Abstract
Induced sputum has been used to study airway inflammation. We sought to determine whether markers of infection and inflammation in induced sputum were a useful and safe outcome measure in cystic fibrosis. We hypothesized that bacterial density and inflammatory content of induced sputum would decrease after antibiotic therapy. Induced sputum was assayed for bacterial density, cell count, and differential and inflammatory markers before and after treatment with intravenous antibiotics. Fifty-five of the 72 subjects enrolled (mean age ± SD 18.2 ± 7.9 years) completed the study. FEV1 increased by an average 0.3 ± 0.3 L (10.4 ± 8.7% predicted FEV1), p < 0.0001; density of Pseudomonas aeruginosa and Staphylococcus aureus decreased by 2.4 ± 3.1 log10 cfu/g (p < 0.0005) and 4.0 ± 2.3 log10 cfu/ml (p < 0.0001), respectively; neutrophil count decreased by 0.4 ± 0.6 log10 cells/ml (p < 0.0001), interleukin-8 concentration by 0.5 ± 1.3 log10 pg/ml (p < 0.05), and neutrophil elastase by 0.4 ± 0.7 log10 μg/ml (p < 0.005). Seven of 127 (6%) sputum induction procedures showed a decrease in FEV1 of 20% or more. We conclude that markers in induced sputum may be useful, noninvasive outcome measures to assess response to therapies in cystic fibrosis studies.
Chronic endobronchial infection and neutrophil-dominated lower airway inflammation are hallmarks of cystic fibrosis (CF) (1). Currently, investigators are focused on developing new medications to modify the bacterial and inflammatory environment of the CF airway to slow the progression of CF lung disease. There is a need for validated, noninvasive outcome measures in CF to study the safety and efficacy of new therapies. Several surrogates for clinical outcomes have been used in CF clinical trials, such as measurements of pulmonary function (2–6), bacterial density (4, 5), and measurements of mediators of systemic and pulmonary inflammation (7–11). Most published studies use improvement in pulmonary function, number of days spent in the hospital, and decreased requirement for antibiotics as clinically relevant outcome measures. Markers of airway infection and inflammation may be more sensitive than pulmonary function and antibiotic use to determine the efficacy of new therapies and may help determine the mechanistic action of novel drugs.
Bacterial density and airway inflammation have been measured from either expectorated sputum or bronchoalveolar lavage specimens (12–19). The application of either method is limited in studies of new CF therapies. Bronchoscopy requires general anesthesia or conscious sedation making it less acceptable to study subjects and clinical investigators. Moreover, there is little or no data on the reproducibility of inflammatory markers in bronchoalveolar lavage fluid. Although easier to obtain and generally more acceptable to subjects, the cytopathologic quality of expectorated sputum samples is poor. In addition, subjects with mild disease and children are often unable to produce spontaneously expectorated sputum.
Sputum induction is a method of sampling lower airway secretions that may be useful to study airway infection and inflammation in CF. Induced sputum is used to detect pathogens in human immunodeficiency virus–infected patients and has been validated for studies of airway inflammation in asthma (20) and chronic obstructive pulmonary disease (21–23). Recently, pilot studies have demonstrated the feasibility of sputum induction in CF (24–30). Preliminary studies in stable, adult patients with CF suggested that induced sputum samples appeared to be equally sensitive compared with expectorated sputum or bronchoalveolar lavage samples in detecting bacterial pathogens (26). Bronchospasm, the only significant adverse effect of the procedure, occurred in up to 7% of the subjects in these pilot studies (24, 25, 27, 29).
Determination of the usefulness of microbiologic and inflammatory markers in induced sputum as outcome measures in CF requires that they be studied during an established, effective therapeutic intervention. Treatment of acute pulmonary exacerbations in CF is known to reduce bacterial density and inflammatory markers in expectorated sputum (31, 32). The hypothesis of this study was that markers of infection and inflammation in induced sputum samples would be reduced after intravenous antibiotic therapy for a pulmonary exacerbation. Safety of the sputum induction procedure was also investigated. The results of these studies have been reported previously in the form of an abstract (33).
Subjects were recruited from eight CF Foundation Therapeutics Development Network centers. Adult subjects and parents of those less than 18 years of age signed consents approved by Institutional Review Boards. Inclusion criteria were a diagnosis of CF, age greater than 6 years, experience of pulmonary exacerbation (defined as having at least three of the following: increased cough or sputum, fever, weight loss, absenteeism from school or work due to illness, tachypnea, new findings on chest examination, decreased exercise tolerance, decreased pulmonary function tests or oxyhemoglobin saturation, or a new finding on chest radiograph) (34), treatment with at least two intravenous antibiotics (recommended by the treating physician) for a minimum of 9 days, and ability to perform spirometry reproducibly. Exclusion criteria included FEV1 less than 40% predicted, oxyhemoglobin saturation less than 92% on room air, pneumothorax, hemoptysis, and history of Burkholderia cepacia in sputum. Initially, subjects were excluded if tobramycin inhalation was prescribed during the study, later this criterion was removed to facilitate enrollment.
This was a prospective study in subjects with CF who received intravenous antibiotics in addition to routine therapies for an acute pulmonary exacerbation. Objectives were to examine changes in microbiology, white cell count, neutrophil count, and interleukin (IL)-8 and neutrophil elastase concentrations in induced sputum after intravenous antibiotic therapy and to determine safety of sputum induction. Sputum induction occurred before administration of the second dose of each intravenous antibiotic and within 2 days after completion of antibiotics. Subjects were defined as expectorators (if they could spontaneously expectorate sputum at the end of intravenous antibiotic therapy) or as nonexpectorators. Oximetry and spirometry were obtained before and after sputum induction. Adverse events were recorded. Subjects were withdrawn if systemic steroids were added or if changes in their chronic systemic steroid dose occurred during the study, if oxyhemoglobin saturation decreased to less than 88% or FEV1 decreased to 80% or less of baseline after sputum induction, or if the induced sputum sample was inadequate.
Inhaled antibiotics were withheld for 8 hours before sputum induction. Induced sputum samples were obtained using 3% hypertonic saline, as described previously, with minor modifications (25–27). At each 2-minute interval, subjects were instructed to expectorate sputum into one of two containers (one for microbiology and another for inflammatory markers) so that dithiothreitol was not present in the aliquot for microbiology.
The aliquot used to measure bacterial density was shipped overnight to the Therapeutics Development Network (TDN) Microbiology Core Laboratory and processed within 48 hours, as described previously (35). At each site, the aliquot used to measure inflammatory markers was liquefied, as described previously (25), and an aliquot used for total cell count. Cell differentials were performed at the TDN Cytology Core Laboratory, as described previously, with minor modifications (36). IL-8 concentration and neutrophil elastase activity were measured at the TDN Inflammatory Mediator Core Laboratory, as described previously (27).
Subjects who completed both sputum induction procedures were included in the analyses involving changes in pulmonary function, microbiology, and inflammatory markers. All subjects who performed a sputum induction procedure were included in the safety analyses. Data were log-transformed for analysis, as appropriate. Paired one-sample t tests were used to determine whether the changes in each end point were statistically significant. Data are presented as mean ± SD. Statistical tests were two sided, and significance was determined at the 0.05 level. Analyses were performed using SAS 8.02 and SPLUS 2000.
Seventy-two subjects with CF (38 females; 40 expectorators) were enrolled in the study. Of these, 55 (76%) completed both study visits (31 females; 30 expectorators). The 55 subjects who completed the study were 18.2 ± 7.9 years old (range from 8 to 43 years), and the baseline FEV1 was 2.1 ± 0.8 L (% predicted FEV1 was 71.4 ± 17.4%). Seventeen subjects (24%) did not complete the study because they were unable to produce an adequate sample of induced sputum at Visit 1 (n = 5), corticosteroid therapy was started or changed (n = 3), and FEV1 decreased by greater than 20% during the first sputum induction procedure (n = 3). The clinical characteristics of the excluded subjects did not differ from those of the included subjects. Other subjects withdrew because of hemoptysis (n = 1), subject decision (n = 1), tobramycin inhalation was prescribed for treatment of exacerbation (n = 1), inability to perform spirometry (n = 1), and time constraint (n = 2). Duration of intravenous antibiotic therapy was 14.8 ± 4.7 days (range 9–34 days), and there were 15.2 ± 4.7 days between study visits. Of the 55 subjects who completed both study visits, all received two to three intravenous antibiotics, six (11%) had additional oral antibiotics, and six (11%) had additional aerosolized antibiotics. Of the 55 subjects treated with intravenous antibiotics, 49 (89%) were treated with an aminoglycoside, 17 (31%) with an anti-Pseudomonal penicillin, 31 (56%) with a cephalosporin, 13 (24%) with a carbapenem, 4 (7%) with a monobactam, 4 (7%) with a fluoroquinone, 3 (5%) with a vancomycin, 6 (11%) with a colistin, 4 (7%) with an anti-Staphylococcal penicillin, and 2 (4%) with a clindamycin, 2 (4%). Of the six subjects treated with oral antibiotics, three (5%) were treated with a fluoroquinone, two (4%) with a sulfonamide, and one (2%) with a tetracycline. Of the subjects treated with aerosolized antibiotics, four (7%) were treated with inhaled tobramycin and two (4%) with colistin.
The change in FEV1 with therapy was 0.3 ± 0.3 L, with a range of −0.2 to 1.1 L (change in % predicted FEV1 was 10.4 ± 8.7%, range −5.8 to 32.2%), p < 0.0001.
Bacterial density results from both sputum induction procedures are available in 40 of 55 subjects (73%). A total of 29 of 40 subjects (73%) had Pseudomonas aeruginosa isolated from induced sputum before administration of intravenous antibiotics. Among these 29 subjects, the density of P. aeruginosa in induced sputum decreased significantly from 7.3 ± 1.5 log10 cfu/g before intravenous antibiotics to 4.9 ± 3.2 log10 cfu/g after intravenous antibiotics, p < 0.0005 (Figure 1A)
Figure 1. Decrease in density of (A) Pseudomonas aeruginosa (n = 29) and (B) Staphylococcus aureus (n = 18) in induced sputum after 15 days of intravenous antibiotic therapy. In each box plot, the bottom and the top of the box denote the 25th and 75th percentiles, respectively, the solid line the median, and the brackets the 10th and 90th percentiles, respectively; the dot within the box denotes the mean.
[More] [Minimize]Mean indices of all inflammatory markers including total white cell count, neutrophil count, percent neutrophils, and levels of IL-8 and neutrophil elastase in induced sputum decreased significantly after intravenous antibiotic therapy (Table 1)
Visit 1 Before Therapy (Mean ± SD) | Visit 2 After Therapy (Mean ± SD) | Change Before and After Therapy (Mean ± SD) | |
|---|---|---|---|
| White cell count, log10 cells/ml (n = 45) | 7.0 ± 0.4 | 6.7 ± 0.6 | 0.4 ± 0.6* |
| Neutrophil count, log10 cells/ml (n = 45) | 6.9 ± 0.4 | 6.6 ± 0.6 | 0.4 ± 0.6* |
| Neutrophils, % (n = 47) | 70.4 ± 19.7 | 61.7 ± 24.4 | 8.7 ± 21.3† |
| IL-8, log10 pg/ml (n = 42) | 4.8 ± 0.8 | 4.3 ± 1.5 | 0.5 ± 1.3‡ |
| Free elastase, log10 μg/ml (n = 38) | 1.9 ± 0.7 | 1.5 ± 0.9 | 0.4 ± 0.7§ |
All 72 subjects on whom at least one sputum induction was performed were included in the safety analyses (n = 127 sputum induction procedures). The most frequently reported adverse events were: wheezing in 12 (17%) subjects, coughing in 7 (10%) subjects, and chest tightness in 6 (8%) subjects. FEV1 decreased by 20% or more in three (4%) subjects at Visit 1 and in four (7%) subjects at Visit 2. The greatest decrease in FEV1 observed among these subjects was 46%. Decreases in FEV1 improved after bronchodilators in all instances. Sputum induction produced increases in FEV1: +0.1 ± 0.2 L (% predicted FEV1 4.6 ± 8.4%) and +0.2 ± 0.2 L (% predicted FEV1 6.8 ± 6.0%) at Visits 1 and 2, respectively. Oxyhemoglobin saturation did not change significantly in any subject.
We found that treatment of a pulmonary exacerbation in CF resulted in improvements in FEV1 that were associated with changes in several markers in induced sputum. Specifically, we observed reductions in P. aeruginosa and S. aureus density, neutrophil count, IL-8 concentration, and neutrophil elastase activity. We also found that sputum induction was relatively safe in subjects with CF even during an acute pulmonary exacerbation.
There are limited data on pulmonary function and markers in sputum before and after treatment of a pulmonary exacerbation in CF. Redding and coworkers (37) first showed that FVC and FEV1 improved significantly in 17 subjects after approximately 2 weeks of intravenous antibiotics, chest physiotherapy, and bronchodilators. Smith and coworkers (31) found that after approximately 2 weeks of intravenous antibiotic therapy, FEV1 improved by 26% in 11 subjects with systemic manifestations of infection (fever and leukocytosis) and by 12% in 64 subjects with a pulmonary exacerbation and no systemic manifestations while density of P. aeruginosa decreased significantly by 1.8 log10 cfu/g in both groups. Regelmann and coworkers (32) found that a 16% increase in FEV1 and decrease in density of P. aeruginosa in expectorated sputum of 2 log10 cfu/g was seen in seven subjects treated with 14 days of intravenous antibiotics, whereas no improvement in FEV1 or change in bacterial density was seen in the placebo group. Thus, our study supports the use of markers in induced sputum as outcome measures in CF studies by demonstrating that a significant improvement in pulmonary function is associated with a decrease in quantitative microbiology, as one would expect from intravenous antibiotics in a CF population. Our study provides important additional data on the effect of intravenous antibiotics on pulmonary function in the treatment of a pulmonary exacerbation in CF.
This is the first study to examine changes in neutrophil count and concentrations of IL-8 and neutrophil elastase in lower airway secretions obtained by sputum induction from subjects with CF before and after intravenous antibiotic therapy. Smith and coworkers (31) found that the concentration of DNA in expectorated sputum decreased significantly by approximately 2.0 mg/g. This reduction in sputum DNA is consistent with our observed reduction in total neutrophil count and other markers of airway inflammation. Alternatively, Wolter and coworkers (38) found no change in the levels of IL-8 in sputum obtained from subjects with CF at the beginning and during the course of a pulmonary exacerbation, though they did find a trend toward decreased levels of neutrophil elastase complex with α-1–protease inhibitor. The small sample size (n = 10), the lack of a definitive time point for collection of the second sample (range 2 to 10+ days), and the failure to use a paired t test in the analysis might explain the discrepancies between our findings and those of that study. Although the subjects in our study experienced an improvement in symptoms and pulmonary function, this was associated with a modest change in inflammatory markers in induced sputum. The approximate 0.5 log decrease in markers of inflammation in induced sputum may serve as the benchmark for the evaluation of other antibacterial and antiinflammatory therapies. It is possible that potent antiinflammatory therapies or immunomodulators could result in greater decreases in lower airway inflammation compared with this trial of antibacterial therapy.
Although the absolute total cell count decreased, the percent neutrophil count remained high (62%) despite a decrease in bacterial load. Controversy exists over the role of infection and inflammation in the CF lung. Some investigators have shown that airway inflammation is present without evidence of ongoing infection (39) and that airway infection has been associated with changes in lung function, whereas airway inflammation is not (40). In addition, antibiotic therapy in young children with CF has been associated with decreased bacterial infection but not with change in airway inflammation (5). Other investigators have shown that bacterial infection precedes development of airway inflammation (41) and that bacterial density correlates with the degree of inflammation (42). Regardless of whether infection or inflammation comes first, it is thought that the CF epithelium has an exaggerated response to infection and that it persists long after the stimulus is withdrawn (43). Thus, the persistence of neutrophil-dominated airway inflammation was not unexpected in our study of subjects with CF with well established infection.
Induced sputum is superior to expectorated sputum because not all subjects with CF were able to expectorate sputum. A volume of approximately 1 ml of sputum is required for both quantitative microbiology and for inflammatory markers. Sputum induction allows the potential enrollment of a larger number of study subjects. In our study, almost half of the subjects completing both visits did not expectorate regularly, and thus we had almost twice the number of subjects for analysis.
Bronchospasm, defined as a greater than 20% decline in FEV1, occurred in 7% of the sputum induction procedures and was the primary complication. Bronchodilator therapy reversed the bronchospasm on all occasions. The incidence of bronchospasm is similar to that reported previously in pilot studies with stable subjects with CF (24, 25, 27, 29). Subjects with poor lung function (FEV1 40–60% predicted value) tolerated the procedure as well as those with better lung function (FEV1 > 60% predicted value). Wheezing and cough were also observed, and there were no serious adverse events. We conclude that sputum induction is relatively safe in subjects with CF and does not appear to be associated with larger falls in spirometric values than observed in asthmatic populations.
Some induced sputum samples were insufficient to allow measurement of all study end points. It is important for the personnel coaching study subjects to understand that the sputum induction procedure is not a passive one for either the investigator or the study subject. The subject has to be encouraged and coached to produce adequate samples. In the design of studies using sputum induction, investigators should prioritize the assays performed on induced sputum samples in the eventuality that it may not be possible to collect an adequate volume of sputum from subjects.
These results should be interpreted with several limitations. First, only subjects with an FEV1 of 40% or greater were eligible to participate in this study. Thus, the safety of the procedure is not known for subjects with a lower FEV1. Although sputum induction appears relatively safe in CF, caution must still be exercised in its application. Second, we did not verify whether the intravenous antibiotics chosen were ones to which the subject's bacterial organism(s) had demonstrated in vitro susceptibility. We relied on clinicians to choose antibiotics appropriate for the subject's bacterial organisms. This omission would bias the study toward a negative result, not a positive one as seen in this study. Third, there was no control group that did not receive antibiotics. Our study was designed as an observational study. However, if the change we detected in induced sputum was related to the inherent variability of the markers measured, we would not have expected to find a statistically significant difference in all the markers measured.
In summary, we demonstrated that sputum induction is a useful, noninvasive technique to measure biomarkers in assessing response to an antibacterial intervention. Furthermore, our findings suggest that sputum induction may also be useful in assessing a response to antiinflammatory interventions in CF clinical trials. The procedure is relatively well tolerated by subjects with CF and can be used to obtain lower airway secretions from subjects with a wide range of lung disease severity. Given the advantages of induced sputum over other methods to measure biomarkers, we are hopeful that it will improve the implementation of trials evaluating new CF therapies.
The authors acknowledge the support provided by the TDN Microbiology Core Laboratory at Children's Hospital and Regional Medical Center, Seattle, WA, the TDN Inflammatory Mediators Core Laboratory, University of Colorado, Denver, CO, the CFF TDN Statistical Analysis Unit and the TDN Cytology Core Laboratory, Case Western Reserve University, Cleveland, OH; the technical assistance of Jay Hilliard, B.S., Iris Osberg, M.T., Maricela Pier, B.S., Heidi Caron, M.S.; and the coordinating services of Mary Haelsen, B.A., and the research coordinators at each site. Most importantly, the authors thank the subjects and their families for taking the time to participate in this study.
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