METHODS

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A more detailed description of the Methods is provided in the online supplement.

CT Reporting

Images were reported by an experienced pediatric thoracic radiologist (C.M.) blind to BAL results. Scans were read in batches, in random order, on a soft copy reporting station (Agfa ImpaxTM v. 5.2; Agfa-Gevaert, Mortsel, Belgium) using standard lung settings. Lungs were considered in six zones (upper, mid, and lower; right and left) corresponding to each axial slice. The presence of bronchial dilatation, bronchial wall thickening, and gas trapping was recorded in a binary fashion for each zone. The standard radiologic definition of bronchiectasis (bronchus to pulmonary artery diameter ratio >1 [19]) was used to define bronchial dilatation for the purpose of this study. Bronchial wall thickening was assessed subjectively. Gas trapping was defined as geographic foci of reduced density on the expiratory images (20) (Figure 1). The extent of each abnormality was graded by determining the proportion of each zone affected (<50% = 1; >50% = 2) and calculated by adding the scores (0,1) for each zone multiplied by the extent (1, 2) to give sums of 0 to 12 for bronchial dilatation, bronchial wall thickening, and gas trapping, respectively.

Figure 1. Representative images from chest computed tomography scans showing (A) normal scan, (B) bronchial dilatation (arrow), (C) bronchial wall thickening (arrows), and (D) gas trapping (arrows). Images in A, B, and C were obtained at full inspiration (inspiratory pressure, 25 cm H₂O), and the image in D was obtained at end expiration.

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Inflammation.

Total and differential cell counts were performed using identical protocols (11) at each center, with interoperator variability <10%. Inflammatory markers were performed in Perth to ensure consistency.

RESULTS

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Fifty-seven infants (31 male infants, 54.4%) diagnosed with CF at a median age of 28 days had a chest CT and BAL performed at a median age of 3.6 months (Table 1). The mode of presentation and presence of clinically apparent disease at diagnosis are given in Table E1 (see the online supplement). At the time of assessment, 9 infants (15.8%) had respiratory symptoms, and 33 (57.9%) were receiving antistaphylococcal prophylaxis with amoxicillin clavulanate. Despite being diagnosed with CF after detection by NBS, 49 (86.0%) infants had pancreatic insufficiency and showed signs of inadequate nutrition, with a group mean body mass index (Z score) of −1.19 (25th to 75th percentile, −1.78 to −0.50).

DISCUSSION

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The results of the present study demonstrate that a substantial proportion of infants diagnosed with CF after detection by NBS have active pulmonary inflammation, 30% have detectable NE activity, 20% have pulmonary infection, and 80% have evidence of structural lung disease on chest CT at 3 months of age. Furthermore, although infants with respiratory symptoms are more likely to have lung disease, the majority with detectable lung disease are asymptomatic. The inflammatory marker most associated with structural lung disease was free NE activity, suggesting that new therapeutic approaches are required for preventing structural lung disease in infants with CF, including assessment of therapies, such as macrolides and intravenous antibiotics, that appear to be effective in older children.

The data from the present study may provide some explanation as to why a clear improvement in respiratory outcomes in populations with NBS has been difficult to demonstrate. In our population of infants, 21% had pulmonary infection, including four infected with S. aureus and three with P. aeruginosa; neutrophilic inflammation was common with raised neutrophil counts, and 30% had detectable levels of free NE in BAL. In addition, 80% had an abnormal chest CT, with 19% having bronchial dilatation, 45% having bronchial wall thickening, and 67% having gas trapping. These findings demonstrate that clinically significant lung disease is present very early in infants with CF in the absence of clinically apparent respiratory symptoms in the majority of infants.

There are some limitations to our study. First, we do not have comparative data from a control group of healthy 3-month-old children; there is a paucity of such data in the literature. We are aware of only one report of BAL findings in healthy children (21), in which neutrophil counts were reported from seven healthy infants with median (25%, 75%) absolute count of 32 (11.3, 46.5) × 10³ cells per ml, representing 8% (4.0%, 11.5%) of the total cell count. No data are available on NE or proinflammatory cytokines; however, free NE activity is not expected in the lungs of healthy individuals (22). Thus, the levels of inflammation in the present study in uninfected children with CF are higher than expected. However, the levels of inflammation are similar to previous reports in children with CF, especially when not infected (8, 13, 14, 23, 24). In addition, we do not have chest CTs from healthy control children. Where chest CTs are performed for assessment of lung disease or for cancer staging, very different scanning protocols are used. Specifically, oncology or nonrespiratory chest CTs are performed with much higher radiation doses (in our institutions), so the resolution is very different. In addition, because airway dilatation or gas trapping are not usually the focus of the examination in other diseases, the controlled breathing maneuvers are not used, and expiratory images are not obtained. Finally, high-resolution scan protocols are generally used, and these protocols are not comparable to those used in our CF program. Another limitation of our CT data is that we have a single radiologist reporting the scans. Although a second independent report would be ideal, we were not able to achieve this. Instead, we have relied on the reports that we would receive clinically from a senior experienced thoracic radiologist.

Second, we have used a conventional microbiological diagnosis (>10⁴ cfu/ml) to define pulmonary infection. Although we can be confident that those we define as infected are infected, it is more difficult to be certain that those we classify as uninfected are indeed free of infection. Tables E2 and E3 examine this issue in more detail by showing all bacterial counts and the associations of inflammation and structural lung disease with lower bacterial densities. The majority of children in the present study had a BAL performed in a single lobe only. Variations of infection, both the presence of and the infecting organism, have been reported in children with CF (25, 26). Although we did not see any differences in cultures in the 16 children with more than one lobe sampled (see the online supplement), we may be underestimating the true proportion of our population with pulmonary infection at diagnosis, and our data may represent a “best case” scenario.

In the present study, 33 (57.9%) of the infants were receiving anti-staphylococcal prophylaxis at the time of the BAL. These children were somewhat younger (22 vs. 31 d; P = 0.045) than those not on prophylaxis. According to clinic protocol, children with CF are given antistaphylococcal prophylaxis for the first 1 to 2 years of life; however, physician preference and consultation with the parents determines the practice in individual patients. There were no differences in cellular or cytokine profiles in the BAL between children on and children not on prophylaxis. These data need to be interpreted with caution; we were not able to determine retrospectively reasons why individual children were or were not on prophylaxis. Thus, a randomized clinical trial using BAL-based assessments of inflammation and infection needs to been performed to determine whether antistaphylococcal prophylaxis is of benefit in infants with CF.

The concept of significant lung disease early in life in CF is not new. Khan and colleagues (10) performed BAL on 16 infants (mean age, 6 mo) with CF and demonstrated increased levels of neutrophils, free NE activity, and IL-8 when compared with 11 disease-free control infants. Inflammation was present in infants without evidence of infection (n = 7) and in infants as young as 4 weeks old. Similar data have been reported by others (8, 9, 11, 13, 14, 24); however, the debate as to whether pulmonary inflammation can be a primary event or only follows infection has not been settled. The data from the present study are compatible with the notion that inflammation can be a primary event in the lungs of infants with CF. Although the level of inflammation is greater in infected infants, the levels of neutrophils, proinflammatory cytokines, and free NE activity in uninfected infants are greater than would be expected in the lungs of healthy infants. Unfortunately, our data cannot shed light on the mechanism(s) underlying inflammation in the absence of detectable infection.

Similarly, radiologic evidence of structural lung disease has been reported in children as young as 10 weeks old with CF (16–18). Long and colleagues (17), reporting data from HRCT performed in 34 children with CF (mean age, 2.4 y), demonstrated that the mean airway wall thickness and airway lumen diameter were greater than in scans from healthy control children. Davis and colleagues (16) performed HRCT in 17 children with CF (aged 2–44 mo) scheduled for bronchoscopy during respiratory exacerbations and used a modified Brody score (27) to characterize structural lung disease. Disease was greatest in the right lung; however, it is difficult to ascertain from their report whether all scans were abnormal (16). Scans were repeated after treatment with intravenous antibiotics. Using a modified Brody score, they demonstrated an improvement post treatment in the total score (18.2 pretreatment vs. 12.2 post-treatment; P < 0.01). The component subscores for bronchiectasis/bronchial dilatation and gas trapping improved, but those for mucous plugging and peribronchial thickening did not. These studies highlight that none of the current scoring systems used for assessing chest CTs is suitable for use in infants with mild, early lung disease. In the present study, we have used a binary classification (present:absent) for the three indicators of structural lung disease (bronchial dilatation, bronchial wall thickening, and gas-trapping). We have not attempted to create a total score by summing components because we do not believe that sufficient data are available to allow this to be done. Ours is the first study to describe structural changes in the lung in infants with CF soon after diagnosis by NBS. We chose to use the term “bronchial dilatation” even though the radiologic criterion used is in routine clinical practice to denote bronchiectasis. We took this stance because the term “bronchiectasis” generally implies permanent and irreversible airway damage. Longitudinal data are required from a cohort of children studied from early infancy through childhood before the implications of such early structural changes can be understood, in particular whether these early changes are predictive of bronchiectasis in later childhood.

We have recently reported that lung function measured using the raised volume rapid thoracic compression technique (RVRTC) was normal in infants with CF in the first 6 months of life but became progressively abnormal after this time (28). Some of the infants in that earlier report are also in the present study population; however, there are too few to make a formal assessment of the relationship between lung function and lung structural abnormalities. Martinez and colleagues (18) measured lung function by RVRTC in 11 of 13 clinically stable children with CF aged 8 to 33 months and reported lower FEF (Z-scores: FEF₅₀ = −2.0; FEF₇₅ = −1.6) and FEV values (FEV_0.5 = −2.1). In addition, they reported significant negative relationships between these variables and the ratio between airway wall thickness to airway lumen (FEF₅₀: r² = −0.66, P < 0.002; FEF₇₅: r² = −0.41, P < 0.002; FEV_0.5: r² = −0.40, P < 0.002). Reports from older children show a lack of association between measurements of forced expiration and structural abnormalities on CT (29). Thus, the true relationship between radiologic indications of structural lung disease and lung function, measured with tests of forced expiration or with other lung function tests, such as the lung clearance index or forced oscillation, remains to be determined.

A major finding of the present study is the association between structural lung disease and free NE activity in the BAL. It may be surprising that the percentage of neutrophils in the BAL is only weakly associated with bronchial dilatation. There is a relation between the number of neutrophils per milliliter of BAL fluid and the percentage of neutrophils; however, the absolute number of activated neutrophils and the products they release, such as NE, are more likely to be associated with tissue damage. Neutrophil elastase is a serine protease found mainly in azurophil granules in neutrophil cytoplasm that is capable of degrading a variety of proteins, including collagen, proteoglycans, fibronectin, and elastin, in the extracellular matrix and bacterial outer membrane proteins (22). When released from activated neutrophils, NE is “inactivated” by being bound to a variety of antiproteases, including α1 antitrypsin, secretory leukocyte protease inhibitor, monocyte/neutrophil protease inhibitor, and elafin/prelafin (22, 30). The assay used in the present study measures free NE activity, the presence of which implies that the amount of NE present in the BAL has exceeded the antiprotease binding capacity in the lung. The association of free NE activity with structural lung disease in infants warrants further investigation but suggests that new therapeutic approaches are required to prevent the onset of progressive lung damage and that such approaches need to be implemented in infancy.

The comprehensive early disease surveillance program run by AREST CF has the potential to contribute to the clinical management of individual patients in our clinics. Through this program, we have identified that the median age of acquisition of pulmonary infection with P. aeruginosa is approximately 26 months of age and that this infection can be eradicated by intensive treatment in approximately 80% of children (31). In addition, our routine practice is to prescribe antistaphylococcal prophylaxis for the first 12 to 24 months of life. This would normally be stopped for children with no evidence of structural abnormality of chest CT and no evidence of pulmonary infection on BAL. Conversely, children with radiologic or BAL evidence of established lung disease will be treated more aggressively, including earlier use of intravenous antibiotics for respiratory exacerbations.

In summary, we have presented data demonstrating that a substantial proportion of infants with CF diagnosed after detection by NBS have significant pulmonary inflammation, pulmonary infection, and radiologic evidence of structural lung disease at diagnosis. Although only 30% of infants had free NE activity detectable in BAL at 3 months of age, free NE activity was most strongly associated with bronchial dilatation. Although lung disease is greater in those with clinically significant respiratory symptoms, the absence of clinically apparent lung disease cannot be taken as an indication of “normal lungs.” The optimal treatment for preventing or delaying the onset of structural lung disease remains to be determined.