Human Airway Basal Cells Undergo Reversible Squamous Differentiation and Reshape Innate Immunity.

Histological and lineage immunofluorescence examination revealed that healthy conducting airways of humans and animals harbor sporadic poorly differentiated epithelial patches mostly in the dorsal noncartilage regions that remarkably manifest squamous differentiation. In vitro analysis demonstrated that this squamous phenotype is not due to intrinsic functional change in underlying airway basal cells. Rather, it is a reversible physiological response to persistent Wnt signaling stimulation during de novo differentiation. Squamous epithelial cells have elevated gene signatures of glucose uptake and cellular glycolysis. Inhibition of glycolysis or a decrease in glucose availability suppresses Wnt-induced squamous epithelial differentiation. Compared with pseudostratified airway epithelial cells, a cascade of mucosal protective functions is impaired in squamous epithelial cells, featuring increased epithelial permeability, spontaneous epithelial unjamming, and enhanced inflammatory responses. Our study raises the possibility that the squamous differentiation naturally occurring in healthy airways identified herein may represent "vulnerable spots" within the airway mucosa that are sensitive to damage and inflammation when confronted by infection or injury. Squamous metaplasia and hyperplasia are hallmarks of many airway diseases, thereby expanding these areas of vulnerability with potential pathological consequences. Thus, investigation of physiological and reversible squamous differentiation from healthy airway basal cells may provide critical knowledge to understand pathogenic squamous remodeling, which is often nonreversible, progressive, and hyperinflammatory.

Working towards consensus in the management of pediatric chronic rhinosinusitis in cystic fibrosis.

OBJECTIVE: The prevalence of chronic rhinosinusitis (CRS), defined by mucosal thickening on imaging, approaches 100% in the cystic fibrosis (CF) population. CRS is associated with significant morbidity in CF, including its ability to trigger pulmonary exacerbations. CRS in CF is typically managed by pediatricians, otolaryngologists and pulmonologists. This survey evaluates the variance in practice patterns of CRS in CF amongst specialists. METHODS: This is a cross-sectional, electronic survey in which maximum variation purposive sampling was used by a multi-disciplinary group of pediatric, otolaryngology and pulmonology providers in order to select a survey population with expertise in CRS in CF patients. The survey was distributed to 381 practitioners from September to October 2019. RESULTS: 175 participants responded (45% response rate). Ten (of 54) statements achieved 75% consensus agreement. Consensus statements included: The decision to pursue surgical intervention for CRS in CF is a multi-disciplinary approach (94%; n = 146); maximal medical management should include nasal saline irrigation (93%; n = 142), topical steroids (75%; n = 117), maximal medical management should not include intravenous steroids (79%; n = 122); image guidance in surgery is necessary for all surgery involving the frontal sinuses (77%; n = 43), and all revision surgery(80%, n = 45); the appropriate setting for sinus surgery in a CF patient varies depending on patient presentation (89%; n = 133); post-operative regimen should include nasal saline (93%; n = 137); but does depend on the severity of disease discovered intra-operatively (84%; n = 124); post-operative antibiotics should be guided by intra-operative culture data (82%; n = 121). CONCLUSIONS: There is a great deal of variation amongst specialists in the treatment of CRS in CF, however 10 statements met consensus criteria and should be considered when forming clinical care guidelines in this population.

An open-label extension study of ivacaftor in children with CF and a CFTR gating mutation initiating treatment at age 2-5 years (KLIMB).

BACKGROUND: KIWI (NCT01705145) was a 24-week, single-arm, pharmacokinetics, safety, and efficacy study of ivacaftor in children aged 2 to 5 years with cystic fibrosis (CF) and a CFTR gating mutation. Here, we report the results of KLIMB (NCT01946412), an 84-week, open-label extension of KIWI. METHODS: Children received age- and weight-based ivacaftor dosages for 84 weeks. The primary outcome was safety. Other outcomes included sweat chloride, growth parameters, and measures of pancreatic function. RESULTS: All 33 children who completed KIWI enrolled in KLIMB; 28 completed 84 weeks of treatment. Most adverse events were consistent with those reported during KIWI. Ten (30%) children had transaminase elevations >3 × upper limit of normal (ULN), leading to 1 discontinuation in a child with alanine aminotransferase >8 × ULN. Improvements in sweat chloride, weight, and body mass index z scores and fecal elastase-1 observed during KIWI were maintained during KLIMB; there was no further improvement in these parameters. CONCLUSIONS: Ivacaftor was generally well tolerated for up to 108 weeks in children aged 2 to 5 years with CF and a gating mutation, with safety consistent with the KIWI study. Improvements in sweat chloride and growth parameters during the initial 24 weeks of treatment were maintained for up to an additional 84 weeks of treatment. Prevalence of raised transaminases remained stable and did not increase with duration of exposure during the open-label extension.

SMAD Signaling Restricts Mucous Cell Differentiation in Human Airway Epithelium.

Mucin-secreting goblet cell metaplasia and hyperplasia (GCMH) is a common pathological phenotype in many human respiratory diseases, including asthma, chronic obstructive pulmonary disease, cystic fibrosis, primary ciliary dyskinesia, and infections. A better understanding of how goblet cell quantities or proportions in the airway epithelium are regulated may provide novel therapeutic targets to mitigate GCMH in these devastating diseases. We identify canonical SMAD signaling as the principal pathway restricting goblet cell differentiation in human airway epithelium. Differentiated goblet cells express low levels of phosphorylated SMAD. Accordingly, inhibition of SMAD signaling markedly amplifies GCMH induced by mucous mediators. In contrast, SMAD signaling activation impedes goblet cell generation and accelerates the resolution of preexisting GCMH. SMAD signaling inhibition can override the suppressive effects imposed by a GABAergic receptor inhibitor, suggesting the GABAergic pathway likely operates through inhibition of SMAD signaling in regulating mucous differentiation. Collectively, our data demonstrate that SMAD signaling plays a determining role in mucous cell differentiation, and thus raise the possibility that dysregulation of this pathway contributes to respiratory pathophysiology during airway inflammation and pulmonary diseases. In addition, our study also highlights the potential for SMAD modulation as a therapeutic target in mitigating GCMH.

Effects of a primary palliative care intervention on quality of life and mental health in cystic fibrosis.

BACKGROUND: Despite the significant impact of chronic symptoms on quality of life with cystic fibrosis (CF), the role of palliative care in management of this disease is not well defined. The coping, goal assessment, and relief from evolving CF symptoms (CF-CARES) model is a primary palliative care intervention designed to provide chronic symptom management at all stages of the disease. The goal of this pilot study was to estimate the effectiveness of the CF-CARES intervention on improving chronic symptoms and quality of life for people living with CF. METHODS: A structured assessment was used to guide referral to supportive services intended to address burdensome symptoms. Follow-up assessments were performed approximately 3 and 6 months later. Longitudinal regression analyses of changes in symptoms and quality of life were performed for all participants regardless of utilization of supportive services. Subgroup analyses were performed for subjects participating in mental health and alternative health services. RESULTS: Forty-one subjects completed assessment and referral processes. The mean number of CF-associated symptoms decreased over time, as did respiratory symptom-related distress and depressive symptoms. Subjects utilizing alternative health services reported less psychological distress at follow-up. Among subjects with severe disease, mental health, and quality of life improved, especially for those using mental health services. CONCLUSIONS: The CF-CARES model resulted in significant mental health and quality-of-life benefits, suggesting the value of integrating symptom management interventions into routine CF care. Moreover, mental health services can play a key role in CF-specific primary palliative care, especially for those with advanced disease.

Dual SMAD Signaling Inhibition Enables Long-Term Expansion of Diverse Epithelial Basal Cells.

Functional modeling of many adult epithelia is limited by the difficulty in maintaining relevant stem cell populations in culture. Here, we show that dual inhibition of SMAD signaling pathways enables robust expansion of primary epithelial basal cell populations. We find that TGFß/BMP/SMAD pathway signaling is strongly activated in luminal and suprabasal cells of several epithelia, but suppressed in p63+ basal cells. In airway epithelium, SMAD signaling promotes differentiation, and its inhibition leads to stem cell hyperplasia. Using dual SMAD signaling inhibition in a feeder-free culture system, we have been able to expand airway basal stem cells from multiple species. Expanded cells can produce functional airway epithelium physiologically responsive to clinically relevant drugs, such as CFTR modulators. This approach is effective for the clonal expansion of single human cells and for basal cell populations from epithelial tissues from all three germ layers and therefore may be broadly applicable for modeling of epithelia.

Safety, pharmacokinetics, and pharmacodynamics of ivacaftor in patients aged 2-5 years with cystic fibrosis and a CFTR gating mutation (KIWI): an open-label, single-arm study.

BACKGROUND: Ivacaftor has been shown to be a safe, effective treatment for cystic fibrosis in patients aged 6 years or older with a CFTR gating mutation. We aimed to assess the safety, pharmacokinetics, and pharmacodynamics of ivacaftor in children aged 2-5 years. METHODS: In the two-part KIWI study, we enrolled children aged 2-5 years weighing 8 kg or more with a confirmed diagnosis of cystic fibrosis and a CFTR gating mutation on at least one allele from 15 hospitals in the USA, UK, and Canada. Participants received oral ivacaftor 50 mg (if bodyweight <14 kg) or 75 mg (if bodyweight ≥14 kg) every 12 h for 4 days in part A (to establish the short-term safety of doses for subsequent assessment in part B), and then for 24 weeks in part B (to assess safety and longer-term pharmacodynamics). Children could participate in both or just one part of the study. Primary outcomes were pharmacokinetics and safety, analysed in all patients who received at least one dose of ivacaftor. Secondary outcomes were absolute change from baseline in sweat chloride concentrations and bodyweight, body-mass index (BMI), and height Z scores, and pharmacokinetic parameter estimation of ivacaftor. This study is registered with ClinicalTrials.gov, number NCT01705145. FINDINGS: Between Jan 8, 2013, and March 1, 2013, nine patients were enrolled onto part A of the study, all of whom completed the 4 day treatment period, and eight of whom took part in part B. Between June 28, 2013, and Sept 26, 2013, 34 patients were enrolled in part B, 33 of whom completed the 24 week treatment period. All patients received at least one dose of ivacaftor. Results of ivacaftor pharmacokinetics suggested that exposure was similar to that reported in adults (median Cmin were 536 ng/mL for the 50 mg dose; 580 ng/mL for the 75 mg dose; median ivacaftor AUC values were 9840 ng × h/mL and 10 200 ng × h/mL, respectively). Common adverse events in part B included cough (in 19 [56%] of 34 patients) and vomiting (in ten [29%]). Five (15%) patients had liver function test (LFT) results that were more than eight times higher than the upper limit of normal, four of whom had study drug interrupted, and one of whom had study drug discontinued. Six (18%) of 34 patients had seven serious adverse events; a raised concentration of transaminases was the only serious adverse event regarded as related to ivacaftor and the only adverse event that resulted in study treatment discontinuation. At week 24, in patients for whom we had data, sweat chloride had changed from baseline by a mean of -46·9 mmol/L (SD 26·2, p<0·0001), weight Z score by 0·2 (0·3; p<0·0001), BMI Z score by 0·4 (0·4, p<0·0001), and height Z score by -0·01 (0·3; p=0·84). INTERPRETATION: Ivacaftor at doses of 50 mg and 75 mg seems to be safe in children aged 2-5 years with cystic fibrosis with a gating mutation followed up for 24 weeks, although the frequency of elevated LFTs suggests that monitoring should be frequent in young children, particularly those with a history of elevated LFTs. Results of an ongoing extension study assessing durability of these effects and longer-term safety are warranted. FUNDING: Vertex Pharmaceuticals Incorporated.

Development and evaluation of a palliative care curriculum for cystic fibrosis healthcare providers.

BACKGROUND: Primary palliative care refers to basic skills that all healthcare providers can employ to improve quality of life for patients at any stage of disease. Training in these core skills is not commonly provided to clinicians caring for cystic fibrosis (CF) patients. The objective of this study was to assess change in comfort with core skills among care team members after participation in CF-specific palliative care training focused on management of burdensome symptoms and difficult conversations. METHODS: A qualitative needs assessment was performed to inform the development of an 18-hour curriculum tailored to the chronicity and complexity of CF care. A 32-question pre- and post-course survey assessed CF provider comfort with the targeted palliative care skills in 5 domains using a 5-point Likert scale (1=very uncomfortable, 3=neutral, 5=very comfortable). RESULTS: Among course participants (n=16), mean overall comfort score increased by 0.9, from 3 (neutral) to 3.9 (comfortable) (p<0.001). Mean comfort level increased significantly (range 0.8 to 1.4) in each skill domain: use of supportive care resources, pain management, non-pain symptom management, communication, and psychosocial skills. CONCLUSIONS: CF-specific palliative care training was well received by participants and significantly improved self-assessed comfort with core skills.

Fathers and Asthma Care: Paternal Involvement, Beliefs, and Management Skills.

OBJECTIVE: To compare asthma care roles of maternal and paternal caregivers, and examine associations between caregiver involvement and the outcomes of adherence, morbidity, and parental quality of life (QoL). METHODS: Mothers and fathers in 63 families of children, ages 5-9 years, with persistent asthma completed semistructured interviews and questionnaires. Adherence was measured via electronic monitoring. Paired t tests compared parental asthma care roles, and analysis of covariance, controlling for socioeconomic status, evaluated associations of asthma outcomes with caregiver involvement scores. RESULTS: Mothers had higher scores on measures of involvement, beliefs in medication necessity, and on four subscales of the Family Asthma Management System Scale interview (Asthma Knowledge, Relationship with Provider, Symptom Assessment, and Response to Symptoms). Maternal QoL was lowest when both maternal and paternal involvement was high. Paternal involvement was associated with increased morbidity. CONCLUSIONS: There is room for enhancement of fathers' asthma care roles. Higher levels of paternal involvement may be driven by family need.

Trends in bone mineral density in young adults with cystic fibrosis over a 15 year period.

BACKGROUND: Improvements in clinical care have led to increased life expectancy in patients with cystic fibrosis (CF) over the past several decades. Whether these improvements have had significant effects on bone health in patients with CF is unclear. METHODS: This is a cross-sectional study comparing clinical characteristics and bone mineral density (BMD) measured by dual energy X-ray absorptiometry (DXA) in adults with CF evaluated in 1995-1999 to age-, race-, and gender-matched patients with CF evaluated in 2011-2013 at the same center on calibrated DXA machines. RESULTS: The cohorts were similar in terms of age, BMI, pancreatic insufficiency, presence of F508del mutation, and reproductive history. In the most recent cohort, pulmonary function was superior, and fewer patients had vitamin D deficiency or secondary hyperparathyroidism. Areal BMD measures of the PA spine, lateral spine, and distal radius were similarly low in the two cohorts. CONCLUSIONS: Although pulmonary function and vitamin D status were better in patients in the present-day cohort, areal BMD of the spine was reduced in a significant number of patients and was no different in patients with CF today than in the late 1990s. Further attention to optimizing bone health may be necessary to prevent CF-related bone disease.

Compromised bone microarchitecture and estimated bone strength in young adults with cystic fibrosis.

CONTEXT: Young adults with cystic fibrosis (CF) are at risk for low bone density and fractures, but the underlying alterations in bone microarchitecture that may contribute to their increased fracture risk are currently unknown. OBJECTIVE: The main goal of this study was to use high-resolution peripheral quantitative computed tomography (HR-pQCT) to characterize the bone microarchitecture, volumetric bone mineral density (vBMD), and estimated strength of the radius and tibia in young adults with CF compared with healthy volunteers. DESIGN AND SETTING: This was a cross-sectional study at an outpatient clinical research center within a tertiary academic medical center. PARTICIPANTS: Thirty young adults with CF, 18 to 40 years of age, were evaluated and compared with 60 healthy volunteers matched by age (±2 years), gender, and race. MAIN OUTCOME MEASURES: The primary outcomes were HR-pQCT-derived cortical and trabecular vBMD, bone microarchitecture, and estimates of bone strength. RESULTS: At the radius and tibia, young adults with CF had smaller bone cross-sectional area and lower vBMD. Cortical and trabecular microarchitecture were compromised at both sites, most notably involving the trabecular bone of the tibia. These differences translated into lower estimated bone strength both at the radius and tibia. After accounting for body mass index differences, young adults with CF had lower bone area and estimated bone strength at the radius and had compromised trabecular microarchitecture and lower total and trabecular vBMD and estimated bone strength at the tibia. Alterations in trabecular bone density and microarchitecture and estimated strength measures of the tibia were also greater than expected based on dual-energy x-ray absorptiometry-derived areal BMD differences. CONCLUSIONS: Young adults with CF have compromised bone microarchitecture and lower estimated bone strength at both the radius and tibia, even after accounting for their smaller body size. These skeletal deficits likely explain the higher fracture risk observed in young adults with CF.

Pooled analysis of two large randomised phase III inhaled mannitol studies in cystic fibrosis.

BACKGROUND: To evaluate safety and efficacy of inhaled mannitol treatment in subgroups of a large global CF population. METHODS: Data were pooled from two multicentre, double-blind, randomised, controlled, parallel group phase III studies in which 600 patients inhaled either mannitol (400 mg) or control (mannitol 50 mg) twice a day for 26 weeks. RESULTS: Both the mean absolute change in FEV(1) (mL) and relative change in FEV(1) by % predicted from baseline for mannitol (400 mg) versus control were statistically significant (73.42 mL, 3.56%, both p<0.001). Increases in FEV(1) were observed irrespective of rhDNase use. Significant improvements in FEV1 occurred in adults but not children (6-11) or adolescents (aged 12-17). Pulmonary exacerbation incidence was reduced by 29% (p=0.039) in the mannitol (400 mg) group. CONCLUSIONS: Sustained six-month improvements in lung function and decreased pulmonary exacerbation incidence indicate that inhaled mannitol is an important additional drug in the treatment of CF.

Generation of multipotent lung and airway progenitors from mouse ESCs and patient-specific cystic fibrosis iPSCs.

Deriving lung progenitors from patient-specific pluripotent cells is a key step in producing differentiated lung epithelium for disease modeling and transplantation. By mimicking the signaling events that occur during mouse lung development, we generated murine lung progenitors in a series of discrete steps. Definitive endoderm derived from mouse embryonic stem cells (ESCs) was converted into foregut endoderm, then into replicating Nkx2.1+ lung endoderm, and finally into multipotent embryonic lung progenitor and airway progenitor cells. We demonstrated that precisely-timed BMP, FGF, and WNT signaling are required for NKX2.1 induction. Mouse ESC-derived Nkx2.1+ progenitor cells formed respiratory epithelium (tracheospheres) when transplanted subcutaneously into mice. We then adapted this strategy to produce disease-specific lung progenitor cells from human Cystic Fibrosis induced pluripotent stem cells (iPSCs), creating a platform for dissecting human lung disease. These disease-specific human lung progenitors formed respiratory epithelium when subcutaneously engrafted into immunodeficient mice.

Long-term inhaled dry powder mannitol in cystic fibrosis: an international randomized study.

RATIONALE: New treatment strategies are needed to improve airway clearance and reduce the morbidity and the time burden associated with cystic fibrosis (CF). OBJECTIVES: To determine whether long-term treatment with inhaled mannitol, an osmotic agent, improves lung function and morbidity. METHODS: Double-blind, randomized, controlled trial of inhaled mannitol, 400 mg twice a day (n = 192, "treated" group) or 50 mg twice a day (n = 126, "control" group) for 26 weeks, followed by 26 weeks of open-label treatment. MEASUREMENTS AND MAIN RESULTS: The primary endpoint was absolute change in FEV(1) from baseline in treated versus control groups, averaged over the study period. Secondary endpoints included other spirometric measurements, pulmonary exacerbations, and hospitalization. Clinical, microbiologic, and laboratory safety were assessed. The treated group had a mean improvement in FEV(1) of 105 ml (8.2% above baseline). The treated group had a relative improvement in FEV(1) of 3.75% (P = 0.029) versus the control group. Adverse events and sputum microbiology were similar in both treatment groups. Exacerbation rates were low, but there were fewer in the treated group (hazard ratio, 0.74; 95% confidence interval, 0.42-1.32; P = 0.31), although this was not statistically significant. In the 26-week open-label extension study, FEV(1) was maintained in the original treated group, and improved in the original control group to the same degree. CONCLUSIONS: Inhaled mannitol, 400 mg twice a day, resulted in improved lung function over 26 weeks, which was sustained after an additional 26 weeks of treatment. The safety profile was also acceptable, demonstrating the potential role for this chronic therapy for CF. Clinical trial registered with www.clinicaltrials.gov (NCT 00630812).

Cystic fibrosis newborn screening: using experience to optimize the screening algorithm.

Newborn screening (NBS) for cystic fibrosis (CF) offers the opportunity for early diagnosis and improved outcomes in patients with CF and has been universally available in the state of Massachusetts since 1999 using an immunoreactive trypsinogen (IRT)-DNA algorithm. Ideally, CF NBS is incorporated as part of an integrated NBS system that allows for comprehensive and coordinated education, laboratory screening, clinical follow-up, and evaluation so that evidence-based data can be used to maximize quality improvements and optimize the screening algorithm. The New England Newborn Screening Program (NENSP) retrospectively analyzed Massachusetts's CF newborn screening data that yielded decisions to eliminate a screen-positive category, maintain the IRT cutoff value that prompts the second tier DNA testing, and communicate CF relative risk to primary care providers (PCPs) based on categorization of positive CF NBS results.

Nutrient status of adults with cystic fibrosis.

Nutrition is thought to influence disease status in patients with cystic fibrosis (CF). This cross-sectional study sought to evaluate nutrient intake and anthropometric data from 64 adult outpatients with cystic fibrosis. Nutrient intake from food and supplements was compared with the Dietary Reference Intakes for 16 nutrients and outcomes influenced by nutritional status. Attention was given to vitamin D and calcium given potential skeletal implications due to cystic fibrosis. Measurements included weight, height, body composition, pulmonary function, and serum metabolic parameters. Participants were interviewed about dietary intake, supplement use, pulmonary function, sunlight exposure, and pain. The participants' mean body mass index (+/-standard deviation) was 21.8+/-4.9 and pulmonary function tests were normal. Seventy-eight percent used pancreatic enzyme replacement for malabsorption. Vitamin D deficiency [25-hydroxyvitamin D (25OHD)<37.5 nmol/L] was common: 25 (39%) were deficient despite adequate vitamin D intake. Lipid profiles were normal in the majority, even though total and saturated fat consumption represented 33.0% and 16.8% of energy intake, respectively. Reported protein intake represented 16.9% of total energy intake (range 10%-25%). For several nutrients, including vitamin D and calcium, intake from food and supplements in many participants exceeded recommended Tolerable Upper Intake Levels. Among adults with cystic fibrosis, vitamin D deficiency was common despite reported adequate intake, and lipid profiles were normal despite a relatively high fat intake. Mean protein consumption was adequate, but the range of intake was concerning, as both inadequate or excessive intake may have deleterious skeletal effects. These findings call into question the applicability of established nutrient thresholds for patients with cystic fibrosis.

Population-based newborn screening for genetic disorders when multiple mutation DNA testing is incorporated: a cystic fibrosis newborn screening model demonstrating increased sensitivity but more carrier detections.

OBJECTIVES: Newborn screening for cystic fibrosis (CF) provides a model to investigate the implications of applying multiple-mutation DNA testing in screening for any disorder in a pediatric population-based setting, where detection of affected infants is desired and identification of unaffected carriers is not. Widely applied 2-tiered CF newborn screening strategies first test for elevated immunoreactive trypsinogen (IRT) with subsequent analysis for a single CFTR mutation (DeltaF508), systematically missing CF-affected infants with any of the >1000 less common or population-specific mutations. Comparison of CF newborn screening algorithms that incorporate single- and multiple-mutation testing may offer insights into strategies that maximize the public health value of screening for CF and other genetic disorders. The objective of this study was to evaluate technical feasibility and practical implications of 2-tiered CF newborn screening that uses testing for multiple mutations (multiple-CFTR-mutation testing). METHODS: We implemented statewide CF newborn screening using a 2-tiered algorithm: all specimens were assayed for IRT; those with elevated IRT then had multiple-CFTR-mutation testing. Infants who screened positive by detection of 1 or 2 mutations or extremely elevated IRT (>99.8%; failsafe protocol) were then referred for definitive diagnosis by sweat testing. We compared the number of sweat-test referrals using single- with multiple-CFTR-mutation testing. Initial physician assessments and diagnostic outcomes of these screened-positive infants and any affected infants missed by the screen were analyzed. We evaluated compliance with our screening and follow-up protocols. All Massachusetts delivery units, the Newborn Screening Program, pediatric health care providers who evaluate and refer screened-positive infants, and the 5 Massachusetts CF Centers and their affiliated genetic services participated. A 4-year cohort of 323 506 infants who were born in Massachusetts between February 1, 1999, and February 1, 2003, and screened for CF at approximately 2 days of age was studied. RESULTS: A total of 110 of 112 CF-affected infants screened (negative predictive value: 99.99%) were detected with IRT/multiple-CFTR-mutation screening; 2 false-negative screens did not show elevated IRT. A total of 107 (97%) of the 110 had 1 or 2 mutations detected by the multiple- CFTR-mutation screen, and 3 had positive screens on the basis of the failsafe protocol. In contrast, had we used single-mutation testing, only 96 (87%) of the 110 would have had 1 or 2 mutations detectable by single-mutation screen, 8 would have had positive screens on the basis of the failsafe protocol, and an additional 6 infants would have had false-negative screens. Among 110 CF-affected screened-positive infants, a likely "genetic diagnosis" was made by the multiple-CFTR-mutation screen in 82 (75%) versus 55 (50%) with DeltaF508 alone. Increased sensitivity from multiple-CFTR-mutation testing yielded 274 (26%) more referrals for sweat testing and carrier identifications than testing with DeltaF508 alone. CONCLUSIONS: Use of multiple-CFTR-mutation testing improved sensitivity and postscreening prediction of CF at the cost of increased referrals and carrier identification.