Patients
Patients participated in the screening program
The study population comprised of infants who were born between 01.07.2009 and 31.12.2019 in Lodz Voivodship and were referred to our CF center as a part of NBS for CF and performed CFTR gene analysis according to IRT/DNA protocol described below. The infants had been evaluated at least once per 3 months. They were divided to three groups according to laboratory tests and further clinical evaluation as CF NBS Group, CF SPID and false positive NBS. Cystic fibrosis (CF NBS Group) was diagnosed in infants based on the level of sweat chloride in addition to evidence of CFTR dysfunction. The designation CF SPID was established to asymptomatic infants if they presented a positive CF NBS test plus: sweat chloride < 59 mmol/L and 2 CFTR mutations with 0–1 CF-causing CFTR mutations. The asymptomatic CF NBS positive infant with presence of no CFTR mutation or mutation in one allele plus a negative sweat test, referred as false positive NBS (including carriers).
Patients diagnosed with CF before introduction of screening program
We also analyzed data of 52 patients (24 females and 28 males) with established CF diagnosis born between 1996 and 2009 (before the newborn screening in Poland) attending our Cystic Fibrosis Outpatient Center in Copernicus Hospital in Lodz, Poland. Patients in this group had been referred to the Center due to occurrence of symptoms suggesting CF (CF Group).
Next, we compared both groups CF NBS and CF. We have chosen the interval - between 5 and 8 years of age to compare both groups, because at that time the most data were available.
Data assessment
In both groups the data were obtained retrospectively after a review of patient charts. The socio-economic data were analysed. These included a place of living defined as a large city above 100,000 inhabitants, a small city 10,000–100,000 inhabitants, and a village below 10,000 inhabitants. The perinatal period contained data: delivery data and weight, Apgar score and presence of meconium ileus (MI), time of CF diagnosis. We also analysed body weight and percentile of body weight at 5 and 8 years of age.
Clinical evaluation
Respiratory status was assessed by the forced expiratory volume in 1 s (FEV1) in spirometry, expressed as percentages of predicted values for height, weight, age, and gender. All analyzed measurements were done in fifth and 8 year of life, only in stable periods (using a Jaeger MasterScreen Body spirometer; E Jaeger GmbH; Wurzburg, Germany). The tests were performed according to American Thoracic Society standards [10].
The number of bronchopulmonary exacerbations and number of hospitalizations (due to pulmonary disease, longer than 3 days) between five and 8 years of life were evaluated. Pulmonary exacerbation was defined as excessive sputum expectoration, general malaise, and need for antibiotics. Chronic bacteria colonization Pseudomonas aeruginosa (PA) or methicillin-resistant Staphylococcus aureus (MRSA) were also collected, and defined as three positive consecutive sputum cultures over a period of 6 months in fifth and 8 year of life.
CF NBS protocol
IRT was measured in dried blood samples from 3 to 5 days old neonates. The IRT concentration cut-off was established as > 99.4 percentile (according to the pilot CF NBS program [6]). The elevated IRT caused further genetic testing from the same dried blood samples. In neonates with meconium ileus (MI), the DNA analysis was performed regardless of IRT level. The measurements of IRT (by using IRT Neonatal Screening ELISA colorimetric assay - IBL International) and DNA analysis from sampling paper (processing with the Extract Blood PCR Kit) were performed in the Genetic Department, Institute of Mother and Child, Warsaw, Poland. Since September 2011, the extended DNA analysis panel has been used and comprised 95% of mutated alleles in the Polish population.
Neonates with one or two mutations in CFTR detected due to NBS examinations were directed to CF Centres for clinical assessment and sweat tests (measured by the quantitative pilocarpine iontophoresis method and Nanoduct method parallel) [6]. CF NBS was conducted as a pilot programme in four Polish districts in the period 1999–2003. In 2006 CF NBS started again in the same regions and was gradually extended across the country. In June 2009 covered all of newborn population in Poland. In the 1980s, the prevalence of CF for Caucasians was estimated at 1: 2500 live births. The disease incidence was more accurately calculated to 1: 4000–5000 thanks to the extensive implementation of CF NBS in the world. In Poland due to data from CF NBS in the years 2006–2010 it is believed that one child every 4394 live births is born with CF.
The study was approved by the Ethical Committee of the Medical University of Lodz, Poland (nr RNN/145/20/KE).
Statistical analysis
Numerical traits were described by way of the arithmetic mean, standard deviation (SD), standard error (SE), when applicable, and their minimum-to-maximum values. Categorical variables were depicted by using counts (n) and percentages (%).
Multiple logistic (for binary dependent variables), multiple linear (for numerical traits), and multiple Poisson (for count data) regression models were fitted in order to test statistical co-dependencies. When dealing with non-normally distributed variables, robust standard errors (i.e. sandwich estimators) were used in the regression equations. All the regression models were controlled for the studied patients’ characteristics such as sex, gender, place of residence, birth weight, APGAR and also for crucial infections (PA, MRSA) before their 5th year of age. Missing data were case-wise deleted.
A level of P < 0.05 was deemed statistically significant. All the computations were performed by using of Stata/Special Edition, release 14.2 (StataCorp LP, College Station, Texas, USA).