Cohort. In our observational cohort study, adult patients over the age of 18 with biopsy-proven pulmonary sarcoidosis were enrolled at the University of Iowa Interstitial Lung Disease clinic. This study has obtained ethics approval from the IRB review board-01 review board #IRB00000099. Study protocol followed the University of Iowa’s Institutional Review Board requirements (IRBs: #201709743, 201909726, 202009330, Approved by IRB Chair). Subjects completed informed written consent prior to study participation for all study procedures. Homes were located in Iowa and surrounding states (e.g., Illinois, Wisconsin). Control subject homes were from a similar geographical area. Subjects with sarcoidosis completed a survey interrogating daily habits and exposure sources (N=55, survey included in Appendix A, Supplemental Material).
Pulmonary Function Tests (PFTs). PFT measurements (pre-bronchodilator percent predicted forced vital capacity, FVC, forced expiratory volume in one second FEV1, FVC/FEV1 ratio, and diffusing capacity of carbon monoxide, DLCO) temporally nearest bioaerosol measurement were extracted from the medical records.
Disease severity assessment and quantification. Patient medical records were reviewed to extract demographics and clinical indicators of disease severity, including details of confirmed or probable extrapulmonary organ involvement, disease duration (according to diagnostic guidelines [16]), and the use of immunosuppression (IS). This information was used to calculate a Wasfi Severity Score according to the multivariate model derived by Wafsi et al. [17] (for details, see Appendix A, Supplemental Methods). The presence or absence of lung fibrosis was qualitatively assessed by a clinical radiologist using clinical chest CT scans and confirmed by expert sarcoidosis physician (AKG).
Additionally global disease severity was assessed using a tool applied in various diseases [17,18]. Patients were rated for disease severity by visual analog scale (VAS). An expert clinician (AKG) blinded to exposure data marked a 5-inch horizontal line, whereby 0 indicated incidentally discovered asymptomatic disease and 10 denoted severe end-stage organ dysfunction (e.g., progressive fibrotic lung disease requiring consideration for transplant). The distance from 0 to the mark (in inches), multiplied by 2 defined a patient’s VAS score. We included sarcoidosis health questionnaire (SHQ) data regarding daily, emotional and physical function [19] in those who completed these clinical assessments.
Residential bioaerosol exposure assessment. Electrostatic dust collectors (EDCs) were placed at approximate breathing zone height using sterile endotoxin-free gloves in the primary household occupied room for 2 weeks [20], and subsequently mailed back to our University. EDC cloths were then removed and stored at -20°C. Field and lab blanks (7% of samples) were collected and analyzed identically to the samples.
EDC β-(1,3)-d-glucan extraction. β-(1,3)-d-glucan (BDG) was determined using the Glucatell® assay (Associates of Cape Cod, Inc., East Falmouth, MA, USA) of air samples collected on the EDC cloths, based on a modification of the Limulus Amebocyte Lysate factor G pathway. Room temperature samples were eluted into LAL reagent water (Lonza RTP, Durham, NC, USA), with vortexing and shaking on an orbital shaker (30 min.) followed by sonication (30 min., 22°C), and further shaking (10 min.). Extracts were then centrifuged (15 min., 600 x g, 4°C) and mixed with NaOH (final 0.3N), then shaken (4°C, 30 min.), centrifuged (15 min., 600 x g), and analyzed. BDG was quantified at 37°C using a multimode microplate reader (Molecular Devices M5) at 550 nm. The reagent does not react to other polysaccharides, including β glucans with different glycosidic linkages and is a well-established β-(1,3)-d-glucan measurement method [21].
EDC endotoxin extraction. Endotoxin was extracted from EDCs by established kinetic chromogenic Limulus amebocyte lysate assay methodology [22,23], with a minor modification (additional 30 min. sonication of suspended cloths).
Phlebotomy and serum cytokine processing. Venipuncture was performed on consented individuals within approximately one year of residential measurement. Of our case cohort, serum was obtained from 36 individuals (no pulmonary fibrosis, N=22; with pulmonary fibrosis N=14). Serum and plasma separator tubes were centrifuged (10 min., 2000 RPM), deidentified and stored at -80°C. Serum was centrifuged (10 min., 1000g, 4°C) then assayed using the Human Cytokine/Chemokine 71-Plex Discovery Assay® Array and the Human Supplemental Biomarker 10-Plex Discovery Assay® Array (Eve Technologies, Alberta, CA, for details, see Appendix A, Supplemental Methods).
Statistical analyses. Population size was determined by power analysis (G*Power version 3.1.9.7) based on a publication of a Slovenian population with sarcoidosis – the study measure units of non-specific NAHA from 4hr. filter measurements [12]. Control subject homes had a mean NAHA concentration of 10 U/m3 (N=30, SD=6) and homes of subjects with newly diagnosed sarcoidosis (N=55, SD=37) was 33.6 U/m3. Our two-tailed a priori t-test analysis included α=0.05 and a power of 0.8. We allocated 2 patients:1 control subject, for a total sample size of 48 (32 with sarcoidosis, 16 without). To assure sufficient power, we recruited 84 subjects (59 with sarcoidosis, 25 without).
Residential characteristics were assessed using R (v.4.3.3) by comparing raw BDG values across various strata via Kruskal-Wallis rank sum tests and Wilcoxon rank sum tests. Comparisons between subjects with sarcoidosis and control subjects were made using Fisher’s exact test for categorical variables and using Welch two sample T-tests for continuous variables. Right-skewed data were log-transformed before T-tests were implemented to properly meet T-test assumptions. The same tests were used to compare characteristics of non-fibrotic and fibrotic patients. Remaining statistical analyses were performed using GraphPad Prism (v.10.2.3). Log-transformed bioaerosol concentrations (BDG, endotoxin) were compared to lung function and significance and correlation determined by Pearson correlation. Trend lines were determined by simple linear regression. Differences in residential bioaerosol levels between those with and without significant pulmonary fibrosis were analyzed by unpaired T-tests. One-way ANOVA with Tukey’s adjustment for multiple comparisons was used to assess the effect of season on bioaerosol exposures. Next, BDG and endotoxin concentrations were log transformed and compared to lung function and log-transformed houseplant number +0.1 by simple linear regression. Serum cytokine data were checked for outliers by ROUT, and definitive outliers removed (Q=0.1%), then their association with log-transformed BDG concentrations was evaluated by Pearson r. Herein we present data with a P value ≤0.11 and considered significance at P≤0.05.