skin prick test status. The analyses were performed with the exposure and outcome measured on the same day (lag 0), and with the exposure lagged by 1 day with respect to the outcome (lag 1). Results The analysis cohort comprised 20 men and 16 women with a mean (standard deviation) age of 60 (8) years. No significant or consistent relationships were observed between fungal spore counts and either PEF or self-reported symptom scores, regardless of skin prick test status and lag time between exposure and outcome. In a linear mixed model, the effect size of total fungal spore count on morning PEF was negligible (−0.000011, p = 0.343 for lag 0; −0.000002, p = 0.847 for lag 1). Conclusion In this retrospective analysis we found no evidence of a significant link between fungal spore counts and either PEF or symptoms in patients with Aspergillus-associated asthma. Further research is required to confirm this result in a prospective study and to identify whether aeroallergen levels relate to other important asthma outcomes such as exacerbations.

S90 Effectiveness of Voriconazole In the Treatment of Aspergillus fumigatus Associated Asthma

Background IgE sensitisation to Aspergillus fumigatus and a positive sputum fungal culture are common in refractory asthma. It is not clear whether these patients would benefit from anti-fungal treatment. Objectives To determine if a three-month course of voriconazole improved asthma related outcomes in people with asthma who are IgE sensitised to A. fumigatus. Methods Asthmatics IgE sensitised to A fumigatus with a history of at least two severe exacerbations in the previous twelve months were treated for three months with voriconazole two hundred milligrams twice daily, followed by observation for nine months, in a double blind, placebo controlled, randomised design. Primary outcomes were improvement in quality of life at the end of the treatment period and a reduction in the number of severe exacerbations over the twelve months of the study. Results 65 patients were randomised. 59 patients started treatment (32 voriconazole and 27 placebo) and were included in an intention to treat analysis. 56 patients took the full three months of medication. There was no significant difference in the number of severe exacerbations between the voriconazole and placebo groups (1.25 vs 1.52/patient/year; mean difference 0.27; 95% CI 0.24 to 0.31) respectively, quality of life (change in AQLQ 0.44 vs 0.35, mean difference between groups 0.08; 95% CI 0.07–0.09), or in any of our secondary outcome measures between the two groups. Conclusion We were unable to show a beneficial effect of three months treatment with voriconazole in people with moderate to severe asthma who were IgE sensitised to A fumigatus on either the rate of severe exacerbations, quality of life or other markers of asthma control. Abstract S90 Figure 1. References Fairs A, Agbetile J, Hargadon B, Bourne M, Monteiro WR, Brightling CE, et al. IgE sensitisation to Aspergillus fumigatus is associated with reduced lung function in asthma. Am J Respir Crit Care Med. 2010;182(11):1362–8.

S91 Aspergillus fumigatus sensitisation in patients with chronic obstructive pulmonary disease

Background Bacteria and viruses have been implicated in exacerbations of chronic obstructive pulmonary disease (COPD) and bacteria are often isolated in stable state. Whether fungi are also commonly present and associated with clinical and pathological features of disease is uncertain. Objectives To determine the frequency of filamentous fungal culture and sensitisation to Aspergillus fumigatus in COPD and its relationship to clinical outcomes. Methods Subjects with COPD were recruited from a single centre into a 1-year observational study. Assessments of lung function, allergen testing, and sputum analysis for inflammation, bacterial and fungal cultures were undertaken in COPD subjects and in smoking healthy controls. Results Fungi were cultured at baseline in 63/128 subjects of which 47/63 were A fumigatus. A fungus was cultured in 2/11 controls (both were A fumigatus). The total sputum cell count, sputum neutrophil % and inhaled corticosteroid dosage were significantly increased in COPD patients with a positive fungal culture compared to those without a fungal culture (p<0.05), but the within subject repeatability of fungal culture between stable visits was low (K=−0.04). Sensitisation to A fumigatus was present in 13% of COPD subjects and was associated with worse lung function (FEV1 % predicted 39% vs 51%; p=0.01), but not related to fungal culture. Positive fungal cultures were present in 42/110 exacerbations and were not associated with bacterial culture or severity of exacerbation. Conclusions A fumigatus sensitisation is related to poor lung function. Positive fungal culture is a common feature of COPD. The clinical significance of this remains uncertain.

S136 Fungal sputum culture in patients with severe asthma is associated with a reduced post bronchodilator FEV1

Introduction and objectives IgE sensitisation to fungal allergens is common in severe asthma, but the clinical relevance of this, and the relationship to airway colonisation with fungi, is not known. Many of the fungi that can grow at body temperature are filamentous moulds from the genera Aspergillus and Pencillium. We report here the relationship between lung function and fungal sputum culture in patients with severe asthma. Methods We recruited 126 patients attending a tertiary referral centre with a diagnosis of asthma and 18 healthy volunteers. 93% of patients were on GINA treatment step 4 or higher. At a single stable visit subjects underwent: spirometry with reversibility to 200 μg salbutamol; sputum fungal culture and a sputum cell differential count; skin prick testing to both common aeroallergens and an extended fungal panel (+ve ≥3 mm); specific IgE to Aspergillus fumigatus by CAP (positive >0.35 kU/l). Fungi were identified by morphology and species identity confirmed by sequencing regions of the nuclear ribosomal operon. Results Patients had a mean age of 56 years (21–84 years); 48% were males with median ICS dose of 800 μg Fluticasone equivalent. 60% were atopic to common aeroallergens, 45% were IgE sensitised to one fungal allergen and 27% to ≥2 fungal allergens. 64% of patients cultured a mould in their sputum, 7% more than one species. This compared with three healthy subjects (17%) culturing any mould (p<0.01). Aspergillus species were most frequently cultured (n=58) followed by Penicillium species (n=15) and Thermoascus species (n=2), others (n=8). Four fungal genera were cultured from healthy volunteers sputum,-Aspergillus, Penicillium, Coprinus and one other. Post bronchodilator FEV1% predicted was 71% in those with a positive fungal culture vs 84% in those who were culture negative, (p<0.01). There were no differences in the sputum cell differential between culture positive and negative patients. Conclusions In addition to IgE fungal sensitisation, sputum culture focused towards detection of moulds is frequently positive and associated with impaired post-bronchodilator FEV1. Colonisation of the airways with mould in asthma could be responsible for the development of fixed airflow obstruction.