Abstract
Our aim was to study the effect of lower airway infection on clinical parameters, pulmonary function tests, and inflammation in clinically stable infants and young children with cystic fibrosis (CF). To accomplish this goal, a prospective cohort of screened CF patients under 4 years of age were studied, using elective anesthesia and intubation for: passive respiratory mechanics (single breath occlusion passive deflation) and lung volumes (nitrogen washout), under neuromuscular blockade; and bronchoalveolar lavage (BAL) of 3 main bronchi for cytology, cytokine interleukin (IL)-8, and quantitative microbiology. There were 22 children studied, with a mean age of 23.2 months (6.7–44 months). A greater relative risk of lower airway pathogens was associated with prior respiratory admission (3.60, 95% confidence interval [CI] 2.87–4.51), history of asthma (1.75, 95% CI 1.52–2.03), and chronic symptoms (1.50, 95% CI 1.23–1.83), especially wheeze (1.88, 95% CI 1.61–2.19). Lower respiratory pathogens ( ⩾ 10 cfu/ml BAL) were found in 14 out of 22, and greater than 105 cfu/ml in 8 out of 22 subjects. The level of pathogens in BAL (log10 cfu/ml) explained 78% of the variability in percent neutrophils and 34% of the variability in IL-8 levels. Pathogen level also correlated with pulmonary function tests of specific respiratory system compliance (r − 0.49, p = 0.02) and functional residual capacity over total lung capacity (r 0.49, p = 0.03). We conclude that the presence of pathogens in the lower airways correlated with levels of inflammation, respiratory system compliance, and degree of air trapping.
Keywords: cystic fibrosis; infants; respiratory function tests
The pathogenesis of lung disease in cystic fibrosis, particularly in early life, has not been fully characterized. It is known that pulmonary disease is the cause of most of the morbidity and mortality in cystic fibrosis (1) and that the lungs are essentially normal at birth (2, 3); however, the sequence of events at the onset of pulmonary infection and inflammation are controversial.
The origin of airway inflammation in cystic fibrosis (CF) has been the subject of debate, following the finding of neutrophil dominated inflammation in the absence of bacterial or viral pathogens on bronchial lavage in some studies on CF infants (4-8). This finding has led to the proposal that inflammation (or, alternatively, the failure to downregulate inflammation) is intrinsic to CF (9). This theory has been disputed by Armstrong and colleagues (10) in a longitudinal bronchoalveolar lavage (BAL) study of infants with CF, who found that airway inflammation always followed respiratory infection.
It is possible that the methods used in previous studies were insufficiently sensitive to detect infection. In particular the practice of sampling only one lobe (or rarely two) may have been inadequate, because Meyer and Sharma (11) demonstrated significant regional variability in infection and inflammation on BAL, especially in those subjects with the mildest disease, with the most commonly infected area being the right upper lobe. This regional variability was also described by Wilmott and colleagues (4). Falsely negative cultures could also have been induced by the practice of using lignocaine during the lavage. The findings of studies in which unscreened infants were tested or infants were tested during clinical exacerbations would also result in potential selection and measurement bias.
No study has addressed all three elements of inflammation, infection, and pulmonary function simultaneously in one group of infants. Clearly, a better understanding of these relationships in early lung disease has clinical implications with the potential to intervene to improve clinical outcomes, such as the early detection and aggressive treatment of Pseudomonas infection (12).
The aims of this study were to study the interaction between infection, inflammation and pulmonary function in clinically stable infants and young children with CF and to determine if there are measurable differences between those with pathogens cultured in BAL fluid (BALF) and those without with regard to pulmonary function, BALF cytology, and BALF cytokines. The hypothesis was that bacterial lower respiratory tract infection would be associated with the presence of inflammation and abnormal pulmonary function.
The study design was a prospective cohort study of unselected infants and young children with CF, aged less than 4 years. A diagnosis of CF was the exposure variable and detection of lower airway pathogens the disease outcome. CF was identified by the newborn screening program in the state of New South Wales, which is based on an estimate of the immunoreactive trypsin from a blood spot Guthrie card, followed by genetic analysis for the ΔF508 deletion. A sweat chloride concentration of > 60 mEq/ml was used to confirm the diagnosis for heterozygotes and those with undetectable mutations on further genetic testing.
The study was conducted between March 1997 and June 2000. From birth until the episode of testing, clinical measures such as respiratory symptoms, signs, documented viral infections, and treatment were recorded at each outpatient visit.
Children were noted to have a history of chronic symptoms if they were found during outpatient review to have cough, wheeze, hyperinflation, or increased work of breathing (on observation) in the absence of a clinically evident concurrent viral respiratory infection. A history of asthma was noted if there were episodes of bronchodilator-responsive wheeze. Likewise, the presence of gastroesophageal reflux (GOR) was recorded if it was a clinical problem requiring medical or surgical treatment.
Each child underwent a single episode of testing performed electively when he or she was clinically well (with no cough, fever, or symptoms of upper airways infection). Any oral or nebulized antibiotics were ceased for 48 hours before testing. Length (by Infantometer; Holtain, Dyfed, UK) and weight (Electronic chair scale BWB-600, 0–200 kg in 50-g increments; Tanita, Wedderburn, Australia) were recorded and expressed as z-scores. Anesthetic delivery was standardized and designed to optimize conditions for testing and minimize effects on pulmonary function testing. At induction sevoflurane was administered for intubation and then replaced by intravenous propofol maintenance. Muscle relaxation (atracurium in standard doses) was used for the duration of pulmonary function tests (PFTs) to minimize variability by avoiding reliance on the Hering-Breuer reflex (13), avoiding the effect of stress-relaxation (14), and also preventing premature inspiration (15). Inspired oxygen concentration (Fi O2 ) was held constant throughout the PFTs in the range of 21–40%, and was accounted for in calculating PFTs. The pneumotach readings (Hans-Rudolf 4500; Hans-Rudolf, Kansas City, MO) were corrected for Fi O2 . Measurement of respiratory mechanics was not commenced until exhaled sevoflurane content was less than 0.2%. Ventilation was at zero positive end-expiratory pressure (PEEP) throughout. Once PFTs were measured, the infant was switched to a gaseous anaesthetic agent for bronchial lavage, with change of endotracheal tube (ETT) to a laryngeal mask if the ETT was smaller than a 5.0 mm size.
Respiratory mechanics were assessed with the child in the supine position using a single breath occlusion passive deflation flow–volume technique (15) to measure respiratory system compliance (Crs) and respiratory system resistance (Rrs). An automated system (Sensormedics 2600 Pediatric Pulmonary System, Yorba Linda, CA) was used for all measurements. This entailed the occlusion of the airway by a shutter valve for 200–300 milliseconds at end-inspiration with transduction of the end-inspiratory pressure (Validyne MP45; Validyne, Northridge, CA), followed by passive exhalation through a pneumotach. The flow–volume relationship was obtained by integrating the passive expiratory flow signal. This was displayed graphically and stored numerically, at incremental volume points of 0.95–1.00 ml for each curve. A minimum of eight flow–volume loops were collected per patient. Data points for each curve were retrieved and transferred into the SPSS statistical package (SPSS version 6.1.4; SPSS Inc, Chicago, IL) for integration to yield a volume–time curve for analysis using a single or double compartment model.
Analysis of the curves of those patients with linear flow–volume relationships over at least 50% of the exhaled volume entailed the use of a single compartment model using the simplified Meads equation (16). The curves of those patients with nonlinear flow–volume relationships were analyzed using a two-compartment model, as described by Jarriel and coworkers (17). In this model, two independent compartments with different volumes and time constants empty in parallel: “fast” and “slow.” For each patient the values for compliance and resistance obtained from each set of flow–volume curves were averaged to obtain values for mean and coefficient of variation of each parameter.
The lung volumes of functional residual capacity (FRC) and total lung capacity (TLC) were measured using the automated, modified technique based on standard open-circuit nitrogen washout technique as described by Sivan and coworkers (18). This required the use of a second volume-controlled intermittent flow washout ventilator, also set at zero PEEP. TLC was measured by the initiation of N2 washout after inflation pressure of 40 cm H2O for 3 seconds, as previously described (19). For both volumes, two to three measurements were made and the values averaged. Values were accepted if they were within 10% of each other (20).
BAL was performed on three lung lobes, to maximize the potential for detection of inflammation and infection (11). Lavage was preceded by suctioning 10 ml sterile normal saline (NS) through the bronchoscope into a suction trap, for microbiologic testing to confirm the sterility of the bronchoscope. The 3.5-mm bronchoscope was then introduced into the lower airways via a laryngeal mask or endotracheal tube. The tip of the bronchoscope was wedged in the right lower lobe, right middle lobe or lingula, and the right upper lobe in turn. Room temperature sterile nonbacteriostatic NS was instilled in a total of 3–4 aliquots through the suction channel over 3–5 seconds and immediately aspirated into a plastic suction set over 10–20 seconds. There was a total maximum lavaged volume of 3 ml/kg of sterile NS in equal aliquots. Only one aliquot per lobe was used to maximize sampling from endobronchial sites (21). Lignocaine was not used. Specimens were pooled and analyzed for: cytology (viability, total cell count [TCC], differential count); cytokine IL-8 and IL-10 (Human quantikine elisa kits D8050 & HS100; R&D Systems, Minneapolis, MN); and quantitative microbiology (using the techniques described by Armstrong and colleagues 1996 [22]). Standard microbiologic techniques were used to identify respiratory bacterial pathogens.
Statistical calculations were performed using SPSS 10.0.5 statistical package (SPSS Inc, Chicago, IL) and Epi2000 (Center for Disease Control and Prevention, Atlanta, GA). Cohort data was compared with infection outcomes using chi squared (Fisher's exact), with calculation of relative risk and adjustment for age where appropriate. Interval data was described using mean, range, and distribution. The t test and analysis of variance (ANOVA) were used to analyze group differences in inflammatory markers and lung function for different levels of pathogen cultured from BAL. Relationships between infection, degree of inflammation, and lung function were analyzed using two-tailed Pearson correlation coefficients (at significance < 0.05). Linear regression analysis was used to predict the effect of infection on inflammation and lung function. To allow log transformation for correlation and regression analysis all negative cultures were designated as having 1 (100) cfu/ml, because the lower level of the sensitivity of microbiologic culture was < 100 cfu/ml. To determine if there was a significant influence of assigning values of 1 cfu/ml to negative values, these relationships were also tested for assigned negative culture values of 10, 20, and 30 cfu/ml.
This study was approved by the South Eastern Sydney Area Health Service Ethics Committee, with individual consent from the parents of each child.
Twenty-two out of twenty-four eligible infants and young children were studied, with an age range of 6.7 to 44 months. The parents of two children refused to participate. The investigations were well tolerated, with high fever post procedure being the only complication in three patients. No child had increased cough beyond 24 hours after the procedure.
The characteristics of the cohort at testing are summarized in Table 1. The majority (73%) had chronic symptoms and most (55%) had been admitted to hospital for respiratory problems before the investigations. A high proportion (45%) had a history of asthma (bronchodilator-responsive wheeze, assessed clinically).
Sterility of the bronchoscope was confirmed in each study. Lower respiratory pathogens were cultured from the BALF at a level of ⩾ 10 cfu/ml BALF in 14 out of 22 (64%) and > 105 cfu/ml BALF in 8 out of 22 subjects (36%) (Figure 1). Upper respiratory flora, such as α-haemolytic streptococci, were present in 16 out of 22 subjects.
Staphylococcus aureus was cultured in all three children not on prophylactic flucloxacillin, with a relative risk reduction of culturing staph in those on flucloxacillin of relative risk (RR) 0.21 (95% CI 0.09–0.50). The use of prophylactic flucloxacillin did not alter the risk of culturing Pseudomonas (RR 1.11, 95% CI 0.20–6.08).
Pseudomonas aeruginosa was cultured in seven (32%) children, with ages ranging from 7.5 months to 37 months (median 23 months), with Pseudomonas species in one child. Culture of Pseudomonas was not associated with any significant age or genotype relationship. While Pseudomonas cultures were examined as to whether they were mucoid in three of seven samples, all cultures examined were nonmucoid.
There was no significant difference between those with and those without pathogens in BALF with regard to age, weight, or CF mutation (Table 2). Male sex, prior respiratory admission, chronic symptoms (including wheeze and cough), GOR, and a history of asthma were associated with a significantly increased risk of culturing pathogens in BALF. In addition, all of those with BAL pathogen level greater than 105 cfu/ml had chronic symptoms: all had chronic cough and most had chronic wheeze (six out of eight).
The cytology and cytokine results for the group are summarized in Table 3. The total cell count, percentage, and absolute count of neutrophils (PMN), macrophages, and IL-8 levels in BALF were significantly different in those samples with greater than or less than 105 cfu/ml BALF. In particular, there was a maximum of 50% neutrophils in all samples with < 105 cfu/ml and a minimum of 79% neutrophils for all samples with > 105 cfu/ml. There were also significant differences between samples with and those without pathogens at any level with regard to neutrophil count and percentage, percent macrophages, and IL-8.
The relationship between the level of inflammatory markers, both neutrophils and cytokine IL-8, and the level of pathogens cultured in BALF is shown in Figures 2 and 3.
Fig. 2. Inflammation (neutrophils) versus infection; r = 0.88, p < 0.0001, r2 = 0.78, %PMN = 15.6 + 10.8 (log10 cfu/ml BALF).
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Fig. 3. Bronchoalveolar lavage: Inflammatory cytokine interleukin-8 versus level of bacterial pathogens; r = 0.61, p < 0.03, r2 = 0.34, IL-8 pg/ml = 1,250 + 625 (log10 cfu/ml).
[More] [Minimize]The level of pathogens cultured explained 78% of the variability in %PMN and 34% of the variability in IL-8 levels. The linear regression equations are given with the figures. The effect of applying the range of assigned values to negative cultures (1–30 cfu/ml BAL) was minimal, with the range of values as follows: for %PMN r2 77–78% (p < 0.001), r 0.88–0.89 (p < 0.001), regression equation constant −0.58 to 15.78, with the slope 10.8–13.5. For IL-8 levels: r = 0.60–0.61 (p = 0.003), r2adj = 0.33–0.34 (p = 0.03), regression constant 468–1,323 and slope 624–751.
The pulmonary function results are shown in Table 4. For the flow–volume curves, the mean intra-individual coefficient of variation for compliance was 4.5 (SD 1.98), with a range of 2.3–9.5. The mean coefficient of variation of resistance was 5.3 (SD 2.11).
The flow–volume curve was of nonlinear (concave) morphology in three children, in whom the resistance has been given separately as the fast and the slow component of the two-compartment model. The model was demonstrated to have an excellent fit with the observed data, with correlation coefficients ranging from 0.9833–0.9999. The mean coefficient of variation of the individual curves was a follows: Rrs fast 5.73 (range 2.28–8.6, SD 3.20) and Rrs slow 6.54 (range 5.20–8.50, SD 1.74). In all three children, P. aeruginosa was cultured in BALF. Nonlinear flow–volume curve morphology was significantly associated with culture of pathogens > 105 cfu/ml BALF (Fisher exact p = 0.04), with RR 3.60 (95% CI 1.71–7.58).
The lung volume ratios correlated negatively with specific compliance values (Pearson two-tailed r −0.75 at p < 0.0001).
The pulmonary function parameters for those infants and children with pathogen levels less than and greater than 105cfu/ml are shown in Table 4. There was a significant difference between the two groups with regard to specific compliance (Spec Crs) and lung volume ratios (FRCN2/TLCN2).
Specific compliance correlated negatively with log pathogen cfu/ml BALF, with Pearson (two-tailed) r −0.49, p 0.02 (Figure 4). There was also positive correlation between FRCN2/TLCN2 and pathogen level, with a Pearson (two-tailed) r of 0.49 at p 0.03. Again, applying the range of assigned values for negative cultures, there was minimal variation: Spec Crs r −0.49 to −0.50, p 0.02–0.03; FRCN2/TLCN2 no difference.
A relationship between inflammatory markers and pulmonary function parameters was seen. Specific compliance and FRCN2/ TLCN2 both correlated with IL-8 and % neutrophils. Spec Crs correlated negatively with % neutrophils (2-tailed Pearson r −0.51 at p 0.02), as shown in Figure 5. FRCN2/TLCN2 correlated positively with % neutrophils (two-tailed Pearson r 0.50, p 0.02) and with IL-8 pg/ml (two-tailed Pearson r 0.45, p 0.04). There was a significant difference between those with less than and those with greater than 50% neutrophils with regard to specific Crs (p = 0.03) and FRCN2/TLCN2 (p = 0.02).
The findings from this study demonstrated a number of important relationships between infection, inflammation, and pulmonary function in screened clinically stable infants and young children with CF. An increased RR of lower respiratory infection was associated with chronic symptoms (especially cough), GOR, and asthma. There was a close relationship between the level of pathogens in the lower airways and the level of inflammatory cells and mediators. Similarly, the level of pathogens cultured correlated with specific respiratory system compliance and air trapping. To complete the relationship, these pulmonary function parameters correlated in the expected directions with the proportion of inflammatory cells and mediator levels.
This study was potentially limited by a number of factors. The sample size precluded subclass analysis of the cohort, particularly with regard to genotype. The timing of testing of the cohort, of necessity resulted in a spread of ages; however, no age relationship with infection was demonstrated. The cohort design might be criticized for the lack of a control group; however, it was the aim to investigate relationships within patients with CF, not to compare these with control subjects, which has been done in prior studies. BALF was not checked for viral infection because the subjects were clinically stable and symptoms of viral infection precluded BAL and measurement of pulmonary function. It has been shown that viruses were rarely detected in stable patients with CF undergoing elective BAL (23). Likewise, because diffusion of urea from serum into BALF results in overestimation of the epithelial lining fluid volume, the results were not normalized to urea (21, 24). Lastly, the methods to measure pulmonary function were impracticable for serial measures, but again this was not the intention of the study.
The relationship demonstrated between lower airway infection and clinically diagnosed asthma, GOR, and chronic symptoms has clinical relevance.
Clearly, the diagnosis of asthma in this age group is problematic. To further complicate this problem, CF itself seems to be associated with an increased incidence of bronchodilator responsiveness, as demonstrated by Hiatt and colleagues (25), who showed that 43% of a similar group of infants and young children had a significant increase in maximum expiratory flow at functional residual capacity (VmaxFRC) following bronchodilator treatment.
In this context, the importance of the diagnosis of asthma was that it was related to infection. The presence of the nonspecific chronic symptoms of cough and wheeze, potentially attributable to asthma or lower airways infection (or other causes), were shown to be significantly related to the presence of pathogens in lower airway fluid.
The majority of those with pathogens in the lower airway had chronic symptoms (12 out of 14), and at a pathogen level > 105 cfu/ml all had some chronic symptoms. Based on this cohort, the use of chronic symptoms as a screening tool for lower airway pathogens (at any level) would be 86% sensitive and 50% specific, with a positive predictive value of 75% and a negative predictive value of 67%. The label of asthma has a higher positive predictive value of 80%, but is less sensitive (57%).
The distinction between infection and colonization in this context is an important one, because the presence of pathogens in the lower airway is often reported as colonization (26, 27), promulgating the concept of a benign condition. It is clearly not the colony count alone which defines the presence of infection, despite the use in many studies of a count > 5 × 104 or 105 cfu/ml to designate significant infection (4, 10, 22, 28, 29). Infection may be defined as the presence of a particular type of microorganism in a part of the body where it is normally absent, and where, if allowed to multiply, it is likely to be harmful (30). P. aeruginosa is not normally detected in the lower airways (8, 22, 28, 31, 32), and it is clear that it contributes to inflammatory lung damage (5, 33-35); therefore, the detection of Pseudomonas at any level in BALF should be termed infection. Although the lower airways are thought to be a normally sterile environment, S. aureus and Haemophilus influenzae have been detected in uninfected control subjects (22, 32), possibly as contaminants from upper airway secretions during bronchoscopy, and it is less clear whether their presence alone in BALF should be termed infection.
Although our findings do not exclude the possibility of a degree of intrinsic inflammation in CF, they do describe a very significant relationship between infection and inflammation, with the level of pathogens cultured explaining most of the variation in inflammatory cells and a third of the variation in cytokine IL-8 level.
A relationship between infection and inflammation has been described by others (6, 8, 28); however, Balough and colleagues (6) failed to show a correlation between bacterial count and IL-8, and Armstrong and coworkers (22) described a nonlinear relationship between infection and IL-8 or %PMN, with no rise until the pathogen level was > 105 cfu/ml. In contrast, Kirchner and colleagues did not demonstrate a relationship between BAL neutrophils and bacterial count (31).
The lavage methods used in this study may have improved the sensitivity to detect infection and inflammation. A “bronchial wash” rather than a BAL has been shown to result in sampling of endobronchial sites and therefore a more concentrated sample with a higher % of PMN (36). In addition, sampling of the right upper lobe has been found to yield a higher concentration of neutrophils than in middle or lower lobe samples (11). Other studies have generally reported findings on samples from the middle or lower lobes.
Although there is no ideal method to measure pulmonary function in infants, the methods used in this study were chosen to maximize the sensitivity for detection of differences between infants by containing error with minimal covariance under controlled settings, in the context of a general anesthetic for the BAL. The use of muscle relaxation for measurement of pulmonary function resulted in a reliable FRC, known to be an unreliable volume landmark in spontaneously breathing infants (37). This enabled accurate volume-standardized mechanics measurements. The Sensormedics 2600 system has been used extensively to assess both infants and young children (38-41), and has been demonstrated to have the lowest relative mean volume error (3.5 +/− 1.75% SD) in a comparison of commercially available devices (42).
The volume standardization of mechanics measurements provides the most accurate means of presenting the data. Because these measures are volume-dependent, it is important to compare measurements between subjects at the lung volume at which they were obtained (43). This is particularly important in infants and young children because the dysanaptic pattern of lung growth between alveoli and airways results in a linear relationship between lung compliance and FRC (or a constant specific compliance), which is not the case for compliance and weight or height (44). The methods of muscle relaxation and measurement at zero PEEP (resulting in a mean reduction in FRC/kg of 4 ml/kg at Crs/kg of 1.0) would explain the lower values for FRCN2/kg seen in this study compared with other studies (range of 16.9–24.4) (19, 39, 45) in which measurement was generally at 4 cm PEEP.
Although it was not the point of this study to compare CF subjects with normal subjects, the mean group values obtained were in the normal range using the same methods (Crs 1.09 cm H2O/ml [39] and Rrs 0.03–0.10 [15, 19, 39]).
Comparison of pulmonary function parameters between infected and uninfected subjects revealed significantly lower compliance and increased air trapping in those with lower airway pathogens (> 105 cfu/ml), with correlation between pathogen level and these pulmonary function parameters. This relationship has not previously been investigated in any detail and is clearly an important one with regard to the pathogenesis of respiratory disease. It further invalidates the concept of colonization in these children. This association is expected, given the correlation demonstrated by Baltimore and colleagues (46) between infection with P. aeruginosa and the active inflammatory and obliterative processes in the lung.
Aspects of this relationship have been studied, but previous studies have had difficulty detecting early degrees of lung disease and have been unable to distinguish between controls and those with CF (25, 45, 47, 48). This may relate to the methods used, or more importantly given the significance of infection in the pathogenic process, to the failure of some previous studies to objectively distinguish (by BAL) between infected and uninfected infants (45, 47, 49).
Studies comparing those with and without symptoms have demonstrated reduced compliance and hyperinflation in children with mild symptoms (50, 51). In addition, Clayton and colleagues (52) demonstrated an increase in compliance and reduction in air trapping following treatment of clinically diagnosed exacerbations. No association between V'maxFRC or FRC and IL-8 concentration, neutrophil density, or consistently with pathogen level was demonstrated by Rosenfeld and colleagues in a longitudinal study of infants with CF. However, not all parameters were measured simultaneously.
In this study, both inflammation and infection were related to reduced compliance and air trapping.
In clinically stable infants and young children with CF, bacterial lower respiratory infection has been demonstrated by the present investigators to have a close relationship with both inflammation and abnormal pulmonary function. This study was unique in investigating this relationship and demonstrated abnormalities in pulmonary function very early in life in association with infection. The authors' findings suggest that the presence of any level of pathogen in BALF is associated with an inflammatory response and is therefore significant. Further longitudinal study concerning the benefits of early diagnosis and treatment of lower airway infection will be important in enabling clinicians to improve the long-term respiratory status of those with cystic fibrosis.
The authors wish to thank the anesthetists involved, who patiently assisted with the complicated regimen required. In addition, the authors would like to thank the children and their parents who were involved in this study.
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Dr. Dakin was partially supported by a Research scholarship from the University of New South Wales.
