Immune Profiles in CF Fungal Infection

While bacteria are predominant agents in CF lung disease, fungi are often isolated in both sputum and bronchoalveolar lavage fluid (BAL), yet their role in CF is not fully understood. Fungal infection in CF has a wide spectrum of presentations, varying from transient detection to persistent infection, acute fungal bronchitis with resulting pulmonary exacerbation, sensitization to fungal allergens, and allergic bronchopulmonary aspergillosis (ABPA). While there are clinical guidelines for the diagnosis and treatment of ABPA in patients with CF, there are few guidelines and recommendations regarding monitoring, clinical care, and antifungal treatment in the various presentations of CF fungal disease; further, the clinical impact of fungi without ABPA is poorly understood. While inflammation in CF is classically considered a neutrophil and macrophage driven process (Th1), eosinophils and allergic cytokines (Th2) have been shown to be elevated in the presence of fungal disease in both clinical and animal CF studies. Studies have also shown decreased lung function (e.g. percent predicted forced expiratory volume in one second, ppFEV1) in individuals with frequent fungal detection in their sputum, and associations with Pseudomonas infection and use of antimicrobials. Adults with CF are also more prone to fungal sensitization when compared to non-CF patients as well as having higher rates of allergic rhinitis and atopy. In a retrospective study performed at our center, we found that children with intermittent or chronic fungal infections experienced more rapid decline in lung function compared to those without fungal infections regardless of ABPA status (see preliminary data). Understanding whether fungal infections are driven by an allergic (Th2) inflammatory process may alter treatment approaches by utilizing steroids and antifungal therapies more readily in patients with fungal infection to combat lung function decline. Limited studies have investigated the frequency of allergic sensitization to fungal pathogens in individuals with CF, the frequency of allergic sensitization in the pediatric CF population in general, and the unique inflammatory profiles and phenotypes of CF fungal infections, both in the sputum and the serum. Hypothesis: We hypothesize that children with CF and fungal infections without ABPA will have elevated allergic inflammatory profiles and increased sensitization to fungal elements compared to those without fungal infection. Specific Aim 1: Compare Th2 inflammation in patients with and without fungal infections in patients with CF and to those with ABPA. * Approach: We will obtain serum and sputum samples from 25 patients with either (1) fungal infection without ABPA, n=10 (defined as ≥ two positive fungal cultures in prior 18 months), (2) no fungal infection, n=10, and (3) ABPA, n=5, and measure specific sputum and serum cytokine measurements to evaluate Th2 and Th1 inflammatory pathways. * Biomarkers measured: Eosinophilic cationic protein (ECP), Interleukin-4 (IL4), Interleukin-5 (IL5), Interleukin-10 (IL10), Interleukin-13 (IL13), and eosinophil count in both serum and sputum. * Expected outcome: Individuals with fungal infection without APBA will have elevated Th2 markers of inflammation compared to those with no fungal infection. We will also compare to those with ABPA as a separate control group. We will also measure Th1 markers to be used as a reference to previously established inflammatory profiles seen in CF, seeing if those with fungal disease deviate towards a more Th2 driven process. Specific Aim 2: Investigate allergic sensitization to fungal elements in patients with CF fungal infection without ABPA compared to those without fungal infection and to those with ABPA. * Approach: We will measure total IgE and serum ImmunoCAP testing of specific IgE levels to fungal proteins of various species (Aspergillus, Candida, etc). to determine fungal sensitization. We will also survey patients regarding environmental exposures to explore relationships with allergic sensitization. * Expected outcome: Individuals with fungal infection without ABPA will be more likely to show allergic sensitization to fungal elements when compared to those with no fungal infection. The results of this study will help characterize the inflammatory profile associated with CF fungal infections contributing to the understanding of both the infectious and allergic nature of disease in the CF population. From this, the contribution to both the pathophysiology and clinical characteristics of CF fungal infections will serve as a step towards understanding management options, care guidelines, and disease progression for this unique set of hard to treat organisms. Recruitment: We propose to recruit 25 patients with CF aged between 8 and 25 years who agree to participate in this cross-sectional study looking at the immune system characteristics of fungal infection and ABPA. We plan to screen individuals coming to their routine CF clinic and approach them regarding their interest in participating in this study. From this, we will explain the concept, hypothesis, and procedures involved with intent to schedule them for a research visit. Patient Selection: Patients will be recruited from the CF Center at CHCO by study investigators or qualified research coordinators at the time of a routine clinic visit. Study Visit: We plan to prospectively recruit 25 subjects with CF during a period of clinical stability. Subjects will have one research clinic visit that will coincide with their clinic visit at CHCO Pulmonary Clinic facilities. The following diagnostic information will be entered onto Case Report Forms: demographic information, diagnostic history and mutations, CF-related co-morbidities, historic microbiology results including fungal culture results, medications including modulator therapy and long-term antibiotic regimens, previous allergy testing, diagnostic criteria for ABPA (if applicable). We will also utilize historical clinical data from the medical record to establish other co-morbidities, lung function trends and baseline, history of other bacterial infections, history of allergy diagnoses, and other pertinent medical history and diagnostic testing (ie previous CT scan results, previous blood work, etc.). The following evaluations will be performed : * Historical data in the medical record. Examples include Co-morbid diagnoses related to CF (such as but not limited to CF related diabetes, CF liver disease, asthma, etc), previous blood work (such as but not limited to serum IgE levels, previous allergy testing, bronchoscopy data and lavage studies, routine and urgent/admission CF labs, etc), previous spirometery and lung function data, previous nutritional and growth data (BMI, vitamin levels, etc), previous microbiology and culture data, etc. * BMI measurements obtained from regular CF clinic visit * Height and weight to be obtained and subsequently calculated. * Pulmonary function testing obtained from regular CF clinic visit. Spirometry will be performed according to American Thoracic Society criteria, while absolute values will be converted to percent predicted using the Global Lung Function initiative reference equations34. * Sputum collection. Spontaneously expectorated sputum will be used as possible.This will be collected at home by the patient following a routine respiratory airway clearance regimen within 12 hours of clinic visit. The sample will be placed in a mailed specimen cup and placed on ice and brought to the clinic visit. If sputum is not able to be expectorated prior to visit, we will attempt to utilize remaining sputum expectorated that is not used for clinical needs for further analysis and storage. Sputum may be collected by sputum induction according to the current CFF Therapeutics Development Network standard operating procedure if 1) unable to expectorate, (2) patient meets clinical criteria for sputum induction and (3) clinic is able to perform research induced sputum based on current COVID-19 guidelines to ensure safety of team member and adequate PPE supply. For safety reasons, the induction procedures will only be performed for subjects who meet the following criteria on the day of the induction: FEV1 ≥ 30% predicted, No history of \> 5 mL hemoptysis within 48 hours prior to the visit, Able to tolerate the sputum induction procedure. Of note, there is little significant difference in specimen quality for analysis between expectorated and induced sputum. Markers to be tested as previously described above. * Environmental fungal exposure questionnaire. Will be performed prior to finish of clinic visit. This questionnaire in entirety is a validated questionnaire used by Dr. Andy Liu. Given time constraints for study participants, we are using a modified questionnaire focused on questions of interested to this study (e.g. pertaining to fungal exposure). This has not been validated but may provide important information in this pilot study for future research * Serum collection. Peripheral venous blood draw will be performed by certified individual using appropriate sterile technique by the CTRC. Samples will be labeled per research protocol standards. Markers to be tested described above. * All sputum samples to be banked and saved for potential microbiome, mycobiome and transcriptome analysis, assessing gene expressions unique to various inflammatory pathways. Sample Size Estimates: Power and sample size for the study proposal is fixed due to the expected number of eligible patients to be recruited into this pilot study during the study window. Based on preliminary data and the described definitions of types of infection, investigators anticipate that over a 1-year recruitment window they will be able to enroll approximately 10 individuals with no fungal infection, 10 patients with a fungal infection but not ABPA and 5 with ABPA. The primary analysis plan focuses on the description and comparison of key plasma and sputum measures described in Table 3. Power analysis provides the detectable effect sizes when comparing the control arm (no fungal infection) to either the non-ABPA group or the ABPA group. Calculations are based on a power of 80% and Type I error rate of 0.05 for two-sided two-sample equal-variance t-test conducted in PASS version 15 statistical software (2). Group sample sizes of 10 and 5 in the fungal infection groups achieve 80% power to reject the null hypothesis of zero effect size when the population effect size is 1.36 and 1.66 respectively. Thus, the study is powered at 80% to detect a difference in the mean markers to be measured greater than 1.66 and 1.32 standard deviations. Given limited data in the literature regarding Th2 markers in CF, we are basing these calculations off of deviations from a mean for the multiple Th2 markers we are testing. Data Collection and Storage: All subjects will be de-identified upon study sample collection and stored with a master key. There will be limited access to this information with the PI and only essential research staff able to monitor and view the data. All hardcopy source documents will be kept in a locked file cabinet in the CF research team's storage facilities. Additionally, to further ensure data integrity, data for this study will be stored in Research Electronic Data Capture system (REDCap), which will allow limited access to only essential personal. REDCap enables in-line validation to minimize transcription errors and provides real-time notifications of data submission and allows immediate central monitoring and feedback. Access to data requires an encrypted secure socket layer (SSL) connection and changes are logged by user ID, time stamp and project. Databases are backed up frequently.