Mucus Removal Is Impaired in Children with Cystic Fibrosis Who Have Been Infected by Pseudomonas aeruginosa Beth L. Laube, PhD, Gail Sharpless, BS, Jane Benson, MD, Kathryn A. Carson, ScM, and Peter J. Mogayzel, Jr, MD, PhD Objective To determine if mucus removal is impaired in children with cystic fibrosis (CF) who have been recently infected with Pseudomonas aeruginosa.
Study design We compared mucociliary clearance (MCC), cough clearance (CC), lung morphology, and forced expiratory volume in 1 second (FEV1) in 7- to 14-year-old children with CF and mild lung disease (FEV1 $ 80%). Children were either P aeruginosa negative (n = 8), or P aeruginosa positive (P aeruginosa obtained from at least 1 airway culture in the preceding 18 months) (n = 10). MCC and CC were quantified from gamma camera imaging of the right lung immediately after inhalation of 99mtechnetium sulfur-colloid (time 0), over the next 60 minutes (average percent clearance over the first 60 minutes [AveMCC60]), 60-90 minutes (average percent clearance between 70 and 90 minutes [AveMCC/CC90]), and after 24 hours (percent clearance after 24 hours [MCC24hrs]). Children coughed 30 times between 60 and 90 minutes. Lung morphology was assessed by high resolution computed tomography (HRCT) scores of both lungs (total score) and of the right lung, using the Brody scale. Percent AveMCC60, AveMCC/CC90, MCC24hrs, FEV1, and HRCT scores were compared across the 2 groups using unpaired t tests. Associations were assessed using Spearman correlation. Results There were no differences between the 2 groups in AveMCC60, MCC24hrs, mean HRCT total scores, right lung HRCT scores, or mean FEV1. AveMCC/CC90 was significantly decreased in children with P aeruginosa compared with those without (16.2% 11.0% vs 28.6% 7.8%, respectively; P = .016). There was a significant negative correlation of AveMCC60 and AveMCC/CC90 with total lung HRCT score (all P < .05) but not with FEV1. Conclusions Infection with P aeruginosa is associated with a significant slowing of MCC/CC in children with mild CF and may be a more sensitive indicator of the effects of P aeruginosa than measurements of FEV1. (J Pediatr 2014;164:839-45). See related article, p 832
A
pproximately 70 000 people worldwide have cystic fibrosis (CF), an inherited, autosomal recessive disease.1,2 Mutations in the CF transmembrane conductance regulator gene lead to loss, or misregulation, of chloride, sodium and water transport, accumulation of viscous secretions in the airways of individuals with CF2-5 and impaired mucus removal.3,4 In healthy lungs, mucus is removed from the lungs within a few hours by the mucociliary clearance (MCC) apparatus, an important aspect of host defense. Cough clearance (CC), which facilitates mechanical removal mucus, takes over when there is mucus overload, or when MCC becomes impaired. Individuals with CF become infected with numerous bacteria and fungi during the course of their disease and chronic infection of the airways is the major cause of morbidity and mortality.6 Common pathogens known to infect the airways of CF patients include Pseudomonas aeruginosa, Aspergillus fumigatus, Aspergillus niger, Haemophilus influenzae, Stenotrophomonas maltophilia, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus. The most common bacterium affecting individuals with CF is P aeruginosa, with the majority being infected by the age of 18.7 Nevertheless, there has been little, if any, research into whether infection with P aeruginosa affects removal From the Johns Hopkins Medical Institutions, Baltimore, MD of mucus from the lung in individuals with CF, or if it contributes to airway AveMCC60 AveMCCCC90 CC CF CT FEV1 FVC HRCT MCC MCC24Hrs O:I
Average percent mucociliary clearance over the first 60 minutes Average percent clearance between 70 and 90 minutes Cough clearance Cystic fibrosis Computed tomography Forced expiratory volume in 1 second Forced vital capacity High resolution computed tomography Mucociliary clearance Percent clearance after 24 hours Outer/inner lung zone ratio
Funded by Gilead Sciences, Inc, which had initial input in study design, but no involvement in the collection, analysis or interpretation of the data, writing of the report, or the decision to submit the manuscript for publication. Supported by Johns Hopkins Institute for Clinical and Translational Research (ICTR), which is funded in part by the National Center for Advancing Translational Sciences (NCATS), a component of the National Institutes of Health (NIH), and NIH Roadmap for Medical Research (UL1 TR 000424-06). Its contents are solely the responsibility of the authors and do not necessarily represent the official view of the Johns Hopkins ICTR, NCATS, or NIH. The authors declare no conflicts of interest. Portions of the study were presented as a poster at the North American Cystic Fibrosis Conference, October 17-19, 2013, Salt Lake City, UT. 0022-3476/$ - see front matter. Copyright ª 2014 Mosby Inc. All rights reserved. http://dx.doi.org/10.1016/j.jpeds.2013.11.031
839
THE JOURNAL OF PEDIATRICS
www.jpeds.com
damage and morphologic changes such as bronchiectasis. For this reason, we compared mucus removal in children who had been recently infected with P aeruginosa with children with mild CF lung disease who had not been infected with P aeruginosa. We hypothesized that recently infected children would demonstrate significantly slower mucus removal and greater lung morphologic abnormalities than children who had not been infected with P aeruginosa.
Methods This study consisted of a screening visit and 2 study visits. Most screening visits and study visits took place over a 4week period. However, because of scheduling conflicts and pulmonary exacerbations, some screening visits occurred between 6 and 19 weeks before the study visits. The protocol was approved by the Johns Hopkins Medicine Institutional Review Board. Written informed consent/assent was obtained from parents and participants. Children with or without P aeruginosa through review of their medical records in the preceding 18 months and who met all of the eligiblity criteria were referred for recruitment by their treating physicians. Eligibility criteria included males and nonpregnant females, age 7-14 years with a diagnosis of CF by sweat chloride $60 meq/L, or the presence of 2 disease-causing CF transmembrane conductance regulator mutations, and a forced expiratory volume in 1 second (FEV1) $80% of predicted values. Children were grouped as P aeruginosa positive if P aeruginosa had been obtained from at least 1 airway culture positive for P aeruginosa in the preceding 18 months, or P aeruginosa grew from the culture obtained at the screening visit. They were grouped as P aeruginosa negative if P aeruginosa had not been obtained from any airway culture in the preceding 18 months, or from the culture obtained at the screening visit. We chose to review each child’s chart over the preceding 18 months because it is unknown how long 1 infection with P aeruginosa can affect MCC. Concomitant Medications Children who were taking inhaled tobramycin or aztreonam were studied during their “off” month of treatment. Children discontinued the use of bronchodilators, anticholinergic drugs, fluticasone propionate, recombinant human DNase, and airway clearance therapy for 12 hours before the study visit and hypertonic saline for 3 days before the study visit. All medications were discontinued for 36 hours thereafter. Our goal in withholding these medications was to minimize their possible effects on MCC measurements. The time period for withholding each medication was somewhat arbitrary because there is little, if any, information in the literature as to how long a specific drug affects MCC once the drug is stopped. Thus, although the bronchodilator effect of albuterol is thought to last only for 2 hours,8 it is unclear how long albuterol continues to affect MCC once it is stopped. In general, we asked patients to withhold medications for as long a time as we thought was medically advisable, while also reducing possible effects on MCC. Children who were 840
Vol. 164, No. 4 treated with oral, or intravenous, antibiotics for a pulmonary exacerbation completed those medications at least 2 weeks before the screening and study visits. Children who were being chronically treated with oral antibacterials such as sulfamethoxazole/trimethoprim were excluded from the study. Children who were being treated with azithromycin continued with their treatment before and throughout the study visits. Screening Visit Children underwent a focused medical history and physical examination and routine pulmonary function measurements of FEV1 and forced vital capacity (FVC). They also underwent an induced sputum test to determine their airway microbiology and a high resolution computed tomography (HRCT) scan to assess lung morphology.
Induced Sputum Test. Children underwent the standardized sputum induction procedure that is utilized by the CF Therapeutic Development Network.9 Sputum expectorated during this procedure was cultured for bacteria and fungi.
HRCT Scan. Children underwent an HRCT scan in the Johns Hopkins Radiology Department. Computed tomography (CT) scanning was performed as described by Brody et al.10
Scoring Lung HRCT Scans. HRCT scans were read by an experienced pediatric radiologist with no clinical information. Each CT scan was scored using a scoring system developed by Dr Alan Brody that evaluates the severity of lung disease in each lobe, with the lingula considered a lobe.10 The severity of bronchiectasis, mucous plugging, peribronchial thickening, air trapping, parenchymal opacities, and overall disease severity was scored for each lung separately. Overall disease severity score for the right lung and both lungs combined was averaged for each group. Pulmonary Function Measurements FVC and FEV1 were measured by a computerized 10-liter Survey III spirometer (Warren E. Collins, Inc, Braintree, Massachusetts). Spirometry was performed in accordance with American Thoracic Society/European Respiratory Society guidelines,11 using equations from Hankinson et al12 and Wang et al13 and a protocol based on the CF Foundation’s recommendations14 to determine percent predicted values. Study Visit 1A Children underwent the following procedures: (1) a focused medical history and physical exam; (2) pulmonary function measurements of FEV1 and FVC; (3) a 133xenon-ventilation scan; (4) inhalation of aerosol generated from a solution of 99m technetium sulfur-colloid; and (5) gamma camera imaging of the lungs for 90 minutes.
Ventilation Scan. Children inhaled 133xenon while sitting with their back to a large-field-of-view 2-dimensional Siemens Orbiter gamma camera (Gammasonics, Des Plains, Illinois). This procedure produced a ventilation image that Laube et al
ORIGINAL ARTICLES
April 2014 was used to identify the right lung border and inner and outer lung zones.
Identification of the Right Lung Region. Delineation of the right lung border has been described previously.15 The right-lung border was first delineated on the 133xenon ventilation image. During computer processing, each subsequent aerosol lung scan was registered with the ventilation scan. After registration, the right-lung border that was delineated on the ventilation scan was superimposed onto the aerosol lung scan.
Radioaerosol Inhalation. Children inhaled aerosol generated from a saline solution containing the radioisotope technetium sulfur-colloid (radioaerosol) while breathing 20-30 times intermittently from the mouthpiece of a 646 nebulizer connected to a PulmoAide compressor (Devilbiss, Somerset, Pennsylvania) through a Spira Electro2 Inhalation Dosimeter (Respiratory Care Center, H€ameenlinna, Finland). The goal was to deliver 50 mCi of radioisotope to the child’s mouth during the inhalation procedure. For some children, the dosing goal was achieved in 20 breaths. Other children required 30 breaths. Variability in the number of breaths resulted from differences in nebulizer dose. Inhalation started from functional residual capacity at a flow rate of 0.5 L/s. The nebulizer was pulsed for 0.7 seconds during each inhalation. There was no breathhold after each inhalation. Following the total inhalation procedure, children rinsed their mouth with water, expectorated the rinse, and drank some water. 99m
Quantification of Mucus Removal during First 90 Minutes. Children underwent lung imaging procedures every 2 minutes with the gamma camera starting immediately after inhalation of the radioaerosol (time 0) through the first 20 minutes and every 10 minutes thereafter for 60 minutes. Counts retained in the right lung field at the various time points were background-corrected and decay-corrected to time 0. Decay-corrected counts were subtracted from time 0 counts. The difference represented radioactivity that had been cleared from the lung at each time period. The number of cleared counts was expressed as a percentage of time 0 counts and reported as percent clearance. Percent clearance at 10, 20, 30, 40, 50, and 60 minutes was averaged as described previously.16 Average percent clearance over the first 60 minutes was expressed as AveMCC60. Children coughed 10 times between 60 and 70 minutes, 70 and 80 minutes, and 80 and 90 minutes. Children were instructed to cough into a Piko-1 electronic peak flow meter (Ferraris, Medical, Inc, Louisville, Colorado) such that the peak flow registered after each cough was approximately 200 mL/s. Images were obtained after each set of coughs. Percent clearance at 70, 80, and 90 minutes was calculated as described above for MCC60 and averaged to obtain average percent clearance between 70 and 90 minutes (AveMCC/CC90). Study Visit 1B Children returned to the laboratory approximately 24 hours after study visit 1A for a 30-minute imaging procedure.
MCC after 24 Hours. Percent clearance after 24 hours (MCC24hrs) was calculated as the difference between 100% retention at time 0 and the background-corrected and decay-corrected percent retention at 24 hours.
Quantification of Right Lung Regional Deposition Pattern. Deposition pattern of the radioaerosol was quantified in terms of inner and outer zones, as described previously.16 Outer/inner lung zone ratios (O:I) at time 0 (first aerosol lung image) for each study group were compared. O:I were also derived for the ventilation lung image. Ratios for the aerosol scan were divided by that of the ventilation scan to correct for lung volume differences. Deposition pattern for the radioaerosol was characterized in terms of the corrected O:I as described previously.17 Statistical Analyses Group comparisons were made using Fisher exact test for categorical data and 2-sample t tests for continuous. Possible associations between percent AveMCC60, AveMCC/CC90, and MCC24hrs and O:I, FEV1, and CT scores were assessed using Spearman correlation. Analyses were performed using SAS v. 9.3 (SAS Institute, Inc, Cary, North Carolina) and Microsoft Office Excel 2003 (Microsoft Corp, Redmond, Washington). All reported P values are 2-sided, and significance was set at P < .05.
Results Eight children who were P aeruginosa negative and 10 children who were P aeruginosa positive completed the screening visit and study visits 1A and 1B. Two children completed the screening visit, but did not complete the study visits. One child moved to another state after the screening visit and the other could not perform the inhalation maneuvers associated with study visit 1A. Baseline characteristics of the 18 participants are shown in the Table. There were no statistically significant differences between the P aeruginosa groups in age, sex, pulmonary function, height, weight, body mass index, or genotype. Four P aeruginosa positive children grew P aeruginosa at screening. The other 6 P aeruginosa positive children grew P aeruginosa from throat cultures over the preceding 18 months. One of the children who grew P aeruginosa on the screening visit also grew A fumigatus. Three other children with P aeruginosa who did not grow P aeruginosa on the screening visit grew A fumigatus. Sixty percent (6 children) of the P aeruginosa positive group grew more than 1 organism at screening, compared with 12% (1 child) in the P aeruginosa negative group. Concomitant Medications Sixteen of the children were regularly treated with albuterol (89%). One child without P aeruginosa and 1 child with P aeruginosa did not use albuterol. Three children with P aeruginosa (30%) and 1 child without P aeruginosa (12.5%) used fluticasone propionate. Nine children with P aeruginosa (90%) and 7 children without P aeruginosa (87.5%) were being treated with dornase alfa. Five of the children with P aeruginosa (50%) and
Mucus Removal Is Impaired in Children with Cystic Fibrosis Who Have Been Infected by Pseudomonas aeruginosa
841
THE JOURNAL OF PEDIATRICS
www.jpeds.com
Vol. 164, No. 4
Table. Characteristics at screening by P aeruginosa status Characteristics Males Age (years)† FVC (% predicted)† FEV1 (% predicted)† Height (percentile)† Weight (percentile)† BMI (percentile)† Genotype: F508del homozygote F508del heterozygote Other Antibiotic medications: Azithromycin Inhaled tobramycin Cayston Organisms cultured at screening: P aeruginosa A fumigatus A niger Mixed respiratory flora Ste maltophilia Sta aureus MRSA
P aeruginosa (n = 8)
P aeruginosa+ (n = 10)*
5 (62%) 10.9 1.9 86.3 11.0 93.9 10.8 49.6 48.4 39.4 31.6 45.6 18.8
5 (50%) 10.2 2.3 91.2 10.2 99.4 11.1 40.6 28.4 41.6 27.5 45.6 23.1
5 (62%) 3 (38%) 0
7 (70%) 1 (10%) 2 (20%)
4 (50%) 0 0
8 (80%) 3 (30%) 3 (30%)
0 0 0 7 (88%) 2 (25%) 4 (50%) 1 (12%)
4 (40%) 4 (40%) 2 (20%) 7 (70%) 1 (10%) 3 (30%) 2 (20%)
BMI, body mass index; MRSA, methicillin-resistant Sta aureus. *1 or more positive cultures for P aeruginosa in previous 18 months. †Values are mean (SD).
5 of the children without P aeruginosa (62.5%) were being treated with hypertonic saline. Eight of the children with P aeruginosa and 4 of the children without P aeruginosa were being treated with azithromycin. Six of the children with P aeruginosa were being treated with inhaled antibiotics, compared with none of the children without (P = .013). Deposition Pattern The O:I for the children with and without P aeruginosa were similar, averaging 0.515 0.147 (range, 0.362-0.820) and 0.547 0.170 (range, 0.374-0.888), respectively, indicating there was no difference in the deposition pattern of the radioaerosol in the lungs of the 2 groups of children. The mean AveMCC60 for the children with P aeruginosa was 12.5% 9.0% (range, 0.6%-29%) compared with 18.8% 8.3% (range, 6.9%-34.1%) for the children without P aeruginosa. This difference was not statistically significant (P = .14; Figure 1). Mean AveMCC/CC90 for the children with P aeruginosa was 16.2% 11.0% (range, 0%-36.9%) compared with 28.6% 7.8% (range, 13.7%-37.9%) for the children without P aeruginosa. The children with P aeruginosa had statistically significantly slower AveMCC/CC90 (P = .016; Figure 1). Mean MCC24hrs was similar for the 2 groups of children (48.7% 23.7%; range, 3.7%-87.4% for the children with P aeruginosa and 50.4% 8.9%; range, 34.6%-60.6% for the children without P aeruginosa) (Figure 1). Mean FEV1 was similar for the children with and without P aeruginosa on the screening visit (Table). Mean FEV1 for the 2 groups was also similar on study visit 1A, with 93.0 8.0 and 100.1 11.6 of the percent predicted, respectively. 842
Figure 1. Average percent mucus removal over 60 minutes (AveMCC60), between 60 and 90 minutes (AveMCC/CC90), and after 24 hours (MCC24hrs) for children with CF, without (white bars) and with P aeruginosa (black bars). AveMMC/ CC90 was significantly slower in the children with P aeruginosa compared with those without (P = .016).
Total and right lung HRCT scores were similar for the 2 groups, with 3.62 5.37 (P aeruginosa negative) and 6.78 6.25 (P aeruginosa positive) and 1.88 2.49 (P aeruginosa negative) and 4.08 4.00 (P aeruginosa positive), respectively. Lower scores indicated less disease severity. With respect to scores directly pertaining to MCC, 5 of 10 children with P aeruginosa were scored for mucus plugging in at least 1 lung, whereas, none of the children without P aeruginosa were scored for mucus plugging. In addition, 6 of 10 children with P aeruginosa (60%) were scored for bronchiectasis (2.32 3.3), whereas only 1 of the 8 of the children without P aeruginosa (12.5%) was scored for bronchiectasis (0.25 0.71). These differences were not statistically significant. Five of 8 children without P aeruginosa (62.5%) had overall disease severity scores of 0-1, compared with 2 of 10 children with P aeruginosa (20%). There was a significant negative correlation between percent AveMCC60 and total lung HRCT score and AveMCC/CC90 and total lung HRCT score for all children (N = 18) (P = .039 and .047, respectively). Lower percent AveMCC60 and AveMCCCC90 were associated with higher HRCT scores and more parenchymal damage (Figures 2 and 3). There were no statistically significant correlations between mean percent AveMCC60, AveMCC/CC90, or MCC24hrs and FEV1, O:I, or right lung HRCT score.
Discussion Although most individuals with CF are infected with P aeruginosa by the age of 18, little is known about the effect of P aeruginosa on MCC, or MCC and CC combined, despite the fact that these are important lung defense mechanisms. Laube et al
April 2014
Figure 2. Correlation between percent AveMCC60 and total lung HRCT score for all children (N = 18). Percent AveMCC60 was significantly negatively correlated with total lung HRCT score (P = .039). The rs value was generated using a Spearman correlation test.
In this study, we found that a history of infection with P aeruginosa was associated with a significant slowing of MCC with CC combined in children with mild CF. We found no significant difference in MCC alone between the 2 groups of children. This lack of differentiation may indicate that infection with P aeruginosa does not affect MCC mechanisms so much as CC mechanisms. This is evident from the significant improvement in mucus removal with the addition of coughing between 60 and 90 minutes in the children without P aeruginosa, and there was little improvement in the children with P aeruginosa. The lack of differentiation in MCC alone may also have been because the study was underpowered for this measurement. The proposed sample
Figure 3. Correlation between percent AveMCC/CC90 and total lung HRCT score for all children (N = 18). Percent AveMCC/CC90 was significantly negatively correlated with total lung HRCT score (P = .047). The rs value was generated using a Spearman correlation test.
ORIGINAL ARTICLES size was initially to be 12 subjects in each group. However, we were able to recruit only 8 and 10 children with and without P aeruginosa, respectively, during the study period. With group sizes of 8 and 10 there was only 69% power to detect a difference of 10% in AveMCC60 using our observed SDs. There was also a lack of differentiation between the 2 groups based on MCC measured after 24 hours. This may have been due to several factors. First, there may be no difference in mucus removal after 24 hours between these 2 groups of children with mild CF disease. Second, there may have been too little radioisotope remaining in the lung after 24 hours for an accurate measurement. However, although residual activity in the lungs after 24 hours was low, in all cases it was above background activity, suggesting that the 24-hour measurement was valid. Finally, there may have been differences between children in terms of the amount of coughing between the last image on study visit 1A and the image acquired on study visit 1B that were not documented. The techniques used to measure MCC in the current study have only recently been standardized, and no direct comparisons are available with other studies in children with CF. One study has been conducted in adults with CF using the same techniques18 and 1 has been conducted in healthy adults.16 Inclusionary criteria for the CF adults included FEV1 $ 50% of predicted. There was no information regarding their infection history. The investigators reported average mucus clearance 60 minutes after placebo of 9.3 1.1% (SE). Average MCC 24 hours later was 40.7 2.5% (SE). In the 1 study that utilized the same techniques for measuring MCC in healthy adults,16 MCC60 averaged 11 6%, MCC/CC90 averaged 16 8%, and MCC24hrs averaged 36 17%. It is unclear if MCC values measured in adults with CF, or in healthy adults, are comparable with those of CF children, because the lengths of ciliated airways in adult lungs are probably significantly greater than those of young children’s ciliated airways as a result of lung growth. This difference could affect rate of mucus removal in the 2 groups. We found that slower MCC and MCC combined with CC were each associated with more parenchymal abnormalities identified using the Brody HRCT scoring system. This is the first time that such a correlation has been reported and suggests that these measurements may be as sensitive as scored HRCT scans in identifying children at risk for development of early irreversible lung damage. Several studies in children with CF have shown that HRCT scans, quantified with the Brody scoring system, are more sensitive in terms of detecting early lung damage than traditional pulmonary function tests.19-21 Our results support these findings because we found significant correlations between MCC and HRCT scores and between MCC with CC and HRCT scores, but no correlations with FEV1. In our study, 60% of children with P aeruginosa had bronchiectasis as detected by the Brody HRCT scoring system. One child without P aeruginosa had bronchiectasis. Children in our study were similar in age and pulmonary function to children in a study reported by Farrell et al. 20 In that study,
Mucus Removal Is Impaired in Children with Cystic Fibrosis Who Have Been Infected by Pseudomonas aeruginosa
843
THE JOURNAL OF PEDIATRICS
www.jpeds.com
the investigators found that 83% of children with CF (n = 82) had bronchiectasis, and bronchiectasis scores were statistically highest in children with mucoid P aeruginosa. We chose to define “infected with P aeruginosa” based on at least 1 positive airway culture in the preceding 18 months. This was because diagnosis of infection based on throat or induced sputum cultures can yield false negatives, and we did not want to rely on an arbitrary number of positive cultures greater than 1 to define “infected with P aeruginosa” when infection might be undetected by current testing methods. In fact, only 4 of the children with P aeruginosa grew P aeruginosa at the screening visit. Results from this study suggest that infection with P aeruginosa is associated with a decrease in mucus removal in children with CF. It is not known if P aeruginosa infection causes the impairment, but, mechanistically, infection with P aeruginosa has the potential to impair mucus removal in these individuals because pyocyanin, a redox-active virulence factor is secreted by P aeruginosa. Pyocyanin has been shown to slow the beating of cilia,22,23 which is critically important to the removal of mucus. Accumulation of mucus in the airways, as a result of infection and impaired MCC could, in turn, overwhelm removal by CC. Another possible mechanism by which P aeruginosa could lead to a delay in MCC is through the development of bronchiectasis. Bronchiectasis is caused by 1 or more infections or inflammatory insults to the lungs and damaged airways associated with bronchiectasis cannot effectively clear mucus. Children who were infected with P aeruginosa were also infected with other organisms that could impair MCC/CC. Overall, 60% of the children with P aeruginosa grew more than 1 organism at screening, compared with 12% in the children without P aeruginosa. More specifically, 5 of the children with P aeruginosa also were infected with A fumigatus, which, similar to P aeruginosa, secretes virulence factors that could affect mucus clearance.24 Others have shown in vitro that the virulence factors and proteases associated with A fumigatus can inhibit ciliary beat frequency25 and damage the epithelial tissue to the point of cell detachment.26 Each of these could reduce MCC/CC in vivo. Additional studies are needed to determine the effect of combined infection with P aeruginosa and A fumigatus vs P aeruginosa alone on MCC. Alternatively, infection with P aeruginosa may be a marker for children with slower MCC/CC and not a cause of the impairment. Children with slower MCC/CC may be unable to clear the bacteria as quickly as children with faster MCC/CC and, therefore, may simply be more vulnerable to the infection. The fact that several children with a history of infection with P aeruginosa also had a history of infection with A fumigatus and A niger supports the notion that these children may have been more vulnerable to infection because they had slower MCC and CC and not vice versa. Prospective studies of the evolution of infection in children with CF who are uninfected with P aeruginosa, but have slower MCC than other uninfected children, could help to answer this question. 844
Vol. 164, No. 4 In this study, we found that a history of infection with P aeruginosa was associated with a significant slowing of MCC with CC combined in children with mild CF. Slowing of mucus removal was significantly associated with more parenchymal abnormalities detected on HRCT but was not associated with pulmonary function changes based on FEV1 measurements. These findings suggest that MCC/CC measurements may be a more sensitive indicator of the effects of P aeruginosa infection in the lungs of children with mild CF disease than measurements of FEV1. In addition, the significant association with HRCT scores suggests that MCC/ CC measurements might be used as an early biomarker of disease severity in young children with CF with reduced radiation exposure. Early and aggressive antibacterial treatment in combination with therapies that improve mucus rheology in children with slow MCC/CC could increase the ability of these children to fight off infections, possibly reducing future declines in pulmonary function. n The authors thank Drs J. Michael Collaco, Pamela Zeitlin, and Beryl Rosenstein for performing the histories and physicals on the screening visit for children in this study. Submitted for publication Jul 18, 2013; last revision received Sep 26, 2013; accepted Nov 13, 2013. Reprint requests: Beth L. Laube, PhD, The Johns Hopkins Medical Institutions, The David M. Rubenstein Building, 200 North Wolfe St, Ste 3015, Baltimore, MD 21287-2533. E-mail: [email protected]
References 1. Morison S, Lewis PA, Coles EC, Geddes D, Russell G, Littlewood JM, et al. Incidence, population, and survival of cystic fibrosis in the UK, 1968-95. UK Cystic Fibrosis Survey Management Committee. Arch Dis Child 1997;77:493-6. 2. Hodson ME, Geddes D, Bush A, eds. Cystic fibrosis. 3rd ed. London: Hodder Arnold; 2007. 3. Matsui H, Grubb BR, Tarran R, Randell SH, Gatzy JT, Davis CW, et al. Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease. Cell 1998;95:1005-15. 4. Matsui H, Verghese MW, Kesimer M, Schwab UE, Randell SH, Sheehan JK, et al. Reduced three-dimensional motility in dehydrated airway mucus prevents neutrophil capture and killing bacteria on airway epithelial surfaces. J Immunol 2005;175:1090-9. 5. Ratjen F, Doring G. Cystic fibrosis. Lancet 2003;361:681-9. 6. Davis PB, Drumm M, Konstan MW. Cystic fibrosis. Am J Respir Crit Care Med 1996;154:1229-56. 7. Cystic Fibrosis Foundation Patient Registry 2002 Annual Data Report to the Center Directors. Bethesda, MD: 2003 Cystic Fibrosis Foundation; 2002. 8. Devalia JL, Sapsford RJ, Rusznak C, Toumbis MJ, Davies RJ. The effects of salmeterol and salbuterol on ciliary beat frequency of cultured human bronchial epithelial cells, in vitro. Pulmonary Pharmacol 1992;5:257-63. 9. Mayer-Hamblett N, Aitken ML, Accurso FJ, Kronmal RA, Konstan MW, Burns JL, et al. Association between pulmonary function and sputum biomarkers in cystic fibrosis. Am J Respir Crit Care Med 2007;175:822-8. 10. Brody AS, Kosorok MR, Li Z, Broderick LS, Foster JL, Laxova A, et al. Reproducibility of a scoring system for computed tomography scanning in cystic fibrosis. J Thorac Imaging 2006;21:14-21. 11. Miller MR, Hankinson J, Brusasco V, Burgos F, Casaburi R, Coates A, et al. Standardisation of spirometry. J Eur Respir 2005;26:319-38. 12. Hankinson JL, Odencrantz JR, Fedan KB. Spirometric reference values from a sample of the general US population. Am J Respir Crit Care Med 1999;159:179-87.
Laube et al
April 2014 13. Wang X, Dockery DW, Wypij D, Gold DR, Speizer FE, Ware JH, et al. Pulmonary function growth velocity in children 6 to 18 years of age. Am Rev Respir Dis 1993;148:1502-8. 14. Rosenfeld M, Coates A, Corey M, Goss CH, Howard MB, Morgan W, et al. Task Force to Evaluate Choice of Spirometric Reference Equations for the National Patient Registry: Summary and Recommendations. Cystic Fibrosis Foundation Registry Committee; Oct 1, 2005. 15. Laube BL, Norman PS, Wagner HN Jr, Adams GK III. The effect of aerosol distribution on airway responsiveness to inhaled methacholine in patients with asthma. J Allergy Clin Immunol 1992;89:510-8. 16. Bennett W, Laube BL, Corcoran T, Mogayzel PJ Jr, Pilewski J, Donaldson S. Multi-site comparison of mucociliary and cough clearance measures using standardized methods. J Aerosol Med Pulm Drug Deliv 2013;26:157-64. 17. Newman S, Bennett WD, Biddiscombe M, Devadason SG, Dolovich M, Fleming J, et al. Standardization of techniques for using planar (2D) imaging for aerosol deposition assessment of orally inhaled products. J Aerosol Med Pulm Drug Deliv 2012;25(Suppl 1):S10-28. 18. Donaldson SH, Bennett WD, Zeman KL, Knowles MR, Tarran R, Boucher RC. Mucus clearance and lung function in cystic fibrosis with hypertonic saline. N Engl J Med 2006;354:241-50. 19. Brody AS, Klein JS, Molina PL, Quan J, Bean JA, Wilmott RW. High resolution computed tomography in young patients with cystic fibrosis: distribution of abnormalities and correlation with pulmonary function tests. J Pediatr 2004;145:32-8.
ORIGINAL ARTICLES 20. Farrell P, Collins J, Broderick L, Rock M, Li Z, Kosorok M, et al. Association between mucoid Pseudomonas infection and bronchiectasis in children with cystic fibrosis. Radiology 2009;252:534-43. 21. Stick S, Brennan S, Murray C, Douglas T, von Ungern-Sternberg B, Garratt L, et al. Australian Respiratory Early Surveillance Team for Cystic Fibrosis (AREST CF). Bronchiectasis in infants and preschool children diagnosed with cystic fibrosis after newboen screening. J Pediatr 2009;155:623-8.e1. 22. Kanthakumar K, Taylor G, Tsang KW, Cundell DR, Rutman A, Smith S, et al. Mechanisms of action of Pseudomonas aeruginosa pyocyanin on human ciliary beat in vitro. Infect Immun 1993;61:2848-53. 23. Munro NC, Barker A, Rutman A, Taylor G, Watson D, McDonaldGibson WJ, et al. Effect of pyocyanin and 1-hydroxyphenazine on in vivo tracheal mucus velocity. J Appl Physiol 1989;67:316-23. 24. Latge JP. The pathobiology of Apergilluis fumigatus. Trends Microbiol 2001;9:382-9. 25. Amitani R, Taylor G, Elezis E-N, Llewellyn-Jones C, Mitchell J, Kuze F, et al. Purification and characterization of factors produced by Aspergillus fumigatus which affect human ciliated respiratory epithelium. Infect Immun 1995;63:3266-71. 26. Tomee JFC, Wierenga AT, Hiemstra PS, Kauffman HF. Proteases from Aspergillus fumigatus induce release of pro-inflammatory cytokines and cell detachment in airway epithelial cell lines. J Infect Dis 1997; 176:300-3.
Mucus Removal Is Impaired in Children with Cystic Fibrosis Who Have Been Infected by Pseudomonas aeruginosa
845
Study design We compared mucociliary clearance (MCC), cough clearance (CC), lung morphology, and forced expiratory volume in 1 second (FEV1) in 7- to 14-year-old children with CF and mild lung disease (FEV1 $ 80%). Children were either P aeruginosa negative (n = 8), or P aeruginosa positive (P aeruginosa obtained from at least 1 airway culture in the preceding 18 months) (n = 10). MCC and CC were quantified from gamma camera imaging of the right lung immediately after inhalation of 99mtechnetium sulfur-colloid (time 0), over the next 60 minutes (average percent clearance over the first 60 minutes [AveMCC60]), 60-90 minutes (average percent clearance between 70 and 90 minutes [AveMCC/CC90]), and after 24 hours (percent clearance after 24 hours [MCC24hrs]). Children coughed 30 times between 60 and 90 minutes. Lung morphology was assessed by high resolution computed tomography (HRCT) scores of both lungs (total score) and of the right lung, using the Brody scale. Percent AveMCC60, AveMCC/CC90, MCC24hrs, FEV1, and HRCT scores were compared across the 2 groups using unpaired t tests. Associations were assessed using Spearman correlation. Results There were no differences between the 2 groups in AveMCC60, MCC24hrs, mean HRCT total scores, right lung HRCT scores, or mean FEV1. AveMCC/CC90 was significantly decreased in children with P aeruginosa compared with those without (16.2% 11.0% vs 28.6% 7.8%, respectively; P = .016). There was a significant negative correlation of AveMCC60 and AveMCC/CC90 with total lung HRCT score (all P < .05) but not with FEV1. Conclusions Infection with P aeruginosa is associated with a significant slowing of MCC/CC in children with mild CF and may be a more sensitive indicator of the effects of P aeruginosa than measurements of FEV1. (J Pediatr 2014;164:839-45). See related article, p 832
A
pproximately 70 000 people worldwide have cystic fibrosis (CF), an inherited, autosomal recessive disease.1,2 Mutations in the CF transmembrane conductance regulator gene lead to loss, or misregulation, of chloride, sodium and water transport, accumulation of viscous secretions in the airways of individuals with CF2-5 and impaired mucus removal.3,4 In healthy lungs, mucus is removed from the lungs within a few hours by the mucociliary clearance (MCC) apparatus, an important aspect of host defense. Cough clearance (CC), which facilitates mechanical removal mucus, takes over when there is mucus overload, or when MCC becomes impaired. Individuals with CF become infected with numerous bacteria and fungi during the course of their disease and chronic infection of the airways is the major cause of morbidity and mortality.6 Common pathogens known to infect the airways of CF patients include Pseudomonas aeruginosa, Aspergillus fumigatus, Aspergillus niger, Haemophilus influenzae, Stenotrophomonas maltophilia, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus. The most common bacterium affecting individuals with CF is P aeruginosa, with the majority being infected by the age of 18.7 Nevertheless, there has been little, if any, research into whether infection with P aeruginosa affects removal From the Johns Hopkins Medical Institutions, Baltimore, MD of mucus from the lung in individuals with CF, or if it contributes to airway AveMCC60 AveMCCCC90 CC CF CT FEV1 FVC HRCT MCC MCC24Hrs O:I
Average percent mucociliary clearance over the first 60 minutes Average percent clearance between 70 and 90 minutes Cough clearance Cystic fibrosis Computed tomography Forced expiratory volume in 1 second Forced vital capacity High resolution computed tomography Mucociliary clearance Percent clearance after 24 hours Outer/inner lung zone ratio
Funded by Gilead Sciences, Inc, which had initial input in study design, but no involvement in the collection, analysis or interpretation of the data, writing of the report, or the decision to submit the manuscript for publication. Supported by Johns Hopkins Institute for Clinical and Translational Research (ICTR), which is funded in part by the National Center for Advancing Translational Sciences (NCATS), a component of the National Institutes of Health (NIH), and NIH Roadmap for Medical Research (UL1 TR 000424-06). Its contents are solely the responsibility of the authors and do not necessarily represent the official view of the Johns Hopkins ICTR, NCATS, or NIH. The authors declare no conflicts of interest. Portions of the study were presented as a poster at the North American Cystic Fibrosis Conference, October 17-19, 2013, Salt Lake City, UT. 0022-3476/$ - see front matter. Copyright ª 2014 Mosby Inc. All rights reserved. http://dx.doi.org/10.1016/j.jpeds.2013.11.031
839
THE JOURNAL OF PEDIATRICS
www.jpeds.com
damage and morphologic changes such as bronchiectasis. For this reason, we compared mucus removal in children who had been recently infected with P aeruginosa with children with mild CF lung disease who had not been infected with P aeruginosa. We hypothesized that recently infected children would demonstrate significantly slower mucus removal and greater lung morphologic abnormalities than children who had not been infected with P aeruginosa.
Methods This study consisted of a screening visit and 2 study visits. Most screening visits and study visits took place over a 4week period. However, because of scheduling conflicts and pulmonary exacerbations, some screening visits occurred between 6 and 19 weeks before the study visits. The protocol was approved by the Johns Hopkins Medicine Institutional Review Board. Written informed consent/assent was obtained from parents and participants. Children with or without P aeruginosa through review of their medical records in the preceding 18 months and who met all of the eligiblity criteria were referred for recruitment by their treating physicians. Eligibility criteria included males and nonpregnant females, age 7-14 years with a diagnosis of CF by sweat chloride $60 meq/L, or the presence of 2 disease-causing CF transmembrane conductance regulator mutations, and a forced expiratory volume in 1 second (FEV1) $80% of predicted values. Children were grouped as P aeruginosa positive if P aeruginosa had been obtained from at least 1 airway culture positive for P aeruginosa in the preceding 18 months, or P aeruginosa grew from the culture obtained at the screening visit. They were grouped as P aeruginosa negative if P aeruginosa had not been obtained from any airway culture in the preceding 18 months, or from the culture obtained at the screening visit. We chose to review each child’s chart over the preceding 18 months because it is unknown how long 1 infection with P aeruginosa can affect MCC. Concomitant Medications Children who were taking inhaled tobramycin or aztreonam were studied during their “off” month of treatment. Children discontinued the use of bronchodilators, anticholinergic drugs, fluticasone propionate, recombinant human DNase, and airway clearance therapy for 12 hours before the study visit and hypertonic saline for 3 days before the study visit. All medications were discontinued for 36 hours thereafter. Our goal in withholding these medications was to minimize their possible effects on MCC measurements. The time period for withholding each medication was somewhat arbitrary because there is little, if any, information in the literature as to how long a specific drug affects MCC once the drug is stopped. Thus, although the bronchodilator effect of albuterol is thought to last only for 2 hours,8 it is unclear how long albuterol continues to affect MCC once it is stopped. In general, we asked patients to withhold medications for as long a time as we thought was medically advisable, while also reducing possible effects on MCC. Children who were 840
Vol. 164, No. 4 treated with oral, or intravenous, antibiotics for a pulmonary exacerbation completed those medications at least 2 weeks before the screening and study visits. Children who were being chronically treated with oral antibacterials such as sulfamethoxazole/trimethoprim were excluded from the study. Children who were being treated with azithromycin continued with their treatment before and throughout the study visits. Screening Visit Children underwent a focused medical history and physical examination and routine pulmonary function measurements of FEV1 and forced vital capacity (FVC). They also underwent an induced sputum test to determine their airway microbiology and a high resolution computed tomography (HRCT) scan to assess lung morphology.
Induced Sputum Test. Children underwent the standardized sputum induction procedure that is utilized by the CF Therapeutic Development Network.9 Sputum expectorated during this procedure was cultured for bacteria and fungi.
HRCT Scan. Children underwent an HRCT scan in the Johns Hopkins Radiology Department. Computed tomography (CT) scanning was performed as described by Brody et al.10
Scoring Lung HRCT Scans. HRCT scans were read by an experienced pediatric radiologist with no clinical information. Each CT scan was scored using a scoring system developed by Dr Alan Brody that evaluates the severity of lung disease in each lobe, with the lingula considered a lobe.10 The severity of bronchiectasis, mucous plugging, peribronchial thickening, air trapping, parenchymal opacities, and overall disease severity was scored for each lung separately. Overall disease severity score for the right lung and both lungs combined was averaged for each group. Pulmonary Function Measurements FVC and FEV1 were measured by a computerized 10-liter Survey III spirometer (Warren E. Collins, Inc, Braintree, Massachusetts). Spirometry was performed in accordance with American Thoracic Society/European Respiratory Society guidelines,11 using equations from Hankinson et al12 and Wang et al13 and a protocol based on the CF Foundation’s recommendations14 to determine percent predicted values. Study Visit 1A Children underwent the following procedures: (1) a focused medical history and physical exam; (2) pulmonary function measurements of FEV1 and FVC; (3) a 133xenon-ventilation scan; (4) inhalation of aerosol generated from a solution of 99m technetium sulfur-colloid; and (5) gamma camera imaging of the lungs for 90 minutes.
Ventilation Scan. Children inhaled 133xenon while sitting with their back to a large-field-of-view 2-dimensional Siemens Orbiter gamma camera (Gammasonics, Des Plains, Illinois). This procedure produced a ventilation image that Laube et al
ORIGINAL ARTICLES
April 2014 was used to identify the right lung border and inner and outer lung zones.
Identification of the Right Lung Region. Delineation of the right lung border has been described previously.15 The right-lung border was first delineated on the 133xenon ventilation image. During computer processing, each subsequent aerosol lung scan was registered with the ventilation scan. After registration, the right-lung border that was delineated on the ventilation scan was superimposed onto the aerosol lung scan.
Radioaerosol Inhalation. Children inhaled aerosol generated from a saline solution containing the radioisotope technetium sulfur-colloid (radioaerosol) while breathing 20-30 times intermittently from the mouthpiece of a 646 nebulizer connected to a PulmoAide compressor (Devilbiss, Somerset, Pennsylvania) through a Spira Electro2 Inhalation Dosimeter (Respiratory Care Center, H€ameenlinna, Finland). The goal was to deliver 50 mCi of radioisotope to the child’s mouth during the inhalation procedure. For some children, the dosing goal was achieved in 20 breaths. Other children required 30 breaths. Variability in the number of breaths resulted from differences in nebulizer dose. Inhalation started from functional residual capacity at a flow rate of 0.5 L/s. The nebulizer was pulsed for 0.7 seconds during each inhalation. There was no breathhold after each inhalation. Following the total inhalation procedure, children rinsed their mouth with water, expectorated the rinse, and drank some water. 99m
Quantification of Mucus Removal during First 90 Minutes. Children underwent lung imaging procedures every 2 minutes with the gamma camera starting immediately after inhalation of the radioaerosol (time 0) through the first 20 minutes and every 10 minutes thereafter for 60 minutes. Counts retained in the right lung field at the various time points were background-corrected and decay-corrected to time 0. Decay-corrected counts were subtracted from time 0 counts. The difference represented radioactivity that had been cleared from the lung at each time period. The number of cleared counts was expressed as a percentage of time 0 counts and reported as percent clearance. Percent clearance at 10, 20, 30, 40, 50, and 60 minutes was averaged as described previously.16 Average percent clearance over the first 60 minutes was expressed as AveMCC60. Children coughed 10 times between 60 and 70 minutes, 70 and 80 minutes, and 80 and 90 minutes. Children were instructed to cough into a Piko-1 electronic peak flow meter (Ferraris, Medical, Inc, Louisville, Colorado) such that the peak flow registered after each cough was approximately 200 mL/s. Images were obtained after each set of coughs. Percent clearance at 70, 80, and 90 minutes was calculated as described above for MCC60 and averaged to obtain average percent clearance between 70 and 90 minutes (AveMCC/CC90). Study Visit 1B Children returned to the laboratory approximately 24 hours after study visit 1A for a 30-minute imaging procedure.
MCC after 24 Hours. Percent clearance after 24 hours (MCC24hrs) was calculated as the difference between 100% retention at time 0 and the background-corrected and decay-corrected percent retention at 24 hours.
Quantification of Right Lung Regional Deposition Pattern. Deposition pattern of the radioaerosol was quantified in terms of inner and outer zones, as described previously.16 Outer/inner lung zone ratios (O:I) at time 0 (first aerosol lung image) for each study group were compared. O:I were also derived for the ventilation lung image. Ratios for the aerosol scan were divided by that of the ventilation scan to correct for lung volume differences. Deposition pattern for the radioaerosol was characterized in terms of the corrected O:I as described previously.17 Statistical Analyses Group comparisons were made using Fisher exact test for categorical data and 2-sample t tests for continuous. Possible associations between percent AveMCC60, AveMCC/CC90, and MCC24hrs and O:I, FEV1, and CT scores were assessed using Spearman correlation. Analyses were performed using SAS v. 9.3 (SAS Institute, Inc, Cary, North Carolina) and Microsoft Office Excel 2003 (Microsoft Corp, Redmond, Washington). All reported P values are 2-sided, and significance was set at P < .05.
Results Eight children who were P aeruginosa negative and 10 children who were P aeruginosa positive completed the screening visit and study visits 1A and 1B. Two children completed the screening visit, but did not complete the study visits. One child moved to another state after the screening visit and the other could not perform the inhalation maneuvers associated with study visit 1A. Baseline characteristics of the 18 participants are shown in the Table. There were no statistically significant differences between the P aeruginosa groups in age, sex, pulmonary function, height, weight, body mass index, or genotype. Four P aeruginosa positive children grew P aeruginosa at screening. The other 6 P aeruginosa positive children grew P aeruginosa from throat cultures over the preceding 18 months. One of the children who grew P aeruginosa on the screening visit also grew A fumigatus. Three other children with P aeruginosa who did not grow P aeruginosa on the screening visit grew A fumigatus. Sixty percent (6 children) of the P aeruginosa positive group grew more than 1 organism at screening, compared with 12% (1 child) in the P aeruginosa negative group. Concomitant Medications Sixteen of the children were regularly treated with albuterol (89%). One child without P aeruginosa and 1 child with P aeruginosa did not use albuterol. Three children with P aeruginosa (30%) and 1 child without P aeruginosa (12.5%) used fluticasone propionate. Nine children with P aeruginosa (90%) and 7 children without P aeruginosa (87.5%) were being treated with dornase alfa. Five of the children with P aeruginosa (50%) and
Mucus Removal Is Impaired in Children with Cystic Fibrosis Who Have Been Infected by Pseudomonas aeruginosa
841
THE JOURNAL OF PEDIATRICS
www.jpeds.com
Vol. 164, No. 4
Table. Characteristics at screening by P aeruginosa status Characteristics Males Age (years)† FVC (% predicted)† FEV1 (% predicted)† Height (percentile)† Weight (percentile)† BMI (percentile)† Genotype: F508del homozygote F508del heterozygote Other Antibiotic medications: Azithromycin Inhaled tobramycin Cayston Organisms cultured at screening: P aeruginosa A fumigatus A niger Mixed respiratory flora Ste maltophilia Sta aureus MRSA
P aeruginosa (n = 8)
P aeruginosa+ (n = 10)*
5 (62%) 10.9 1.9 86.3 11.0 93.9 10.8 49.6 48.4 39.4 31.6 45.6 18.8
5 (50%) 10.2 2.3 91.2 10.2 99.4 11.1 40.6 28.4 41.6 27.5 45.6 23.1
5 (62%) 3 (38%) 0
7 (70%) 1 (10%) 2 (20%)
4 (50%) 0 0
8 (80%) 3 (30%) 3 (30%)
0 0 0 7 (88%) 2 (25%) 4 (50%) 1 (12%)
4 (40%) 4 (40%) 2 (20%) 7 (70%) 1 (10%) 3 (30%) 2 (20%)
BMI, body mass index; MRSA, methicillin-resistant Sta aureus. *1 or more positive cultures for P aeruginosa in previous 18 months. †Values are mean (SD).
5 of the children without P aeruginosa (62.5%) were being treated with hypertonic saline. Eight of the children with P aeruginosa and 4 of the children without P aeruginosa were being treated with azithromycin. Six of the children with P aeruginosa were being treated with inhaled antibiotics, compared with none of the children without (P = .013). Deposition Pattern The O:I for the children with and without P aeruginosa were similar, averaging 0.515 0.147 (range, 0.362-0.820) and 0.547 0.170 (range, 0.374-0.888), respectively, indicating there was no difference in the deposition pattern of the radioaerosol in the lungs of the 2 groups of children. The mean AveMCC60 for the children with P aeruginosa was 12.5% 9.0% (range, 0.6%-29%) compared with 18.8% 8.3% (range, 6.9%-34.1%) for the children without P aeruginosa. This difference was not statistically significant (P = .14; Figure 1). Mean AveMCC/CC90 for the children with P aeruginosa was 16.2% 11.0% (range, 0%-36.9%) compared with 28.6% 7.8% (range, 13.7%-37.9%) for the children without P aeruginosa. The children with P aeruginosa had statistically significantly slower AveMCC/CC90 (P = .016; Figure 1). Mean MCC24hrs was similar for the 2 groups of children (48.7% 23.7%; range, 3.7%-87.4% for the children with P aeruginosa and 50.4% 8.9%; range, 34.6%-60.6% for the children without P aeruginosa) (Figure 1). Mean FEV1 was similar for the children with and without P aeruginosa on the screening visit (Table). Mean FEV1 for the 2 groups was also similar on study visit 1A, with 93.0 8.0 and 100.1 11.6 of the percent predicted, respectively. 842
Figure 1. Average percent mucus removal over 60 minutes (AveMCC60), between 60 and 90 minutes (AveMCC/CC90), and after 24 hours (MCC24hrs) for children with CF, without (white bars) and with P aeruginosa (black bars). AveMMC/ CC90 was significantly slower in the children with P aeruginosa compared with those without (P = .016).
Total and right lung HRCT scores were similar for the 2 groups, with 3.62 5.37 (P aeruginosa negative) and 6.78 6.25 (P aeruginosa positive) and 1.88 2.49 (P aeruginosa negative) and 4.08 4.00 (P aeruginosa positive), respectively. Lower scores indicated less disease severity. With respect to scores directly pertaining to MCC, 5 of 10 children with P aeruginosa were scored for mucus plugging in at least 1 lung, whereas, none of the children without P aeruginosa were scored for mucus plugging. In addition, 6 of 10 children with P aeruginosa (60%) were scored for bronchiectasis (2.32 3.3), whereas only 1 of the 8 of the children without P aeruginosa (12.5%) was scored for bronchiectasis (0.25 0.71). These differences were not statistically significant. Five of 8 children without P aeruginosa (62.5%) had overall disease severity scores of 0-1, compared with 2 of 10 children with P aeruginosa (20%). There was a significant negative correlation between percent AveMCC60 and total lung HRCT score and AveMCC/CC90 and total lung HRCT score for all children (N = 18) (P = .039 and .047, respectively). Lower percent AveMCC60 and AveMCCCC90 were associated with higher HRCT scores and more parenchymal damage (Figures 2 and 3). There were no statistically significant correlations between mean percent AveMCC60, AveMCC/CC90, or MCC24hrs and FEV1, O:I, or right lung HRCT score.
Discussion Although most individuals with CF are infected with P aeruginosa by the age of 18, little is known about the effect of P aeruginosa on MCC, or MCC and CC combined, despite the fact that these are important lung defense mechanisms. Laube et al
April 2014
Figure 2. Correlation between percent AveMCC60 and total lung HRCT score for all children (N = 18). Percent AveMCC60 was significantly negatively correlated with total lung HRCT score (P = .039). The rs value was generated using a Spearman correlation test.
In this study, we found that a history of infection with P aeruginosa was associated with a significant slowing of MCC with CC combined in children with mild CF. We found no significant difference in MCC alone between the 2 groups of children. This lack of differentiation may indicate that infection with P aeruginosa does not affect MCC mechanisms so much as CC mechanisms. This is evident from the significant improvement in mucus removal with the addition of coughing between 60 and 90 minutes in the children without P aeruginosa, and there was little improvement in the children with P aeruginosa. The lack of differentiation in MCC alone may also have been because the study was underpowered for this measurement. The proposed sample
Figure 3. Correlation between percent AveMCC/CC90 and total lung HRCT score for all children (N = 18). Percent AveMCC/CC90 was significantly negatively correlated with total lung HRCT score (P = .047). The rs value was generated using a Spearman correlation test.
ORIGINAL ARTICLES size was initially to be 12 subjects in each group. However, we were able to recruit only 8 and 10 children with and without P aeruginosa, respectively, during the study period. With group sizes of 8 and 10 there was only 69% power to detect a difference of 10% in AveMCC60 using our observed SDs. There was also a lack of differentiation between the 2 groups based on MCC measured after 24 hours. This may have been due to several factors. First, there may be no difference in mucus removal after 24 hours between these 2 groups of children with mild CF disease. Second, there may have been too little radioisotope remaining in the lung after 24 hours for an accurate measurement. However, although residual activity in the lungs after 24 hours was low, in all cases it was above background activity, suggesting that the 24-hour measurement was valid. Finally, there may have been differences between children in terms of the amount of coughing between the last image on study visit 1A and the image acquired on study visit 1B that were not documented. The techniques used to measure MCC in the current study have only recently been standardized, and no direct comparisons are available with other studies in children with CF. One study has been conducted in adults with CF using the same techniques18 and 1 has been conducted in healthy adults.16 Inclusionary criteria for the CF adults included FEV1 $ 50% of predicted. There was no information regarding their infection history. The investigators reported average mucus clearance 60 minutes after placebo of 9.3 1.1% (SE). Average MCC 24 hours later was 40.7 2.5% (SE). In the 1 study that utilized the same techniques for measuring MCC in healthy adults,16 MCC60 averaged 11 6%, MCC/CC90 averaged 16 8%, and MCC24hrs averaged 36 17%. It is unclear if MCC values measured in adults with CF, or in healthy adults, are comparable with those of CF children, because the lengths of ciliated airways in adult lungs are probably significantly greater than those of young children’s ciliated airways as a result of lung growth. This difference could affect rate of mucus removal in the 2 groups. We found that slower MCC and MCC combined with CC were each associated with more parenchymal abnormalities identified using the Brody HRCT scoring system. This is the first time that such a correlation has been reported and suggests that these measurements may be as sensitive as scored HRCT scans in identifying children at risk for development of early irreversible lung damage. Several studies in children with CF have shown that HRCT scans, quantified with the Brody scoring system, are more sensitive in terms of detecting early lung damage than traditional pulmonary function tests.19-21 Our results support these findings because we found significant correlations between MCC and HRCT scores and between MCC with CC and HRCT scores, but no correlations with FEV1. In our study, 60% of children with P aeruginosa had bronchiectasis as detected by the Brody HRCT scoring system. One child without P aeruginosa had bronchiectasis. Children in our study were similar in age and pulmonary function to children in a study reported by Farrell et al. 20 In that study,
Mucus Removal Is Impaired in Children with Cystic Fibrosis Who Have Been Infected by Pseudomonas aeruginosa
843
THE JOURNAL OF PEDIATRICS
www.jpeds.com
the investigators found that 83% of children with CF (n = 82) had bronchiectasis, and bronchiectasis scores were statistically highest in children with mucoid P aeruginosa. We chose to define “infected with P aeruginosa” based on at least 1 positive airway culture in the preceding 18 months. This was because diagnosis of infection based on throat or induced sputum cultures can yield false negatives, and we did not want to rely on an arbitrary number of positive cultures greater than 1 to define “infected with P aeruginosa” when infection might be undetected by current testing methods. In fact, only 4 of the children with P aeruginosa grew P aeruginosa at the screening visit. Results from this study suggest that infection with P aeruginosa is associated with a decrease in mucus removal in children with CF. It is not known if P aeruginosa infection causes the impairment, but, mechanistically, infection with P aeruginosa has the potential to impair mucus removal in these individuals because pyocyanin, a redox-active virulence factor is secreted by P aeruginosa. Pyocyanin has been shown to slow the beating of cilia,22,23 which is critically important to the removal of mucus. Accumulation of mucus in the airways, as a result of infection and impaired MCC could, in turn, overwhelm removal by CC. Another possible mechanism by which P aeruginosa could lead to a delay in MCC is through the development of bronchiectasis. Bronchiectasis is caused by 1 or more infections or inflammatory insults to the lungs and damaged airways associated with bronchiectasis cannot effectively clear mucus. Children who were infected with P aeruginosa were also infected with other organisms that could impair MCC/CC. Overall, 60% of the children with P aeruginosa grew more than 1 organism at screening, compared with 12% in the children without P aeruginosa. More specifically, 5 of the children with P aeruginosa also were infected with A fumigatus, which, similar to P aeruginosa, secretes virulence factors that could affect mucus clearance.24 Others have shown in vitro that the virulence factors and proteases associated with A fumigatus can inhibit ciliary beat frequency25 and damage the epithelial tissue to the point of cell detachment.26 Each of these could reduce MCC/CC in vivo. Additional studies are needed to determine the effect of combined infection with P aeruginosa and A fumigatus vs P aeruginosa alone on MCC. Alternatively, infection with P aeruginosa may be a marker for children with slower MCC/CC and not a cause of the impairment. Children with slower MCC/CC may be unable to clear the bacteria as quickly as children with faster MCC/CC and, therefore, may simply be more vulnerable to the infection. The fact that several children with a history of infection with P aeruginosa also had a history of infection with A fumigatus and A niger supports the notion that these children may have been more vulnerable to infection because they had slower MCC and CC and not vice versa. Prospective studies of the evolution of infection in children with CF who are uninfected with P aeruginosa, but have slower MCC than other uninfected children, could help to answer this question. 844
Vol. 164, No. 4 In this study, we found that a history of infection with P aeruginosa was associated with a significant slowing of MCC with CC combined in children with mild CF. Slowing of mucus removal was significantly associated with more parenchymal abnormalities detected on HRCT but was not associated with pulmonary function changes based on FEV1 measurements. These findings suggest that MCC/CC measurements may be a more sensitive indicator of the effects of P aeruginosa infection in the lungs of children with mild CF disease than measurements of FEV1. In addition, the significant association with HRCT scores suggests that MCC/ CC measurements might be used as an early biomarker of disease severity in young children with CF with reduced radiation exposure. Early and aggressive antibacterial treatment in combination with therapies that improve mucus rheology in children with slow MCC/CC could increase the ability of these children to fight off infections, possibly reducing future declines in pulmonary function. n The authors thank Drs J. Michael Collaco, Pamela Zeitlin, and Beryl Rosenstein for performing the histories and physicals on the screening visit for children in this study. Submitted for publication Jul 18, 2013; last revision received Sep 26, 2013; accepted Nov 13, 2013. Reprint requests: Beth L. Laube, PhD, The Johns Hopkins Medical Institutions, The David M. Rubenstein Building, 200 North Wolfe St, Ste 3015, Baltimore, MD 21287-2533. E-mail: [email protected]
References 1. Morison S, Lewis PA, Coles EC, Geddes D, Russell G, Littlewood JM, et al. Incidence, population, and survival of cystic fibrosis in the UK, 1968-95. UK Cystic Fibrosis Survey Management Committee. Arch Dis Child 1997;77:493-6. 2. Hodson ME, Geddes D, Bush A, eds. Cystic fibrosis. 3rd ed. London: Hodder Arnold; 2007. 3. Matsui H, Grubb BR, Tarran R, Randell SH, Gatzy JT, Davis CW, et al. Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease. Cell 1998;95:1005-15. 4. Matsui H, Verghese MW, Kesimer M, Schwab UE, Randell SH, Sheehan JK, et al. Reduced three-dimensional motility in dehydrated airway mucus prevents neutrophil capture and killing bacteria on airway epithelial surfaces. J Immunol 2005;175:1090-9. 5. Ratjen F, Doring G. Cystic fibrosis. Lancet 2003;361:681-9. 6. Davis PB, Drumm M, Konstan MW. Cystic fibrosis. Am J Respir Crit Care Med 1996;154:1229-56. 7. Cystic Fibrosis Foundation Patient Registry 2002 Annual Data Report to the Center Directors. Bethesda, MD: 2003 Cystic Fibrosis Foundation; 2002. 8. Devalia JL, Sapsford RJ, Rusznak C, Toumbis MJ, Davies RJ. The effects of salmeterol and salbuterol on ciliary beat frequency of cultured human bronchial epithelial cells, in vitro. Pulmonary Pharmacol 1992;5:257-63. 9. Mayer-Hamblett N, Aitken ML, Accurso FJ, Kronmal RA, Konstan MW, Burns JL, et al. Association between pulmonary function and sputum biomarkers in cystic fibrosis. Am J Respir Crit Care Med 2007;175:822-8. 10. Brody AS, Kosorok MR, Li Z, Broderick LS, Foster JL, Laxova A, et al. Reproducibility of a scoring system for computed tomography scanning in cystic fibrosis. J Thorac Imaging 2006;21:14-21. 11. Miller MR, Hankinson J, Brusasco V, Burgos F, Casaburi R, Coates A, et al. Standardisation of spirometry. J Eur Respir 2005;26:319-38. 12. Hankinson JL, Odencrantz JR, Fedan KB. Spirometric reference values from a sample of the general US population. Am J Respir Crit Care Med 1999;159:179-87.
Laube et al
April 2014 13. Wang X, Dockery DW, Wypij D, Gold DR, Speizer FE, Ware JH, et al. Pulmonary function growth velocity in children 6 to 18 years of age. Am Rev Respir Dis 1993;148:1502-8. 14. Rosenfeld M, Coates A, Corey M, Goss CH, Howard MB, Morgan W, et al. Task Force to Evaluate Choice of Spirometric Reference Equations for the National Patient Registry: Summary and Recommendations. Cystic Fibrosis Foundation Registry Committee; Oct 1, 2005. 15. Laube BL, Norman PS, Wagner HN Jr, Adams GK III. The effect of aerosol distribution on airway responsiveness to inhaled methacholine in patients with asthma. J Allergy Clin Immunol 1992;89:510-8. 16. Bennett W, Laube BL, Corcoran T, Mogayzel PJ Jr, Pilewski J, Donaldson S. Multi-site comparison of mucociliary and cough clearance measures using standardized methods. J Aerosol Med Pulm Drug Deliv 2013;26:157-64. 17. Newman S, Bennett WD, Biddiscombe M, Devadason SG, Dolovich M, Fleming J, et al. Standardization of techniques for using planar (2D) imaging for aerosol deposition assessment of orally inhaled products. J Aerosol Med Pulm Drug Deliv 2012;25(Suppl 1):S10-28. 18. Donaldson SH, Bennett WD, Zeman KL, Knowles MR, Tarran R, Boucher RC. Mucus clearance and lung function in cystic fibrosis with hypertonic saline. N Engl J Med 2006;354:241-50. 19. Brody AS, Klein JS, Molina PL, Quan J, Bean JA, Wilmott RW. High resolution computed tomography in young patients with cystic fibrosis: distribution of abnormalities and correlation with pulmonary function tests. J Pediatr 2004;145:32-8.
ORIGINAL ARTICLES 20. Farrell P, Collins J, Broderick L, Rock M, Li Z, Kosorok M, et al. Association between mucoid Pseudomonas infection and bronchiectasis in children with cystic fibrosis. Radiology 2009;252:534-43. 21. Stick S, Brennan S, Murray C, Douglas T, von Ungern-Sternberg B, Garratt L, et al. Australian Respiratory Early Surveillance Team for Cystic Fibrosis (AREST CF). Bronchiectasis in infants and preschool children diagnosed with cystic fibrosis after newboen screening. J Pediatr 2009;155:623-8.e1. 22. Kanthakumar K, Taylor G, Tsang KW, Cundell DR, Rutman A, Smith S, et al. Mechanisms of action of Pseudomonas aeruginosa pyocyanin on human ciliary beat in vitro. Infect Immun 1993;61:2848-53. 23. Munro NC, Barker A, Rutman A, Taylor G, Watson D, McDonaldGibson WJ, et al. Effect of pyocyanin and 1-hydroxyphenazine on in vivo tracheal mucus velocity. J Appl Physiol 1989;67:316-23. 24. Latge JP. The pathobiology of Apergilluis fumigatus. Trends Microbiol 2001;9:382-9. 25. Amitani R, Taylor G, Elezis E-N, Llewellyn-Jones C, Mitchell J, Kuze F, et al. Purification and characterization of factors produced by Aspergillus fumigatus which affect human ciliated respiratory epithelium. Infect Immun 1995;63:3266-71. 26. Tomee JFC, Wierenga AT, Hiemstra PS, Kauffman HF. Proteases from Aspergillus fumigatus induce release of pro-inflammatory cytokines and cell detachment in airway epithelial cell lines. J Infect Dis 1997; 176:300-3.
Mucus Removal Is Impaired in Children with Cystic Fibrosis Who Have Been Infected by Pseudomonas aeruginosa
845
