Methods
Participants
Participants were recruited from two medical centers in California. One hundred and twelve children were recruited due to having had a positive CF NBS and a designation of either CRMS/CFSPID or CF. As defined by the IRT/DNA/DNA sequencing algorithm, participants had at least twoCFTR variants identified.8 Protocol was approved by the Institutional Review Boards of Children’s Hospital Los Angeles (CHLA) and Kaiser Permanente Los Angeles Medical Center (KP) and informed consent was obtained from all participating families.
Procedures
As part of this research study, clinical data were collected cross-sectionally and included height, weight, physical exam, and a study-specific questionnaire related to relevant signs and symptoms in the past 12 months (Supplemental Figure 1 ). Sweat chloride concentration (sw[Cl-]) testing (ELItech Macroduct® system) was performed following Cystic Fibrosis Foundation guidelines.9 Spirometry was performed using the CareFusion Vmax Encore device (Care-Fusion, Yorba Linda, California, USA). Forced expiratory volume in one second reported in percent predicted (ppFEV1), forced vital capacity (FVC), and FEV1/ FVC measurements were converted to global lung function initiative (GLI).10 Lung Clearance Index (LCI2.5) derived from nitrogen multiple breath washout testing (MBW) was offered to all subjects ≥3 years old, and used the Exhalyzer D® device (Eco Medics AG, Duernten, Switzerland) with Spiroware software (version 3.1.6). Device calibration was performed daily prior to testing. MBW test was performed per ATS/ERS consensus statement and as described previously.11-13 Cystic Fibrosis Questionnaires-Revised (CFQ-R) were applied for parents and children ≥6 years old.14
Electronic medical records (EMR) were reviewed from birth; data collected included CFTR genotype, immunoreactive trypsinogen (IRT) value, sw[Cl] results, microbiology data (focusing on Pseudomonas aeruginosa [PSA]), fecal elastase values, history of hospitalizations, history of lower respiratory tract infections (Pneumonia) and pulmonary exacerbations, use of oral or intravenous antibiotics in the past 12 months, and provider documentation of crackles, chronic or recurrent respiratory symptoms, constipation, signs of malabsorption, and relevant treatments. Pancreatic insufficiency was defined as fecal elastase <200 µg/g. Chronic PSA was defined as two positive respiratory cultures in the past 12 months.15 Both clinical research sites used diagnostic criteria based on CF Foundation guidelines for CF and CRMS/CFSPID.16 CRMS/CFSPID-to-CF reclassification occurred when an individual’s sw[Cl-] rose to ≥60 mmol/L, one or more of an individual’s CFTR variants changed to a CF-causing interpretation by CFTR2 (https://cftr2.org), and/or an individual developed signs and symptoms of CF according to provider’s discretion.
Nasal epithelial samples were obtained from a subgroup of subjects to explore the utility of Human Nasal Epithelial (HNE) assays in defining baseline CFTR function, which could potentially identify risk for reclassification. Subjects with one CF-causing variant and a copy of R117H;7T, 5T;TG12, or 5T;TG13 were prioritized. Sampling and Ussing chamber testing of planar respiratory cultures were performed as previously described.17, 18
Statistical analysis
Data are described in mean and standard deviation or median and interquartile range for continuous variables, frequency and percentage for categorical variables. Differences in key clinical variables between sites, and demographic and clinical characteristics between diagnostic groups (CF vs. CRMS/CFSPID) were examined using chi-squared or Fisher’s exact tests for categorical variables and Wilcoxon rank-sum or two sample t-tests for continuous variables. A non-additive effect was assessed using Firth logistic regression and generalized linear model to evaluate whether the difference in CF and CRMS/CFSPID varied by site on ever having a positive culture for PSA (PSA-ever), fecal elastase, sw[Cl-], and CFQ-R respiratory domain. Using longitudinal data collected from EMR, sw[Cl-] progression was evaluated using a linear median quantile mixed-effects model which time is modeled as age at measurement and random effect at participant level. Individual sw[Cl-] increases were estimated as a slope that was based on at least three measurements (18 individuals were excluded for not having at least 3 values). The association of clinical characteristics with the risk of reclassification was assessed by Firth logistic regression model to reduce bias due to the small number of reclassified patients. Multivariate Firth logistic regression model further assessed the association of clinical characteristics with the risk of reclassification adjusting for site and demographic characteristics. All statistical computations were performed in Stata 17 (StataCorp, College Station, TX).