All infants diagnosed with CF by neonatal screening programs at the Verona CF Centre (Verona), Italy and The Children's Hospital at Westmead (Westmead), Sydney, Australia from January 1993 to December 2001 were included in the study.
The basic strategy of the two screening programs was similar: 1) an IRT measurement was taken on dried blood spots obtained from infants in the first week of life; 2) if the IRT was elevated (at Verona >100 μg/l until May 1995 and >95 μg/l thereafter, and at Westmead >99th percentile), a mutation analysis was performed; and 3) the diagnosis was confirmed by sweat chloride estimation. The major difference between the two programs pertained to the mutational analysis: at Verona, a region where at least one copy of ΔF508 was present in only 40%–60% of patients, the blood spots were analysed for ΔF508, R1162X and N1303K prior to March 1995 and ten additional mutations (2183AA→G, 3849+10KbC→T, G542X, 1717‐1G→A, R553X, Q552X, G85E, 711+5G→A, 3132delTG, 2789+5G→A)16
thereafter; at Westmead, only ΔF508 was screened for. The other minor difference in the screening strategy was that patients at Verona had a meconium lactase test performed and if either a CF mutation was detected and/or the lactase level was >0.5 U/l, a sweat chloride test was carried out. It is noteworthy that nine of the 13 mutations screened at Verona were severe mutations.7,17,18
At both centres, elevated IRT, the presence of at least one CF mutation (or elevated meconium lactase in infants from Verona) and an elevated sweat chloride
60 mmol/l were diagnostic of CF. Infants at both centres with borderline sweat chloride values of 30–59 mmol/l underwent repeat sweat testing and a more extensive panel of mutations was analysed. At Verona this included denaturing gradient gel electrophoresis of coding regions and at Westmead 16 non‐ΔF508 mutations: Δ1507, R117H, G551D, A455E, G542X, N1303K, W128X, 1717‐1G→A, R560T, R347P, R334W, R553X, R1162X, S549N, 3849+10KbC→T and 621+1G→T.19
Excluding ΔF508, six of the other mutations were common to both centres.
At Verona, IRT was assayed by the Pharmacia (Turku, Finland) standardised Delfia Neonatal IRT kit,20
and a variation was used at Westmead.21
Sweat chloride testing was performed by the Gibson and Cooke method22
at both centres and meconium lactase testing as described previously.23
The procedure used to prepare DNA for PCR from the dried blood spots on Guthrie cards was similar at both centres.24,25
Between January 1993 and February 1995, mutation analysis was performed by restriction enzymes26
or heteroduplex analysis27
and thereafter by a reverse dot blot assay.28
At Westmead, DNA for mutation analysis was extracted from blood spots by isopropanol fractionation.29
Assessment of pancreatic function in both centres
Fat balance studies
In formula‐fed infants, dietary fat intake was recorded for 5 days, and stool was collected in the last 3 days of the 5‐day balance study. In breastfed infants, where dietary fat intake was not estimated, stool was collected over 3 days.
Daily faecal fat content was determined by van de Kamer's technique30
and modified by the Jeejeebhoy method for medium chain triglycerides.31
If the infant was already receiving oral pancreatic enzyme supplements, these were stopped 48 h prior to the fat balance study. Fat malabsorption was defined as a faecal fat loss
10% of fat intake in formula‐fed infants and faecal fat of
2 g/day in breastfed infants.4
Pancreatic stimulation test (PST)
The PST was performed only at Westmead using a quantitative marker perfusion technique.4,32
Pancreatic lipase and colipase were analysed by titrimetric assay33
and infants with colipase secretion rates
1% of mean control values were assessed as PS and those with rates <1% as PI.4,32
Infants designated as PS at diagnosis were evaluated prospectively. Symptoms suggestive of malabsorption, such as oily/fatty stools, weight loss and/or a decrease in weight percentile and/or a change in normal serum fat soluble vitamin levels to subnormal levels (serum vitamin A <0.6 μmol/l, vitamin E <7.0 μg/100 ml), and at Verona faecal chymotrypsin <5 U/g stool, provided indirect evidence of the development of PI which was confirmed by fat balance studies. Patients without a history of malabsorption and/or normal fat balances, normal stool microscopy and pancreatic isoamylase >14 U/l were designated as PS.
The sample size was based on the hypothesis that there would be a 10%–15% reduction in the proportion of PS patients among those diagnosed by the IRT mutation strategy versus the older repeat IRT screening strategy (25% vs 37%, respectively). Therefore, at least 300 patients would be required in the current study group to detect this difference with a power of 80% at a significance of 5%. Thus, the study was conducted over 9 years to meet this requirement.34
Data were analysed both cumulatively and separately for the two centres. The χ2 test was performed to analyse the frequency of PS and PI patients and genotypes between the two phenotypes.
The study was approved by the Ethics Committee at The Children's Hospital at Westmead and the Verona Hospital Ethics Committee.