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Department of Medicine, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA, USACenter for Clinical Epidemiology and Biostatistics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
Presence of Aspergillus fumigatus in sputum is linked with greater respiratory symptoms in CF
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Inhaled corticosteroid use may be associated with Aspergillus fumigatus isolation
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Candida species and Aspergillus fumigatus are the most common fungi in CF sputum.
Abstract
Background
The clinical effects of Aspergillus fumigatus in the cystic fibrosis (CF) airway, with the exception of allergic bronchopulmonary aspergillosis, is unclear.
Methods
CF adolescents and adults (age 14 years and older) underwent bacterial and semi-selective fungal culture testing to determine the prevalence of fungi in the CF respiratory tract and the independent association between the presence of Aspergillus fumigatus and clinical characteristics.
Results
Aspergillus fumigatus (10.3%) and Candida species (57.8%) were the most common filamentous fungi and yeast seen respectively in the sputa of 206 individuals with CF. Inhaled corticosteroid (ICS) use was more common in Aspergillus fumigatus-positive than Aspergillus fumigatusnegative (100% versus 75.8%, p = .01). Aspergillus fumigatus was significantly associated with lower respiratory domain score (β −8.74, 95% CI −16.6, −0.88, p = .03), representing worse respiratory-related quality of life, accounting for demographics, disease characteristics, and the presence of a pulmonary exacerbation.
Conclusion
The presence of Aspergillus fumigatus in CF sputum was associated with worse respiratory quality of life in CF in a crosssectional, single center study. Longitudinal analysis examining the clinical implications of Aspergillus fumigatus on respiratory health over time is needed.
Cystic fibrosis (CF), an autosomal recessive life-limiting genetic disease, is classically characterized by chronic airway infections that lead to progressive respiratory disease and ultimately respiratory failure [
]. The epidemiology of pathogens found in the CF airway has historically focused on the bacterial inhabitants; however, Aspergillus fumigatus (Af), the most common filamentous fungi in CF patients, is becoming more frequently isolated in sputum cultures [
]. Yet, the clinical significance of Af in the CF host in the absence of allergic bronchopulmonary aspergillosis (ABPA) remains unclear.
Previous studies have suggested that Af colonization or persistence in the CF airways is associated with increased pulmonary exacerbations requiring hospitalization, mosaic attenuation and bronchiectasis on chest computed tomography, and failure of clinical improvement of pulmonary exacerbations despite intravenous (IV) antibiotics [
]. The interpretation of these data is limited by retrospective study design, small sample size, and ascertainment bias. We aimed to determine the relationship between Af detected in fungal culture and disease characteristics in individuals with CF. We hypothesized the presence of Af in CF sputum was associated with lower respiratory-related quality of life (QOL). In addition, description of other clinically reported fungi in CF, including Scedosporium boydii complex, other Aspergillus species, Trichosporon mycotoxinivorans, and Exophiala dermatidis were conducted. Preliminary data for this study were presented previously in abstract form [
We conducted a cross-sectional study of adolescents and adults with CF (age 14 years and greater) receiving clinical care at the Children's Hospital of Philadelphia (CHOP) and Hospital of University of Pennsylvania (HUP) from March 2017 through October 2018. We included subjects with a diagnosis of CF according to the CF Foundation diagnosis guidelines [
] and excluded patients who had received solid organ transplantation. The study was approved by the University of Pennsylvania and CHOP institutional review boards (825979).
2.2 Study procedures
All participants (or their parents) provided written informed consent (or assent, when age appropriate). Subjects underwent routine clinical evaluation by a CF physician, performed spirometry, and underwent bacterial culture evaluation of sputum. If subjects were unable to expectorate, sputum induction with 3% hypertonic sodium chloride was performed. Spirometry was performed according to the American Thoracic Society guidelines; forced expiratory volume in 1 s (FEV1) percent predicted reported according to the National Health and Nutrition Examination Survey III prediction equations. The Cystic Fibrosis Questionnaire-Revised (CFQ-R), a validated CF-specific health-related QOL survey, was administered [
]. Demographics, comorbidities, medications, past spirometry, microbiological data, and antibiotic usage for pulmonary exacerbation (PEx) episodes over the 12 months previous to the visit were collected.
2.3 Laboratory processing
The sputum samples underwent bacterial culture and semi-selective fungal culture evaluation within 8 h of collection in the HUP clinical microbiology laboratory using a research protocol. Whole sputum was directly inoculated on MacConkey agar, sheep blood agar, chocolate agar, Burkholderia cepacia selective agar (BCSA), and mannitol salt agar and incubated at 35 degrees Celsius for 3 days (with the exception of BCSA incubation for 5 days). In addition, inhibitory mold agar, brain-heart infusion agar, Mycosel, and CHROMagar (Becton, Dickinson, Sparks, Maryland) were incubated at 30 degrees Celsius for two weeks for the culture of fungi. Morphologic identification of macroscopic and microscopic features of fungal isolates and if necessary, DNA sequencing of the D1/D2 region of the 28S ribosomal subunit were performed when morphologic identification alone was insufficient. Assessments of the cultures and DNA sequencing were performed by individuals blinded to all clinical information.
2.4 Dependent and independent variables
The primary independent variable of interest was Af isolation on culture. The primary dependent variable was the CFQ-R respiratory domain score, which ranges from 0 to 100 with a higher score reflecting better respiratory-related QOL (minimal clinically important difference of 4 points) [
Determination of the minimal clinically important difference scores for the cystic fibrosis questionnaire-revised respiratory symptom scale in two populations of patients with cystic fibrosis and chronic Pseudomonas aeruginosa airway infection.
]. Other dependent variables included FEV1 percent predicted and diagnosis of PEx (defined as clinician diagnosis of PEx at time of study visit). Covariates of interest included age, female sex, white race, F508del cystic fibrosis transmembrane conductance regulator (CFTR) homozygosity, pancreatic insufficiency, body mass index (BMI), CF related diabetes mellitus (CFRD), allergic bronchopulmonary aspergillosis (ABPA), number of PEx (oral and/or IV antibiotic use) in previous 12 months, and number of severe PEx (IV antibiotic use) in previous 12 months. BMI was calculated based in weight and heigh for absolute value in kg/m2. Pancreatic insufficiency, CFRD, and ABPA status were defined by clinician diagnosis in the medical chart. Additional covariates included microbiological data: presence of Pseudomonas aeruginosa (Pa), methicillin-resistant Staphylococcus aureus (MRSA), and Burkholderia cepacia complex at the study visit. In additional secondary analyses, the following independent variables were explored: Scedosporium boydii complex, non-fumigatus Aspergillus species, Trichosporon mycotoxinivorans, Exophiala dermatidis, Candida species, and clinically important fungi defined as isolation of Af, Scedosporium boydii complex, non-fumigatus Aspergillus species, Trichosporon mycotoxinivorans, or Exophiala dermatidis.
Prevalence was determined by fungal isolation and identification on culture. Recurrent Af was defined as detection of Af at the study assessment with a history of positive Af cultures in the previous two years.
2.5 Statistical analysis
Continuous and categorical clinical variables were compared between subjects with and without Af status by Wilcoxon rank sum test and Fisher's exact test respectively. Variables were also compared between Af-positive, Af-negative, and non-expectorators by Kruskal-Wallis analysis of variance and Fisher's exact test. Simple linear regression was performed to explore the unadjusted associations between independent variables and outcomes, CFQ-R respiratory domain score and FEV1 percent predicted. Univariate logistic regression was performed to assess association between Af-positive status and PEx diagnosis. We assessed the independent association between Af and CFQ-R respiratory domain score using multivariable linear regression with adjustment for confounders. Model selection of covariates was based on existing medical knowledge and/or univariate analyses with p < .10. Multiple imputation with chained equations was performed to account for missing data. Additional sensitivity analyses were performed, [
] exploring relationships between other clinically relevant fungi (Scedosporium species, other Aspergillus species, Trichosporon mycotoxinivorans, Exophiala dermatidis) individually and collectively. P values are 2-sided, with alpha of 0.05. Statistical analyses were performed using STATA 15 (StataCorp LP, College Station, TX).
3. Results
3.1 Subject characteristics
A total of 206 participants were enrolled in the study. Twenty-four (11.7%) subjects were unable to expectorate sputum or undergo sputum induction. Af was detected in 21 (10.3%, 95% CI 6.4,15.2%) individuals (Fig. 1). Characteristics of the cohort stratified by Af-status are shown in Table 1. Inhaled corticosteroid use was more commonly seen in participants with Af-positive culture. Lower respiratory domain score was seen in the Af-positive group compared to Af-negative. Age, sex, white race, F508del homozygosity, pancreatic insufficiency, BMI, FEV1 percent predicted, CFRD, and other CF-related medications did not differ between Af-positive and Af-negative groups. Antifungal therapy, chronic prednisone use (greater than one month of continuous use), and history of Pa were more commonly trended in Af-positive group, but were not statistically significantly different. Supplemental Table 1 characterizes the full cohort, including individuals unable to expectorate sputum. Non-expectorators (n = 24) represented an older and potentially healthier group (pancreatic sufficient and lower Pa prevalence).
Thirty-seven individuals (18.0%, 95% CI 13.0, 23.9%) of the study population had a history of Af in the two-year period before enrollment. Yet, recurrent Af was seen in only 11 subjects (5.3%, 95% CI 2.70, 9.35%). Fig. 1 shows prevalence of all fungi recovered in the cohort. Candida species (57.8%) was the most common fungus detected in the study population. We found additional clinically relevant fungi, Scedosporium boydii complex (2.4%), Trichosporon mycotoxinivorans (2.4%), non-fumigatus Aspergillus species (1.9%), and Exophiala dermatidis (2.4%). We detected 22 new cases of clinically relevant fungi using semi-selective fungal culture media in 20 individual subjects without history of fungal isolation (Fig. 2). We identified four subjects with co-infection: two subjects with Af and Exophiala dermatitidis, Af and non-fumigatus Aspergillus, and Af and Scedosporium boydii complex.
Fig. 2Newly detected and prevalent fungal diagnoses.
Associations between clinical characteristics and CFQ-R respiratory domain score are depicted in Table 2 (unadjusted). Table 3 represents the imputed multivariable model of participants who produced sputum for evaluation on the day of the assessment. Af culture positivity was associated with a respiratory domain score 11.7 points lower in the univariate analysis, and 8.43 points lower than individuals without Af after accounting for age, sex, lung function, pancreatic insufficiency, Pa status, and a PEx diagnosis at the time of specimen collection (β −8.43, 95% CI −16.3, −0.53, p = .04). We did not find interactions between Af and Pa-positive culture at visit or history of Pa (defined as any present or previous isolation of Pa on culture).
Table 2Unadjusted association between patient characteristics and CFQ-R respiratory domain score (n = 205).
PEx was diagnosed in 91 (44.2%) of patients at the study visit. Af status was not associated with the clinical diagnosis of a PEx (odds ratio 1.82, 95% CI 0.73, 4.57 p = .20). Af status was not associated with FEV1 percent predicted at visit (β −5.35 95% CI −17.1, 6.36, p = .37) or change in FEV1 percent predicted from average lung function over 12 months previous to visit (β −2.00, 95% CI −5.27, 1.28, p = .23).
3.5 Sensitivity analyses
Several sensitivity analyses were conducted to evaluate the rigor of our findings. We found a similar relationship between Af and poor respiratory quality of life (β −8.47, 95% CI −16.4, 0.53, p = .04) in the complete case analysis (Supplemental Table 2). We then performed multiple imputation with chained equations to all subjects (expectorators and non-expectorators), which exhibited consistent results (Supplemental Table 3). We also forced in ABPA into our final multivariable model described in the primary analysis; Af was still associated with lower respiratory domain score, β −8.06, 95% CI −16.1, −0.05, p = .049 (Supplemental Table 4). With excluding the 15 subjects with ABPA, the effect estimate of the association between Af and respiratory QOL remained unchanged with borderline statistical significance (−8.17, 95% CI −17.0, 0.65, p = .07). Recurrent Af was associated with lower respiratory domain score, β −12.9, 95% CI −24.9, −0.90 p = .04 in univariate analysis; however, when adjusting for covariates, the association was attenuated and was no longer statistically significant (β −9.07, 95% CI −19.8, 1.61, p = .10). Other fungi, including Scedosporium boydii complex, non-fumigatus Aspergillus species, Trichosporon mycotoxinivorans, Exophiala dermatidis, and Candida species were not associated with lower patient reported respiratory QOL (data not shown).
4. Discussion
We found that Af was significantly associated with worse patient-reported respiratory-related QOL in a cross-sectional study of adolescents and adults receiving care in a large CFF-accredited center and semi-selective fungal culture evaluation. Subjects with Af had a clinically meaningful difference of −8.43 points in respiratory domain score compared to Af-negative subjects. A trend between recurrent Af and worse respiratory QOL was seen; though, the classification of recurrent Af should be cautiously interpreted, as subjects' historical sputum cultures may not have used selective fungal culture media. The presence of Af has been postulated to be a marker of disease severity and antibiotic exposure in CF; however, clinical markers of more advanced CF, such as Pa colonization, BMI, and FEV1 percent predicted did not differ between the Af-positive and Af-negative groups. Unlike previous studies [
], chronic inhaled and oral antibiotics were not associated with the presence of Af. Whereas, subjects using inhaled corticosteroid use were more likely to recover Af in sputum; a relationship that has been previously reported [
Changes in CF respiratory-related health status over time are highly correlated with significant changes in the CFQ-R respiratory domain score and have exhibited high discrimination of “stable” and “sick” clinical states [
Determination of the minimal clinically important difference scores for the cystic fibrosis questionnaire-revised respiratory symptom scale in two populations of patients with cystic fibrosis and chronic Pseudomonas aeruginosa airway infection.
]. Although our analysis is cross-sectional and cannot inform causality or changes in health over time, these data suggest that Af isolation may be associated with a perception of overall poorer respiratory health independent of other indicators of illness severity. Furthermore, Af may have significant respiratory impact on patients in the setting of stable lung function and absence of an exacerbation. Increased inflammatory responses (neutrophil elastase and IL-8) have been implicated in children with Aspergillus positive bronchoalveolar lavage cultures, suggesting a biological mechanism for the observed relationship [
]. Yet, the question remains; is Af contributing to poor respiratory health or a consequence of poor respiratory health? Longitudinal analyses of these issues are needed and may yield further insights.
To our knowledge, our study is the first investigation of the relationship between Af and patient-reported outcomes in a general CF population. Lung function decline, pulmonary exacerbations, and radiographic findings of lung disease have been the outcomes of previous studies examining the clinical effects of Aspergillus species [
Chronic Aspergillus fumigatus colonization of the pediatric cystic fibrosis airway is common and may be associated with a more rapid decline in lung function.
]. Aaron et al. conducted a randomized, placebo-controlled trial of itraconazole for chronic Af in CF patients which did not find a difference in change in respiratory domain score of CFQ-R and other clinical endpoints over 24-week treatment period. Subtherapeutic itraconazole drug levels in 43% of patients in the treatment arm of the study and small sample size limit the interpretation of these data [
]. In contrast, Coughlan et al. demonstrated an improvement of respiratory symptoms (CFQ-R) after six-weeks of itraconazole therapy in an observational study of 13 Af-colonized CF patients; however, therapeutic drug monitoring was not described in these data and this study was not randomized or controlled [
]. While we did not exclude ABPA in the primary analysis, sensitivity analysis adjusting for ABPA in the multivariable model were performed and did not alter our findings. With omitting ABPA from the study sample, the relationship between Af isolation and lower QOL remained with borderline statistical significance (β −8.17, 95% CI -17.0, 0.65, p = .07), most likely due to sample size reduction.
There was an unexpectedly high proportion of PEx diagnosed at the time of the research study visit. Individuals were recruited in the outpatient clinic, regardless of a routine follow-up visit versus sick visit. A plausible explanation for this finding could be that an individual would be more likely to have a clinical encounter when sick. The median number of antibiotic courses (oral or IV) for a diagnosis of PEx in the previous 12 months was 2 (interquartile range [IQR] 1,3) and IV antibiotic courses were lower with median 0 (IQR 0,1), which is reflective of the general CF population in the U.S. [
] PEx was significantly associated with lower respiratory QOL, β −15.4 points, 95% CI -20.5, −10.2. Therefore, we adjusted PEx status in our final model and the relationship between Af and worse respiratory symptom score remained.
We investigated the interaction between Af and Pa given the evidence of Pa inhibition of Af growth and mutual antagonism of Af towards Pa in vitro [
Inhibition of Aspergillus fumigatus and its biofilm by Pseudomonas aeruginosa is dependent on the source, phenotype and growth conditions of the bacterium.
Aspergillus fumigatus inhibits Pseudomonas aeruginosa in co-culture: implications of a mutually antagonistic relationship on virulence and inflammation in the CF airway.
]. We also saw a trend for history of Pa colonization to observed more in the Af-group compared to Af-negative group (Table 1). The presence of Pa colonization may reflect more diseased airways environment for inhaled Af conidia to germinate. Also, in-vivo microbial interactions between Pa and Af could be contributing; although this is not well-explained by the known in-vitro interactions. In addition, worse clinical outcomes in children and adults with CF, including lower lung function and greater PEx episodes, are seen in co-infection of Pa and Af [
Co-colonisation with Aspergillus fumigatus and Pseudomonas aeruginosa is associated with poorer health in cystic fibrosis patients: an Irish registry analysis.
]. There was no statistical interaction between Af and Pa on respiratory-related QOL in our data.
Our study has several limitations. The cross-sectional design of the study precludes any inference of directionality in the relationship discovered between Af and respiratory QOL. Therefore, Af may represent a bystander of advanced CF lung disease rather than a factor in the causal pathway of disease progression. Longitudinal studies could shed light on this question. We were also unable to classify Af status as chronic or persistent, previously described as two or more Af-positive culture in one year, due to the study design [
]. Our study attempted to overcome this by including previous microbiological culture data. We found a trend for the association of recurrent Af with decreased respiratory QOL. Yet, the risk of misclassification of recurrent Af exists as selective fungal culture procedures were not conducted for all historical sputum cultures. Based on our previous work, our study protocol inclusive of selective fungal culture media likely facilitates detection and recovery for fungi in CF sputa [
]. Our data also lacked the assessment of Af-specific immunoglobin (Ig) levels, including IgE and IgG, which prevented the differentiation of the previously described Aspergillus bronchitis and Aspergillus sensitization; however, the clinical impact of these phenotypes is poorly defined [
In conclusion, these data support the correlation of Af recovery on culture and poor respiratory health in CF, accounting for individual level factors and acute PEx. The study design prevents from inferring that Af directly causes worse respiratory health in people with CF, but these data suggest a relationship which warrants further investigation. Prospective longitudinal analyses examining the clinical implications of Af culture positivity on respiratory health over time is clearly needed.
Contributions
GH had access to the full data in the study and take responsibility for the integrity of the data and data analysis. GH, KA, SN, VF, CK, RCR, DJD, SMK, and DH contributed to the study design and conduct and writing of the manuscript. DH and SMK contributed to the data analysis and writing of the manuscript.
Financial disclosures
None.
Funding
The work was supported by the grant funding and support from the Cystic Fibrosis Foundation (grant HONG16A0).
Acknowledgements
The authors would like to thank the participants and their families for volunteering their time and information for this study. We also would like to thank Karen Hendricks for the microbiology laboratory processing and analysis of sputum samples and Justin Martin, Lydia Ramharack, Yujia Su, Jean Malpass, and Paige Notarianni for their contributions in the subject recruitment and data collection.
Determination of the minimal clinically important difference scores for the cystic fibrosis questionnaire-revised respiratory symptom scale in two populations of patients with cystic fibrosis and chronic Pseudomonas aeruginosa airway infection.
Chronic Aspergillus fumigatus colonization of the pediatric cystic fibrosis airway is common and may be associated with a more rapid decline in lung function.
Inhibition of Aspergillus fumigatus and its biofilm by Pseudomonas aeruginosa is dependent on the source, phenotype and growth conditions of the bacterium.
Aspergillus fumigatus inhibits Pseudomonas aeruginosa in co-culture: implications of a mutually antagonistic relationship on virulence and inflammation in the CF airway.
Co-colonisation with Aspergillus fumigatus and Pseudomonas aeruginosa is associated with poorer health in cystic fibrosis patients: an Irish registry analysis.
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