JCF-01157; No of Pages 8
Journal of Cystic Fibrosis xx (2015) xxx – xxx www.elsevier.com/locate/jcf
Original Article
Factors associated with response to treatment of pulmonary exacerbations in cystic fibrosis patients Valerie J. Waters a,⁎,1 , Sanja Stanojevic b,⁎, Nicole Sonneveld b , Michelle Klingel b , Hartmut Grasemann b , Yvonne C.W. Yau c , Elizabeth Tullis d , Pearce Wilcox e , Andreas Freitag f , Mark Chilvers g , Felix A. Ratjen b a
Division of Infectious Diseases, Department of Pediatrics, Hospital for Sick Children, University of Toronto, Toronto Division of Respiratory Medicine, Department of Pediatrics, Hospital for Sick Children, University of Toronto, Toronto c Division of Microbiology, Department of Pediatric Laboratory Medicine, Hospital for Sick Children, University of Toronto, Toronto Division of Respirology and Keenan Research Centre of Li Ka Shing Knowledge Institute, Department of Medicine, St. Michael’s Hospital, University of Toronto, Toronto e Division of Respiratory Medicine, Department of Medicine, St Paul’s Hospital, University of British Columbia, Vancouver f Division of Respiratory Medicine, Department of Medicine, Hamilton Health Sciences Center, McMaster University, Hamilton g Division of Respiratory Medicine, Department of Pediatrics, British Columbia Children’s Hospital, Vancouver b
d
Received 8 December 2014; revised 23 January 2015; accepted 23 January 2015 Available online xxxx
Abstract Background: Pulmonary exacerbations are associated with significant lung function decline from baseline in cystic fibrosis (CF) and it is not well understood why some patients do not respond to antibiotic therapy. The objective of this study was to identify factors associated with lung function response to antibiotic treatment of pulmonary exacerbations. Methods: As a secondary analysis of a randomized, controlled trial of intravenous antibiotic treatment for pulmonary exacerbations in CF patients, we investigated whether baseline factors and changes in sputum bacterial density, serum or sputum inflammatory markers were associated with recovery of lung function and risk of subsequent exacerbation. Results: In 36 of the 70 exacerbations (51%), patients’ lung function returned to N 100% of their baseline at day 14 of antibiotic treatment; 34 exacerbations were classified as non-responders. Baseline characteristics were not significantly different between responders and non-responders. Less of a drop in FEV1 from baseline to exacerbation (OR 1.09, 95% CI 1.0, 1.18, p = 0.04) as well as a greater decrease in sputum neutrophil elastase (OR 2.94, 95% CI 1.07, 8.06, p = 0.04) were associated with response to antibiotic treatment at day 14. In addition, higher CRP (HR 1.35 (95% CI: 1.01, 1.78), p = 0.04) and sputum neutrophil elastase (HR 1.71 (95% CI: 1.02, 2.88), p = 0.04) at day 14 of antibiotic therapy were associated with an increased risk of subsequent exacerbation. Conclusions: Inadequate reduction of inflammation during an exacerbation is associated with failure to recover lung function and increased risk of subsequent re-exacerbation in CF patients. © 2015 Published by Elsevier B.V. on behalf of European Cystic Fibrosis Society. Keywords: cystic fibrosis; pulmonary exacerbations; inflammation; antibiotics
⁎ Corresponding author at: Department of Pediatrics, Division of Infectious Diseases, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8. Tel.: +1 416 813 7654x204541. E-mail addresses: [email protected] (V.J. Waters), [email protected] (S. Stanojevic), [email protected] (N. Sonneveld), [email protected] (M. Klingel), [email protected] (H. Grasemann), [email protected] (Y.C.W. Yau), [email protected] (E. Tullis), [email protected] (P. Wilcox), [email protected] (A. Freitag), [email protected] (M. Chilvers), [email protected] (F.A. Ratjen). 1 both authors contributed equally to the manuscript.
http://dx.doi.org/10.1016/j.jcf.2015.01.007 1569-1993/© 2015 Published by Elsevier B.V. on behalf of European Cystic Fibrosis Society. Please cite this article as: Waters VJ, et al, Factors associated with response to treatment of pulmonary exacerbations in cystic fibrosis patients, J Cyst Fibros (2015), http://dx.doi.org/10.1016/j.jcf.2015.01.007
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V.J. Waters et al. / Journal of Cystic Fibrosis xx (2015) xxx–xxx
1. Introduction Cystic fibrosis (CF) is a disease characterized by recurrent flare-ups of respiratory symptoms, or pulmonary exacerbations, which directly contribute to lung function deterioration and ultimate mortality [1,2]. Despite antibiotic and other concomitant therapies for pulmonary infections, one quarter to one third of CF patients do not return to 90% of their baseline lung function following pulmonary exacerbations [3,4]. In addition, in a large cohort study of CF patients followed for up to 10 years, half of the decline in forced expiratory volume in 1 second (FEV1) was attributable to pulmonary exacerbations requiring intravenous (IV) antibiotics [2]. An increasing number of pulmonary exacerbations, specifically more than two per year, is also associated with a higher risk of death or lung transplantation in CF [1,5]. Although exacerbations clearly impact the clinical course of CF, it is not known why some patients respond to therapy for pulmonary exacerbations whereas others do not. Response to antibiotic treatment can be defined in a number of ways, including return of FEV1 to N 90% of baseline [3], full recovery of baseline FEV1 or as the change in FEV1 from the beginning to end of therapy. Risk factors such as female gender, persistent Pseudomonas aeruginosa, Burkholderia cepacia complex or methicillin-resistant Staphylococcus aureus (MRSA) infection and lower FEV1, have been associated with failure to recover lung function after an exacerbation but it is not clear through which mechanisms this occurs [3,6]. Intravenous antibiotic treatment has been shown to decrease sputum bacterial density and inflammatory responses during exacerbations which correlate overall with lung function improvements, but there are still patients that fail to respond to treatment [7,8]. Given that many therapies have overlapping functions (eg. anti-inflammatory, antibacterial), it is crucial to determine the main driver of clinical response in order to more effectively target the management of these exacerbations. Therefore, the aim of this study was to identify the factors, including change in microbiological, systemic and pulmonary markers of inflammation that were associated with clinical response to IV antibiotic treatment of pulmonary exacerbations, as measured by changes in lung function, as well as time to subsequent pulmonary exacerbation.
chronic infection with P. aeruginosa (N 50% of respiratory specimens positive in the 24 months prior to screening) [10] and the ability to produce sputum and to reproducibly perform pulmonary function testing. Exclusion criteria included a sputum culture negative for P. aeruginosa or with a density of less than 105 colony forming units (CFU)/ml at screening, history of B. cepacia positive respiratory culture within 24 months prior to screening, and post lung transplantation or listed for lung transplantation [11]. At the time of pulmonary exacerbation, defined as an increase in respiratory symptoms treated with IV antibiotics [12], each patient was randomized to 14 days of IV antibiotic treatment for P. aeruginosa pulmonary infection based on biofilm or conventional antimicrobial susceptibility results; at this time all other antibiotics were discontinued (including inhaled antibiotics and oral azithromycin). The results of the original randomized control trial (Clinicaltrials.gov NCT 00786513; Supplementary Materials) [9] showed no difference between the two treatment groups therefore data from all the patients with pulmonary exacerbations were pooled for the current analysis. 2.2. Study outcomes
2. Materials and Methods
Lung function measurements were performed according to American Thoracic Society criteria [13], and absolute values were converted to percent predicted using the Global Lung Function Initiative-2012 reference equation [14]. Baseline lung function was defined as the last pulmonary function tests done when the patient was clinically stable (ie. not on any antibiotics) prior to randomization for pulmonary exacerbation. Spirometry was measured on the first day of antibiotic treatment (day 0), after 14 (+ 2) days of antibiotic treatment, and at the follow up clinic visit when the patient was deemed clinically stable and no longer receiving IV antibiotic treatment. Response was defined primarily as recovery of N 100% of baseline FEV1 at day 14 of antibiotic therapy. Maintenance (for responders) or recovery (for non-responders) of lung function at the follow up visit was defined as an FEV1 of N 100% of baseline FEV1. Lung function response was secondarily analyzed as absolute change in FEV1 from day 0 to day 14 of antibiotic treatment. Time to subsequent pulmonary exacerbation was calculated from the last day of IV antibiotic therapy to the first day of re-starting IV antibiotic treatment for a new pulmonary exacerbation.
2.1. Study design and patient population
2.3. Factors associated with response
This was a secondary analysis of a randomized, doubleblind study of IV antibiotic treatment based on biofilm vs planktonic susceptibility testing of pulmonary exacerbations in CF patients with chronic P. aeruginosa infection conducted at five centers in Canada (Hospital for Sick Children, Toronto; St Michael’s Hospital, Toronto; Hamilton Health Sciences Center, Hamilton; British Columbia Children’s Hospital, Vancouver; St Paul’s Hospital, Vancouver) from January 2009 to December 2013 [9]. Patients were screened and enrolled during a stable clinic visit. Inclusion criteria included diagnosis of CF,
Baseline demographic characteristics were collected at the enrollment visit. Blood inflammatory markers (white blood cell count (WBC) (109/L), C-reactive protein (CRP) (mg/L), erythrocyte sedimentation rate (ESR) (mm/h)) were collected at the start of the exacerbation and after 14 days of treatment. Sputum bacterial density and sputum inflammatory markers were also collected at a stable follow-up visit (data not shown). Sputum bacterial density, sputum inflammatory markers (interleukin-8 (IL-8) and neutrophil elastase (NE)) (collected only for patients from Hospital for Sick Children, St Michael’s
Please cite this article as: Waters VJ, et al, Factors associated with response to treatment of pulmonary exacerbations in cystic fibrosis patients, J Cyst Fibros (2015), http://dx.doi.org/10.1016/j.jcf.2015.01.007
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Hospital and Hamilton Health Sciences Center given processing requirements) were performed as previously described [15]. Sputum bacterial density was done on solubilized sputum by mixing 0.5 mL of sputum with 0.5 mL of sputolysin (Calbiochem, La Jolla, CA) [16]. Serial 1:10 dilutions were made in PBS (pH 7.0) and 100 μL were plated onto MacConkey agar (Oxoid, Nepean, ON). Plates were incubated at 42 °C aerobically for 48 hours to select for P. aeruginosa. Sputum density results were reported as log10 colony forming units (CFU)/mL. 2.4. Statistical analysis Prior to analysis, CRP, WBC and ESR values were log transformed using a natural logarithm; IL-8, NE and bacterial density were transformed using log10. Change in each of these variables was defined as the difference of the transformed values (or the ratio of the untransformed values) from day 0 to day 14. The units for each variable were: WBC (109/L), ESR(mm/h), CRP (mg/L), density (CFU/ml), IL-8 (ng/ml) and NE (μg/ml). Lung function responders and non-responders at day 14 were compared using a t-test for continuous variables or a Chi-square for categorical variables (equivalent non-parametric tests, Mann-Whitney for non-normally distributed data or Fisher’s exact test for small sample sizes, were used where appropriate). Univariable logistic regression analysis comparing the mean change in explanatory variables (i.e. sputum P. aeruginosa density and markers of inflammation in blood and sputum) between responders and non-responders was performed using a generalized estimating equation model to adjust for the repeated exacerbations in individual patients; odds ratios with confidence intervals are presented. The odds ratio represents the odds of responding for every unit of change in the explanatory variable (e.g. 1 log decrease in sputum density). A second linear model was also performed with absolute change in FEV1 (L) from day 0 to day 14 as the outcome variable, adjusting for the same covariates as above. Multivariable models were built in a step-wise manner adding variables that were significant on univariable analysis. In addition, factors associated with time to next pulmonary exacerbation in the entire study cohort were examined using Cox-Proportional Hazard Models. Hazard ratios were reported and represent the hazard of developing a subsequent pulmonary exacerbation treated with intravenous antibiotics during the study period for every unit of change in the explanatory variable (eg. 1 log increase in sputum neutrophil elastase). Subjects were censored at the end of the original randomized controlled trial after a maximum follow-up period of 3.7 years. All statistical analyses were performed using STATA V12.
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above baseline at day 14 of IV antibiotic treatment in 36 of the 70 exacerbations (51%). Of the 36 total patients, 16 (44%) had more than 1 exacerbation; of those subjects, 56% (n = 9/16) fulfilled response criteria at the first exacerbation of which, 33% (n = 3/9) fulfilled response criteria with all subsequent exacerbations. The absolute change in FEV1 (L) from baseline to day 14 was 0.20 L (95% CI 0.12, 0.28) in responders compared to -0.27 L (95% CI -0.35, -0.19) among the non-responders (p b 0.0001). Baseline characteristics showed few statistically significant differences between the responders and non-responders for both the first exacerbation and all exacerbations (Table 1 and Table 2). Non-responders had a greater drop in FEV1 from baseline to day 0 of the exacerbation (-12.4 %, range -35.7; -0.9) versus responders: (-5.3%, range -19.1; 7.1, p = 0.05) and a lower FEV1 at exacerbation (34.0%, range 18.6; 79.5) compared to responders (49.8%, range 27.3; 79.5, p = 0.02) (Table 1). In addition, non-responders had a higher serum WBC (p = 0.04) and higher sputum P. aeruginosa bacterial density (p = 0.02) at exacerbation compared to non-responders. Intravenous tobramycin and ceftazidime was the most commonly used combination in both groups. The proportion of exacerbations where antibiotic treatment was changed at day 7 of therapy was similar in both groups (responders 5/36, 14%; non-responders 3/34, 9%, p = 0.51). 3.2. Variables associated with response at day 14 of antibiotic treatment
3. Results
Changes in blood markers of inflammation were not associated with treatment response at day 14 (Table 3). Responders demonstrated a decrease in median log transformed sputum neutrophil elastase (-140.9 μg/mL (range -1106.7; 158.1)), whereas non-responders had only moderate decreases (-30.1 μg/mL (range -826.9; 421.5)) (p = 0.02).In the final multivariable analysis, after adjusting for other covariates significant on univariate analysis, less of a drop in FEV1 from baseline to exacerbation (OR 1.09, 95% CI 1.0, 1.18, p = 0.04) as well as a greater decrease in sputum NE (OR 2.94, 95% CI 1.07, 8.06, p = 0.04) were independently associated with response to IV antibiotic treatment at day 14. In a multivariate analysis using the previously published definition of treatment response as return to N 90% of baseline FEV1 [3], decrease in sputum NE remained significantly associated with treatment response at day 14 (OR 8.11, 95% CI 1.14, 57.60, p = 0.04). Additionally, a linear regression model using absolute change in FEV1 (L) from day 0 to day 14 as the outcome measure, adjusting for similar covariates, was calculated. Similar to the previous results, less of a drop in FEV1 from baseline to exacerbation (p b 0.001) and a greater decrease in sputum NE (p = 0.001) were both significantly associated with greater increases in lung function in the final multivariate model.
3.1. Lung function response and patient characteristics
3.3. Time to Subsequent Exacerbation
A total of 70 pulmonary exacerbations in 36 patients were included in the analysis (Fig. 1). Lung function was at 100% or
The median time to subsequent exacerbation was 132 days (range 0(censored)-1343 days). Higher CRP values at day 14 of
Please cite this article as: Waters VJ, et al, Factors associated with response to treatment of pulmonary exacerbations in cystic fibrosis patients, J Cyst Fibros (2015), http://dx.doi.org/10.1016/j.jcf.2015.01.007
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Fig. 1. Flowchart of clinical outcomes of study cohort after treatment for pulmonary exacerbations. Responders: recovered N 100% of baseline FEV1, n = number of exacerbations, F/U: follow up, FEV1: forced expiratory volume in 1 second, AB txn: antibiotic treatment.
antibiotic therapy (HR 1.35 (95% CI: 1.01, 1.78), p = 0.04) and higher NE levels at day 14 (HR 1.71 (95% CI: 1.02, 2.88), p = 0.04) were associated with a greater risk of subsequent exacerbation during the study period. 4. Discussion In this study, we assessed both blood and sputum measures as markers of response to IV antibiotic therapy during pulmonary exacerbations in CF patients. Greater reduction in sputum neutrophil elastase was independently associated with recovery of lung function after 14 days of IV antibiotic therapy based on two different definitions of response. In addition, higher serum CRP and sputum neutrophil elastase at day 14 were associated with increased risk of subsequent exacerbation. Previous studies have demonstrated that a quarter to a third of CF patients do not recover N 90% baseline FEV1 after exacerbations [3,4]. However, the timing of when this loss of lung function occurs is less clear, as most large epidemiologic studies addressing this question use a large window, typically 3 months, to define post-exacerbation FEV1, based on available measurements in registries [3,4]. We primarily used a more stringent definition of 100% recovery of baseline lung function to assess response to antimicrobial treatment of pulmonary exacerbations; only 50% of exacerbations met this criteria. Interestingly, a significant proportion of patients who did not regain lung function at 14 days, did so at the follow up visit. This demonstrates the difficulty in defining treatment response (or failure thereof) after treatment of pulmonary exacerbations as the response rate changes depending on the definition and
time point chosen. Previous definitions of treatment response based on returning to at least 90% of baseline FEV1, rather than recovering full (100%) baseline lung function, may overestimate responders. However, we also confirmed our findings using the previously published model of treatment response as N 90% of baseline FEV1 [3]. Several investigators have examined the reasons for failing to recover lung function after treatment of pulmonary exacerbations. Female gender, worse nutritional status and greater drop in FEV1 from baseline to exacerbation have all been identified as risk factors for failing to recover lung function, but none of these factors are actually modifiable at the time of treatment of the exacerbation [3,17]. Studies that have investigated mechanisms by which these failures may occur have focused on serum markers of inflammation (e.g. CRP and WBC), measured either at the beginning or the end of antibiotic therapy, and showed that higher levels are associated with treatment failure [6]. Variables predicting response cannot be examined in isolation, however, as IV antibiotic treatment is known to have multiple effects in CF patients during pulmonary exacerbations including decreasing sputum bacterial density, neutrophil counts, IL-8 and neutrophil elastase [7]. Changes in these variables correlate, to varying degrees, with improvements in FEV1 but how the changes in these variables predict failure of treatment in any given patient is not known [8,18]. The current study is the first, to our knowledge, to identify greater decreases in sputum neutrophil elastase as a factor independently associated with lung function recovery after two weeks of IV antibiotic therapy. The effects of treatment of pulmonary exacerbations extend beyond the short term. Elevated levels of both systemic (CRP)
Please cite this article as: Waters VJ, et al, Factors associated with response to treatment of pulmonary exacerbations in cystic fibrosis patients, J Cyst Fibros (2015), http://dx.doi.org/10.1016/j.jcf.2015.01.007
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Table 1 Patient characteristics at first exacerbation. P-values indicate the difference between responders and non-responders and are obtained using a t-test for normally distributed continuous variables; Mann-Whitney test for non-normally distributed continuous variables; a Chi-squared test for binary outcomes and a Fisher’s exact test for binary outcomes with small values.
No. of exacerbations/patient during study period, median (range) Age at exacerbation, yrs, median (range) Female, n (%) Homozygous ΔF508, n (%) FEV1 % pred, median (range) Baseline At exacerbation Δ baseline-exacerbation Days from baseline-exacerbation BMI z-score, median (range) Baseline At exacerbation Serum measures at exacerbation, median (range) WBC (109/L) ESR(mm/h) CRP (mg/L) Sputum measures at exacerbation, median (range) n Density (log10 CFU/ml) IL-8 (ng/ml) NE (μg/ml) Complications at enrollment CFRD, n (%) Liver disease, n (%) Pancreatic insufficiency, n (%) ABPA, n (%) Maintenance treatment at exacerbation Dornase alfa, n (%) Azithromycin, n (%) Inhaled tobramycin, n (%) Hypertonic saline, n (%) Other inhaled antibiotics, n (%)
Total N = 36
Responders N = 16
Non-Responders N = 20
P value
1 (1;5) 23.9 (10.1;49.2) 14 (38.9) 20 (57.1) 53.7 (22.2; 93.3) 41.0 (18.6; 79.5) -10.0 (-35.7; 7.1) 61.5 (12; 423)
2 (1;5) 18.1 (10.1;48.0) 7 (43.8) 10 (62.5) 59.4 (30.3; 88.4) 49.8 (27.3; 79.5) -5.3 (-19.1; 7.1) 83 (12; 423)
1 (1;5) 26.1 (11.3;49.2) 7 (35.0) 10 (52.6) 50.9 (22.2; 93.3) 34.0 (18.6; 79.5) -12.4 (-35.7; -0.9) 54 (23; 309)
0.28 0.33 0.59 0.56 0.44 0.02 0.05 0.49
-0.60 (-4.67; 1.49) -0.57 (-3.37; 1.24)
-0.44 (-4.67; 1.49) -0.55 (-3.37; 1.24)
-0.63 (-2.27; 0.72) -0.87 (-3.07; 0.65)
0.76 0.36
11.6 (3.8; 22.2) 28 (1; 91) 12.8 (1.1; 165.7)
10.5 (3.8; 16.9) 14 (1; 91) 6.1 (1.6; 137.2)
13.0 (5.2; 22.2) 36 (1; 77) 18.2 (1.1; 165.7)
0.04 0.14 0.11
26 7.0 (4.1 - 8.6) 191.5 (39.0; 672.9) 177.0 (42.7;1193.5)
12 7.5 (5.8 - 8.6) 244.1 (82.9; 672.9) 208.0 (49.4;1193.5)
14 6.8 (4.1 - 8.4) 167.9 (39.0; 416.9) 149.4 (42.7;913.7)
N/A 0.02 0.26 0.47
11 (30.6) 3 (8.3) 34 (94.4) 5 (13.9)
5 (31.3) 3 (18.8) 15 (93.8) 3 (18.8)
6 (30.0) 0 (0.0) 19 (95.0) 2 (10.0)
0.94 0.08 0.87 0.64
18 (50.0) 17 (47.2) 29 (80.6) 6 (16.7) 4 (11.1)
8 (50.0) 5 (31.3) 12 (75.0) 2 (12.5) 3 (18.8)
10 (50.0) 12 (60.0) 17 (85.0) 1 (5.0) 1 (5)
0.99 0.09 0.45 0.30 0.30
FEV1: forced expiratory volume in 1 second BMI: body mass index CFRD: cystic fibrosis related diabetes ABPA: allergic bronchopulmonary aspergillosis Δ: change N/A: not applicable
and pulmonary (NE) markers of inflammation after 14 days of IV antibiotics in our study cohort were associated with an increased risk of subsequent exacerbations; shorter time between exacerbations is a significant determinant of overall lung function decline in CF [2]. Previous studies have focused on blood-based biomarkers, especially CRP, as predictors of pulmonary exacerbation but have not analyzed the contribution of pulmonary markers of inflammation [19–21]. These markers are relevant given that multiple studies have shown that increased pulmonary neutrophil elastase activity is the most predictive biomarker for subsequent lung function decline and the development of bronchiectasis in young children with CF [22,23]. The observation that non-responders had higher pulmonary neutrophil elastase activity, associated with negative long-term consequences, raises the question as to whether this indicates ongoing primary inflammation or inflammation secondary to inadequately treated infection. This highlights the difficulty in
developing statistical models with variables that may be on the same causal pathway. It is possible that greater levels of infection lead to persistent pulmonary inflammation and worse lung function, although in our study, higher bacterial density measures at the start of treatment were noted in responders. Whether this reflects exacerbations in which infection is playing a primary role and are thus more likely to respond to antibiotic therapy, is unclear. Although this study cannot directly address the mechanistic role of sputum and serum biomarkers, only changes in sputum neutrophil elastase were independently and significantly associated with FEV1 response in the final multivariate model. There are a limited number of anti-inflammatory therapies available for use in CF patients, with varying degrees of efficacy. Drugs such as such as alpha-1 antitrypsin, an endogenous inhibitor of neutrophil elastase, have been shown to suppress neutrophil elastase in the respiratory epithelial lining fluid (ELF) [24]. Although subsequent randomized,
Please cite this article as: Waters VJ, et al, Factors associated with response to treatment of pulmonary exacerbations in cystic fibrosis patients, J Cyst Fibros (2015), http://dx.doi.org/10.1016/j.jcf.2015.01.007
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Table 2 Characteristics of all exacerbations.
FEV1 % pred, median (range) Baseline At exacerbation Δ baseline-exacerbation Days from baseline-exacerbation BMI z-score, median (range) Baseline At exacerbation Serum measures at exacerbation, median (range) WBC (109/L) ESR(mm/h) CRP (mg/L) Sputum measures at exacerbation, median (range) n Density (log10 CFU/ml) IL-8 (ng/ml) NE (μg/ml)
Total n = 70
Responders n = 36
Non-Responders n = 34
P value
59.4 (22.2; 99.4) 44.7 (17.4; 83.7) -10.0 (-35.7; 7.11) 76.5 (7; 423)
59.4 (30.3; 99.4) 47.6 (26.6; 80.1) -7.0 (-19.3; 7.1) 77.5 (7; 423)
58.7 (22.2; 93.3) 40.0 (17.4; 83.7) -12.4 (-35.7; 3.7) 71 (23; 309)
0.86 0.09 0.004 0.81
-0.67 (-4.7; 1.5) -0.77 (-3.5; 1.2)
-0.78 (-4.7; 1.5) -0.71 (-3.5; 1.2)
-0.58 (-3.5; 1.2) -0.85 (-3.3; 0.9)
0.29 0.67
11.1 (2.8; 22.2) 30 (1; 91) 12.8 (0.8; 165.7)
10.3 (2.8; 16.9) 20 (1; 91) 6.4 (1.6; 137.2)
12.5 (5.2; 22.2) 37 (1; 89) 21.1 (0.8 - 165.7)
0.02 0.35 0.14
49 7.0 (4.1; 8.6) 196.2 (25.6; 672.9) 201.2 (42.7; 1193.5)
26 7.6 (5.5; 8.6) 196.2 (31.6; 672.9) 208.0 (49.4; 1193.5)
23 6.7 (4.1; 8.4) 190.7 (25.6; 416.9) 179.8 (42.7; 986.3)
N/A 0.001 0.09 0.32
P-values indicate the difference between responders and non-responders. P-values for continuous variables are derived from linear and logistic generalized estimating equations (GEE) models to account for the repeated exacerbations in the same subject. Non-normally distributed data was transformed to the natural logarithm or using log10 before statistical analysis. FEV1: forced expiratory volume in 1 second BMI: body mass index CFRD: cystic fibrosis related diabetes ABPA: allergic bronchopulmonary aspergillosis Δ: change N/A: not applicable
placebo-controlled trials of inhaled recombinant human alpha-1 antitrypsin in adult CF patients failed to demonstrate a statistically significant reduction in neutrophil elastase activity [25], phase II studies of this drug, using amore clinically relevant primary outcome of pulmonary exacerbations, are
ongoing. Corticosteroids are another potent anti-inflammatory class of drugs known to inhibit neutrophilic accumulation at sites of inflammation [26]. They have been studied mostly as chronic suppressive medications in CF, however, long-term use has been limited by concerns of side effects [27]. A randomized
Table 3 Univariable logistic regression analysis for recovery of lung function at day 14 of antibiotic treatment.
Blood measures Δ WBC (109/L) Δ ESR(mm/h) Δ CRP (mg/L) Sputum measures n Δ Density(log10 CFU/ml) Δ IL-8 (ng/ml) Δ NE (μg/ml)
Responders n = 36 Median (range)
Non-Responders n = 34 Median (range)
Odds Ratio ⁎ (95% CI)
P value
-2.5 (-10.4; 14.1) -4.0 (-62; 18) -4.8 (-135.4; 5.3)
-1.9 (-14.7; 7.6) -2 (-70; 25) -15.3 (-155.8; 6.7)
0.59 (0.19, 1.85) 1.14 (0.72, 1.82) 1.02 (0.72, 1.45)
0.37 0.57 0.90
26 -2.17 (-8.30; 1.5) -66.5 (-411; 647.4) -140.9 (-1106.7; 158.1)
23 -1.42 (-6.3; 2.9) -6.0 (-299; 287.0) -30.1(-826.9; 421.45)
N/A 1.12 (0.94, 1.25) 3.85 (0.95, 16.67) 4.0 (1.26, 12.50)
N/A 0.21 0.06 0.02
P-values were derived from logistic generalized estimating equations (GEE) models to account for the repeated exacerbations in the same subject. The changes in WBC, ESR, CRP, IL-8 and NE were transformed as described in the method section prior to statistical analysis. CI: confidence intervals Δ: change from day 0 to day 14 of antibiotic treatment CFU: colony forming units WBC: white blood cell count ESR: erythrocyte sedimentation rate CRP: C reactive protein NE: neutrophil elastase N/A : not applicable ⁎ Odds Ratio indicates the odds for responding with greater decrease in values. Please cite this article as: Waters VJ, et al, Factors associated with response to treatment of pulmonary exacerbations in cystic fibrosis patients, J Cyst Fibros (2015), http://dx.doi.org/10.1016/j.jcf.2015.01.007
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controlled trial examining the short-term use of oral prednisone in all CF patients presenting with pulmonary exacerbations demonstrated a modest improvement in lung function [28]. Focusing this anti-inflammatory treatment on CF patients with exacerbations who are not responding to antibiotic treatment may maximize its potential benefit while minimizing harmful side effects. Additional anti-inflammatory agents, such as ibuprofen and azithromycin [29,30], have also been used in individuals with CF but their role in modifying the response during an acute pulmonary exacerbation has not yet been defined. This study had several limitations including the relatively small sample size (70 pulmonary exacerbations) which decreased the power to detect smaller effect sizes. Therefore, absence of statistically significant differences between patient groups did not necessarily mean that they were similar. In addition, incomplete long-term pulmonary function testing prohibited meaningful analysis of the four groups identified at follow up. Thus, although the majority of non-responders were treated with longer antibiotic courses and appeared to have recovered lung function at follow up, the small group sizes precluded any such conclusions. We also included repeated pulmonary exacerbations from the same patient which could bias the results but controlled for this in the analysis using generalized estimating equations accounting for repeated measures from the same individual. Finally, measures of bacterial density were limited to P. aeruginosa in this study population of CF patients with chronic P. aeruginosa infection; it is possible that changes in the composition of other members of the pulmonary microbiome may have influenced the response to antimicrobial treatment. However, none of the patients included in this analysis had B. cepacia complex or MRSA infection, previously identified as risk factors for failing to recover FEV1 following a pulmonary exacerbation. In summary, although limited in power due to a small sample size, this study demonstrates that inadequate reduction of pulmonary inflammation during an exacerbation is associated with failure to recover lung function and increased risk of subsequent re-exacerbation in CF patients. To improve our success in recovering lung function following exacerbations, we may need to target the underlying inflammatory process in the CF lung with adjuvant therapies in individuals not responding to antimicrobial treatment.
Authors’ contributions V Waters, YCW Yau, F Ratjen and E Tullis designed the study, participated in the data collection, analysis and interpretation, and wrote the paper. S Stanojevic, M Klingel and N Sonneveld did the statistical analysis and assisted with the writing of the manuscript. H Grasemann supervised the sputum processing and inflammatory marker measurements and assisted with the writing of the manuscript. P Wilcox, A Freitag and M Chilvers undertook the enrolment of patients, supervised treatment, participated in data collection and interpretation, and assisted with the writing of the manuscript.
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[21] Shoki AH, Mayer-Hamblett N, Wilcox PG, Sin DD, Quon BS. Systematic review of blood biomarkers in cystic fibrosis pulmonary exacerbations. Chest 2013;144(5):1659–70. [22] Sagel SD, Wagner BD, Anthony MM, Emmett P, Zemanick ET. Sputum biomarkers of inflammation and lung function decline in children with cystic fibrosis. Am J Respir Crit Care Med 2012;186(9):857–65. [23] Sly PD, Gangell CL, Chen L, Ware RS, Ranganathan S, Mott LS, et al. Risk factors for bronchiectasis in children with cystic fibrosis. N Engl J Med 2013;368(21):1963–70. [24] McElvaney NG, Hubbard RC, Birrer P, Chernick MS, Caplan DB, Frank MM, et al. Aerosol alpha 1-antitrypsin treatment for cystic fibrosis. Lancet 1991;337(8738):392–4. [25] Martin SL, Downey D, Bilton D, Keogan MT, Edgar J, Elborn JS. Safety and efficacy of recombinant alpha(1)-antitrypsin therapy in cystic fibrosis. Pediatr Pulmonol 2006;41(2):177–83.
[26] Parrillo JE, Fauci AS. Mechanisms of glucocorticoid action on immune processes. Annu Rev Pharmacol Toxicol 1979;19:179–201. [27] Cheng K, Ashby D, Smyth RL. Oral steroids for long-term use in cystic fibrosis. Cochrane Database Syst Rev 2013;6 CD000407. [28] Dovey M, Aitken ML, Emerson J, McNamara S, Waltz DA, Gibson RL. Oral corticosteroid therapy in cystic fibrosis patients hospitalized for pulmonary exacerbation: a pilot study. Chest 2007;132(4):1212–8. [29] Lands LC, Stanojevic S. Oral non-steroidal anti-inflammatory drug therapy for lung disease in cystic fibrosis. Cochrane Database Syst Rev 2013;6 CD001505. [30] Saiman L, Marshall BC, Mayer-Hamblett N, Burns JL, Quittner AL, Cibene DA, et al. Azithromycin in patients with cystic fibrosis chronically infected with Pseudomonas aeruginosa: a randomized controlled trial. JAMA 2003;290(13):1749–56.
Please cite this article as: Waters VJ, et al, Factors associated with response to treatment of pulmonary exacerbations in cystic fibrosis patients, J Cyst Fibros (2015), http://dx.doi.org/10.1016/j.jcf.2015.01.007
Journal of Cystic Fibrosis xx (2015) xxx – xxx www.elsevier.com/locate/jcf
Original Article
Factors associated with response to treatment of pulmonary exacerbations in cystic fibrosis patients Valerie J. Waters a,⁎,1 , Sanja Stanojevic b,⁎, Nicole Sonneveld b , Michelle Klingel b , Hartmut Grasemann b , Yvonne C.W. Yau c , Elizabeth Tullis d , Pearce Wilcox e , Andreas Freitag f , Mark Chilvers g , Felix A. Ratjen b a
Division of Infectious Diseases, Department of Pediatrics, Hospital for Sick Children, University of Toronto, Toronto Division of Respiratory Medicine, Department of Pediatrics, Hospital for Sick Children, University of Toronto, Toronto c Division of Microbiology, Department of Pediatric Laboratory Medicine, Hospital for Sick Children, University of Toronto, Toronto Division of Respirology and Keenan Research Centre of Li Ka Shing Knowledge Institute, Department of Medicine, St. Michael’s Hospital, University of Toronto, Toronto e Division of Respiratory Medicine, Department of Medicine, St Paul’s Hospital, University of British Columbia, Vancouver f Division of Respiratory Medicine, Department of Medicine, Hamilton Health Sciences Center, McMaster University, Hamilton g Division of Respiratory Medicine, Department of Pediatrics, British Columbia Children’s Hospital, Vancouver b
d
Received 8 December 2014; revised 23 January 2015; accepted 23 January 2015 Available online xxxx
Abstract Background: Pulmonary exacerbations are associated with significant lung function decline from baseline in cystic fibrosis (CF) and it is not well understood why some patients do not respond to antibiotic therapy. The objective of this study was to identify factors associated with lung function response to antibiotic treatment of pulmonary exacerbations. Methods: As a secondary analysis of a randomized, controlled trial of intravenous antibiotic treatment for pulmonary exacerbations in CF patients, we investigated whether baseline factors and changes in sputum bacterial density, serum or sputum inflammatory markers were associated with recovery of lung function and risk of subsequent exacerbation. Results: In 36 of the 70 exacerbations (51%), patients’ lung function returned to N 100% of their baseline at day 14 of antibiotic treatment; 34 exacerbations were classified as non-responders. Baseline characteristics were not significantly different between responders and non-responders. Less of a drop in FEV1 from baseline to exacerbation (OR 1.09, 95% CI 1.0, 1.18, p = 0.04) as well as a greater decrease in sputum neutrophil elastase (OR 2.94, 95% CI 1.07, 8.06, p = 0.04) were associated with response to antibiotic treatment at day 14. In addition, higher CRP (HR 1.35 (95% CI: 1.01, 1.78), p = 0.04) and sputum neutrophil elastase (HR 1.71 (95% CI: 1.02, 2.88), p = 0.04) at day 14 of antibiotic therapy were associated with an increased risk of subsequent exacerbation. Conclusions: Inadequate reduction of inflammation during an exacerbation is associated with failure to recover lung function and increased risk of subsequent re-exacerbation in CF patients. © 2015 Published by Elsevier B.V. on behalf of European Cystic Fibrosis Society. Keywords: cystic fibrosis; pulmonary exacerbations; inflammation; antibiotics
⁎ Corresponding author at: Department of Pediatrics, Division of Infectious Diseases, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8. Tel.: +1 416 813 7654x204541. E-mail addresses: [email protected] (V.J. Waters), [email protected] (S. Stanojevic), [email protected] (N. Sonneveld), [email protected] (M. Klingel), [email protected] (H. Grasemann), [email protected] (Y.C.W. Yau), [email protected] (E. Tullis), [email protected] (P. Wilcox), [email protected] (A. Freitag), [email protected] (M. Chilvers), [email protected] (F.A. Ratjen). 1 both authors contributed equally to the manuscript.
http://dx.doi.org/10.1016/j.jcf.2015.01.007 1569-1993/© 2015 Published by Elsevier B.V. on behalf of European Cystic Fibrosis Society. Please cite this article as: Waters VJ, et al, Factors associated with response to treatment of pulmonary exacerbations in cystic fibrosis patients, J Cyst Fibros (2015), http://dx.doi.org/10.1016/j.jcf.2015.01.007
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1. Introduction Cystic fibrosis (CF) is a disease characterized by recurrent flare-ups of respiratory symptoms, or pulmonary exacerbations, which directly contribute to lung function deterioration and ultimate mortality [1,2]. Despite antibiotic and other concomitant therapies for pulmonary infections, one quarter to one third of CF patients do not return to 90% of their baseline lung function following pulmonary exacerbations [3,4]. In addition, in a large cohort study of CF patients followed for up to 10 years, half of the decline in forced expiratory volume in 1 second (FEV1) was attributable to pulmonary exacerbations requiring intravenous (IV) antibiotics [2]. An increasing number of pulmonary exacerbations, specifically more than two per year, is also associated with a higher risk of death or lung transplantation in CF [1,5]. Although exacerbations clearly impact the clinical course of CF, it is not known why some patients respond to therapy for pulmonary exacerbations whereas others do not. Response to antibiotic treatment can be defined in a number of ways, including return of FEV1 to N 90% of baseline [3], full recovery of baseline FEV1 or as the change in FEV1 from the beginning to end of therapy. Risk factors such as female gender, persistent Pseudomonas aeruginosa, Burkholderia cepacia complex or methicillin-resistant Staphylococcus aureus (MRSA) infection and lower FEV1, have been associated with failure to recover lung function after an exacerbation but it is not clear through which mechanisms this occurs [3,6]. Intravenous antibiotic treatment has been shown to decrease sputum bacterial density and inflammatory responses during exacerbations which correlate overall with lung function improvements, but there are still patients that fail to respond to treatment [7,8]. Given that many therapies have overlapping functions (eg. anti-inflammatory, antibacterial), it is crucial to determine the main driver of clinical response in order to more effectively target the management of these exacerbations. Therefore, the aim of this study was to identify the factors, including change in microbiological, systemic and pulmonary markers of inflammation that were associated with clinical response to IV antibiotic treatment of pulmonary exacerbations, as measured by changes in lung function, as well as time to subsequent pulmonary exacerbation.
chronic infection with P. aeruginosa (N 50% of respiratory specimens positive in the 24 months prior to screening) [10] and the ability to produce sputum and to reproducibly perform pulmonary function testing. Exclusion criteria included a sputum culture negative for P. aeruginosa or with a density of less than 105 colony forming units (CFU)/ml at screening, history of B. cepacia positive respiratory culture within 24 months prior to screening, and post lung transplantation or listed for lung transplantation [11]. At the time of pulmonary exacerbation, defined as an increase in respiratory symptoms treated with IV antibiotics [12], each patient was randomized to 14 days of IV antibiotic treatment for P. aeruginosa pulmonary infection based on biofilm or conventional antimicrobial susceptibility results; at this time all other antibiotics were discontinued (including inhaled antibiotics and oral azithromycin). The results of the original randomized control trial (Clinicaltrials.gov NCT 00786513; Supplementary Materials) [9] showed no difference between the two treatment groups therefore data from all the patients with pulmonary exacerbations were pooled for the current analysis. 2.2. Study outcomes
2. Materials and Methods
Lung function measurements were performed according to American Thoracic Society criteria [13], and absolute values were converted to percent predicted using the Global Lung Function Initiative-2012 reference equation [14]. Baseline lung function was defined as the last pulmonary function tests done when the patient was clinically stable (ie. not on any antibiotics) prior to randomization for pulmonary exacerbation. Spirometry was measured on the first day of antibiotic treatment (day 0), after 14 (+ 2) days of antibiotic treatment, and at the follow up clinic visit when the patient was deemed clinically stable and no longer receiving IV antibiotic treatment. Response was defined primarily as recovery of N 100% of baseline FEV1 at day 14 of antibiotic therapy. Maintenance (for responders) or recovery (for non-responders) of lung function at the follow up visit was defined as an FEV1 of N 100% of baseline FEV1. Lung function response was secondarily analyzed as absolute change in FEV1 from day 0 to day 14 of antibiotic treatment. Time to subsequent pulmonary exacerbation was calculated from the last day of IV antibiotic therapy to the first day of re-starting IV antibiotic treatment for a new pulmonary exacerbation.
2.1. Study design and patient population
2.3. Factors associated with response
This was a secondary analysis of a randomized, doubleblind study of IV antibiotic treatment based on biofilm vs planktonic susceptibility testing of pulmonary exacerbations in CF patients with chronic P. aeruginosa infection conducted at five centers in Canada (Hospital for Sick Children, Toronto; St Michael’s Hospital, Toronto; Hamilton Health Sciences Center, Hamilton; British Columbia Children’s Hospital, Vancouver; St Paul’s Hospital, Vancouver) from January 2009 to December 2013 [9]. Patients were screened and enrolled during a stable clinic visit. Inclusion criteria included diagnosis of CF,
Baseline demographic characteristics were collected at the enrollment visit. Blood inflammatory markers (white blood cell count (WBC) (109/L), C-reactive protein (CRP) (mg/L), erythrocyte sedimentation rate (ESR) (mm/h)) were collected at the start of the exacerbation and after 14 days of treatment. Sputum bacterial density and sputum inflammatory markers were also collected at a stable follow-up visit (data not shown). Sputum bacterial density, sputum inflammatory markers (interleukin-8 (IL-8) and neutrophil elastase (NE)) (collected only for patients from Hospital for Sick Children, St Michael’s
Please cite this article as: Waters VJ, et al, Factors associated with response to treatment of pulmonary exacerbations in cystic fibrosis patients, J Cyst Fibros (2015), http://dx.doi.org/10.1016/j.jcf.2015.01.007
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Hospital and Hamilton Health Sciences Center given processing requirements) were performed as previously described [15]. Sputum bacterial density was done on solubilized sputum by mixing 0.5 mL of sputum with 0.5 mL of sputolysin (Calbiochem, La Jolla, CA) [16]. Serial 1:10 dilutions were made in PBS (pH 7.0) and 100 μL were plated onto MacConkey agar (Oxoid, Nepean, ON). Plates were incubated at 42 °C aerobically for 48 hours to select for P. aeruginosa. Sputum density results were reported as log10 colony forming units (CFU)/mL. 2.4. Statistical analysis Prior to analysis, CRP, WBC and ESR values were log transformed using a natural logarithm; IL-8, NE and bacterial density were transformed using log10. Change in each of these variables was defined as the difference of the transformed values (or the ratio of the untransformed values) from day 0 to day 14. The units for each variable were: WBC (109/L), ESR(mm/h), CRP (mg/L), density (CFU/ml), IL-8 (ng/ml) and NE (μg/ml). Lung function responders and non-responders at day 14 were compared using a t-test for continuous variables or a Chi-square for categorical variables (equivalent non-parametric tests, Mann-Whitney for non-normally distributed data or Fisher’s exact test for small sample sizes, were used where appropriate). Univariable logistic regression analysis comparing the mean change in explanatory variables (i.e. sputum P. aeruginosa density and markers of inflammation in blood and sputum) between responders and non-responders was performed using a generalized estimating equation model to adjust for the repeated exacerbations in individual patients; odds ratios with confidence intervals are presented. The odds ratio represents the odds of responding for every unit of change in the explanatory variable (e.g. 1 log decrease in sputum density). A second linear model was also performed with absolute change in FEV1 (L) from day 0 to day 14 as the outcome variable, adjusting for the same covariates as above. Multivariable models were built in a step-wise manner adding variables that were significant on univariable analysis. In addition, factors associated with time to next pulmonary exacerbation in the entire study cohort were examined using Cox-Proportional Hazard Models. Hazard ratios were reported and represent the hazard of developing a subsequent pulmonary exacerbation treated with intravenous antibiotics during the study period for every unit of change in the explanatory variable (eg. 1 log increase in sputum neutrophil elastase). Subjects were censored at the end of the original randomized controlled trial after a maximum follow-up period of 3.7 years. All statistical analyses were performed using STATA V12.
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above baseline at day 14 of IV antibiotic treatment in 36 of the 70 exacerbations (51%). Of the 36 total patients, 16 (44%) had more than 1 exacerbation; of those subjects, 56% (n = 9/16) fulfilled response criteria at the first exacerbation of which, 33% (n = 3/9) fulfilled response criteria with all subsequent exacerbations. The absolute change in FEV1 (L) from baseline to day 14 was 0.20 L (95% CI 0.12, 0.28) in responders compared to -0.27 L (95% CI -0.35, -0.19) among the non-responders (p b 0.0001). Baseline characteristics showed few statistically significant differences between the responders and non-responders for both the first exacerbation and all exacerbations (Table 1 and Table 2). Non-responders had a greater drop in FEV1 from baseline to day 0 of the exacerbation (-12.4 %, range -35.7; -0.9) versus responders: (-5.3%, range -19.1; 7.1, p = 0.05) and a lower FEV1 at exacerbation (34.0%, range 18.6; 79.5) compared to responders (49.8%, range 27.3; 79.5, p = 0.02) (Table 1). In addition, non-responders had a higher serum WBC (p = 0.04) and higher sputum P. aeruginosa bacterial density (p = 0.02) at exacerbation compared to non-responders. Intravenous tobramycin and ceftazidime was the most commonly used combination in both groups. The proportion of exacerbations where antibiotic treatment was changed at day 7 of therapy was similar in both groups (responders 5/36, 14%; non-responders 3/34, 9%, p = 0.51). 3.2. Variables associated with response at day 14 of antibiotic treatment
3. Results
Changes in blood markers of inflammation were not associated with treatment response at day 14 (Table 3). Responders demonstrated a decrease in median log transformed sputum neutrophil elastase (-140.9 μg/mL (range -1106.7; 158.1)), whereas non-responders had only moderate decreases (-30.1 μg/mL (range -826.9; 421.5)) (p = 0.02).In the final multivariable analysis, after adjusting for other covariates significant on univariate analysis, less of a drop in FEV1 from baseline to exacerbation (OR 1.09, 95% CI 1.0, 1.18, p = 0.04) as well as a greater decrease in sputum NE (OR 2.94, 95% CI 1.07, 8.06, p = 0.04) were independently associated with response to IV antibiotic treatment at day 14. In a multivariate analysis using the previously published definition of treatment response as return to N 90% of baseline FEV1 [3], decrease in sputum NE remained significantly associated with treatment response at day 14 (OR 8.11, 95% CI 1.14, 57.60, p = 0.04). Additionally, a linear regression model using absolute change in FEV1 (L) from day 0 to day 14 as the outcome measure, adjusting for similar covariates, was calculated. Similar to the previous results, less of a drop in FEV1 from baseline to exacerbation (p b 0.001) and a greater decrease in sputum NE (p = 0.001) were both significantly associated with greater increases in lung function in the final multivariate model.
3.1. Lung function response and patient characteristics
3.3. Time to Subsequent Exacerbation
A total of 70 pulmonary exacerbations in 36 patients were included in the analysis (Fig. 1). Lung function was at 100% or
The median time to subsequent exacerbation was 132 days (range 0(censored)-1343 days). Higher CRP values at day 14 of
Please cite this article as: Waters VJ, et al, Factors associated with response to treatment of pulmonary exacerbations in cystic fibrosis patients, J Cyst Fibros (2015), http://dx.doi.org/10.1016/j.jcf.2015.01.007
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Fig. 1. Flowchart of clinical outcomes of study cohort after treatment for pulmonary exacerbations. Responders: recovered N 100% of baseline FEV1, n = number of exacerbations, F/U: follow up, FEV1: forced expiratory volume in 1 second, AB txn: antibiotic treatment.
antibiotic therapy (HR 1.35 (95% CI: 1.01, 1.78), p = 0.04) and higher NE levels at day 14 (HR 1.71 (95% CI: 1.02, 2.88), p = 0.04) were associated with a greater risk of subsequent exacerbation during the study period. 4. Discussion In this study, we assessed both blood and sputum measures as markers of response to IV antibiotic therapy during pulmonary exacerbations in CF patients. Greater reduction in sputum neutrophil elastase was independently associated with recovery of lung function after 14 days of IV antibiotic therapy based on two different definitions of response. In addition, higher serum CRP and sputum neutrophil elastase at day 14 were associated with increased risk of subsequent exacerbation. Previous studies have demonstrated that a quarter to a third of CF patients do not recover N 90% baseline FEV1 after exacerbations [3,4]. However, the timing of when this loss of lung function occurs is less clear, as most large epidemiologic studies addressing this question use a large window, typically 3 months, to define post-exacerbation FEV1, based on available measurements in registries [3,4]. We primarily used a more stringent definition of 100% recovery of baseline lung function to assess response to antimicrobial treatment of pulmonary exacerbations; only 50% of exacerbations met this criteria. Interestingly, a significant proportion of patients who did not regain lung function at 14 days, did so at the follow up visit. This demonstrates the difficulty in defining treatment response (or failure thereof) after treatment of pulmonary exacerbations as the response rate changes depending on the definition and
time point chosen. Previous definitions of treatment response based on returning to at least 90% of baseline FEV1, rather than recovering full (100%) baseline lung function, may overestimate responders. However, we also confirmed our findings using the previously published model of treatment response as N 90% of baseline FEV1 [3]. Several investigators have examined the reasons for failing to recover lung function after treatment of pulmonary exacerbations. Female gender, worse nutritional status and greater drop in FEV1 from baseline to exacerbation have all been identified as risk factors for failing to recover lung function, but none of these factors are actually modifiable at the time of treatment of the exacerbation [3,17]. Studies that have investigated mechanisms by which these failures may occur have focused on serum markers of inflammation (e.g. CRP and WBC), measured either at the beginning or the end of antibiotic therapy, and showed that higher levels are associated with treatment failure [6]. Variables predicting response cannot be examined in isolation, however, as IV antibiotic treatment is known to have multiple effects in CF patients during pulmonary exacerbations including decreasing sputum bacterial density, neutrophil counts, IL-8 and neutrophil elastase [7]. Changes in these variables correlate, to varying degrees, with improvements in FEV1 but how the changes in these variables predict failure of treatment in any given patient is not known [8,18]. The current study is the first, to our knowledge, to identify greater decreases in sputum neutrophil elastase as a factor independently associated with lung function recovery after two weeks of IV antibiotic therapy. The effects of treatment of pulmonary exacerbations extend beyond the short term. Elevated levels of both systemic (CRP)
Please cite this article as: Waters VJ, et al, Factors associated with response to treatment of pulmonary exacerbations in cystic fibrosis patients, J Cyst Fibros (2015), http://dx.doi.org/10.1016/j.jcf.2015.01.007
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Table 1 Patient characteristics at first exacerbation. P-values indicate the difference between responders and non-responders and are obtained using a t-test for normally distributed continuous variables; Mann-Whitney test for non-normally distributed continuous variables; a Chi-squared test for binary outcomes and a Fisher’s exact test for binary outcomes with small values.
No. of exacerbations/patient during study period, median (range) Age at exacerbation, yrs, median (range) Female, n (%) Homozygous ΔF508, n (%) FEV1 % pred, median (range) Baseline At exacerbation Δ baseline-exacerbation Days from baseline-exacerbation BMI z-score, median (range) Baseline At exacerbation Serum measures at exacerbation, median (range) WBC (109/L) ESR(mm/h) CRP (mg/L) Sputum measures at exacerbation, median (range) n Density (log10 CFU/ml) IL-8 (ng/ml) NE (μg/ml) Complications at enrollment CFRD, n (%) Liver disease, n (%) Pancreatic insufficiency, n (%) ABPA, n (%) Maintenance treatment at exacerbation Dornase alfa, n (%) Azithromycin, n (%) Inhaled tobramycin, n (%) Hypertonic saline, n (%) Other inhaled antibiotics, n (%)
Total N = 36
Responders N = 16
Non-Responders N = 20
P value
1 (1;5) 23.9 (10.1;49.2) 14 (38.9) 20 (57.1) 53.7 (22.2; 93.3) 41.0 (18.6; 79.5) -10.0 (-35.7; 7.1) 61.5 (12; 423)
2 (1;5) 18.1 (10.1;48.0) 7 (43.8) 10 (62.5) 59.4 (30.3; 88.4) 49.8 (27.3; 79.5) -5.3 (-19.1; 7.1) 83 (12; 423)
1 (1;5) 26.1 (11.3;49.2) 7 (35.0) 10 (52.6) 50.9 (22.2; 93.3) 34.0 (18.6; 79.5) -12.4 (-35.7; -0.9) 54 (23; 309)
0.28 0.33 0.59 0.56 0.44 0.02 0.05 0.49
-0.60 (-4.67; 1.49) -0.57 (-3.37; 1.24)
-0.44 (-4.67; 1.49) -0.55 (-3.37; 1.24)
-0.63 (-2.27; 0.72) -0.87 (-3.07; 0.65)
0.76 0.36
11.6 (3.8; 22.2) 28 (1; 91) 12.8 (1.1; 165.7)
10.5 (3.8; 16.9) 14 (1; 91) 6.1 (1.6; 137.2)
13.0 (5.2; 22.2) 36 (1; 77) 18.2 (1.1; 165.7)
0.04 0.14 0.11
26 7.0 (4.1 - 8.6) 191.5 (39.0; 672.9) 177.0 (42.7;1193.5)
12 7.5 (5.8 - 8.6) 244.1 (82.9; 672.9) 208.0 (49.4;1193.5)
14 6.8 (4.1 - 8.4) 167.9 (39.0; 416.9) 149.4 (42.7;913.7)
N/A 0.02 0.26 0.47
11 (30.6) 3 (8.3) 34 (94.4) 5 (13.9)
5 (31.3) 3 (18.8) 15 (93.8) 3 (18.8)
6 (30.0) 0 (0.0) 19 (95.0) 2 (10.0)
0.94 0.08 0.87 0.64
18 (50.0) 17 (47.2) 29 (80.6) 6 (16.7) 4 (11.1)
8 (50.0) 5 (31.3) 12 (75.0) 2 (12.5) 3 (18.8)
10 (50.0) 12 (60.0) 17 (85.0) 1 (5.0) 1 (5)
0.99 0.09 0.45 0.30 0.30
FEV1: forced expiratory volume in 1 second BMI: body mass index CFRD: cystic fibrosis related diabetes ABPA: allergic bronchopulmonary aspergillosis Δ: change N/A: not applicable
and pulmonary (NE) markers of inflammation after 14 days of IV antibiotics in our study cohort were associated with an increased risk of subsequent exacerbations; shorter time between exacerbations is a significant determinant of overall lung function decline in CF [2]. Previous studies have focused on blood-based biomarkers, especially CRP, as predictors of pulmonary exacerbation but have not analyzed the contribution of pulmonary markers of inflammation [19–21]. These markers are relevant given that multiple studies have shown that increased pulmonary neutrophil elastase activity is the most predictive biomarker for subsequent lung function decline and the development of bronchiectasis in young children with CF [22,23]. The observation that non-responders had higher pulmonary neutrophil elastase activity, associated with negative long-term consequences, raises the question as to whether this indicates ongoing primary inflammation or inflammation secondary to inadequately treated infection. This highlights the difficulty in
developing statistical models with variables that may be on the same causal pathway. It is possible that greater levels of infection lead to persistent pulmonary inflammation and worse lung function, although in our study, higher bacterial density measures at the start of treatment were noted in responders. Whether this reflects exacerbations in which infection is playing a primary role and are thus more likely to respond to antibiotic therapy, is unclear. Although this study cannot directly address the mechanistic role of sputum and serum biomarkers, only changes in sputum neutrophil elastase were independently and significantly associated with FEV1 response in the final multivariate model. There are a limited number of anti-inflammatory therapies available for use in CF patients, with varying degrees of efficacy. Drugs such as such as alpha-1 antitrypsin, an endogenous inhibitor of neutrophil elastase, have been shown to suppress neutrophil elastase in the respiratory epithelial lining fluid (ELF) [24]. Although subsequent randomized,
Please cite this article as: Waters VJ, et al, Factors associated with response to treatment of pulmonary exacerbations in cystic fibrosis patients, J Cyst Fibros (2015), http://dx.doi.org/10.1016/j.jcf.2015.01.007
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Table 2 Characteristics of all exacerbations.
FEV1 % pred, median (range) Baseline At exacerbation Δ baseline-exacerbation Days from baseline-exacerbation BMI z-score, median (range) Baseline At exacerbation Serum measures at exacerbation, median (range) WBC (109/L) ESR(mm/h) CRP (mg/L) Sputum measures at exacerbation, median (range) n Density (log10 CFU/ml) IL-8 (ng/ml) NE (μg/ml)
Total n = 70
Responders n = 36
Non-Responders n = 34
P value
59.4 (22.2; 99.4) 44.7 (17.4; 83.7) -10.0 (-35.7; 7.11) 76.5 (7; 423)
59.4 (30.3; 99.4) 47.6 (26.6; 80.1) -7.0 (-19.3; 7.1) 77.5 (7; 423)
58.7 (22.2; 93.3) 40.0 (17.4; 83.7) -12.4 (-35.7; 3.7) 71 (23; 309)
0.86 0.09 0.004 0.81
-0.67 (-4.7; 1.5) -0.77 (-3.5; 1.2)
-0.78 (-4.7; 1.5) -0.71 (-3.5; 1.2)
-0.58 (-3.5; 1.2) -0.85 (-3.3; 0.9)
0.29 0.67
11.1 (2.8; 22.2) 30 (1; 91) 12.8 (0.8; 165.7)
10.3 (2.8; 16.9) 20 (1; 91) 6.4 (1.6; 137.2)
12.5 (5.2; 22.2) 37 (1; 89) 21.1 (0.8 - 165.7)
0.02 0.35 0.14
49 7.0 (4.1; 8.6) 196.2 (25.6; 672.9) 201.2 (42.7; 1193.5)
26 7.6 (5.5; 8.6) 196.2 (31.6; 672.9) 208.0 (49.4; 1193.5)
23 6.7 (4.1; 8.4) 190.7 (25.6; 416.9) 179.8 (42.7; 986.3)
N/A 0.001 0.09 0.32
P-values indicate the difference between responders and non-responders. P-values for continuous variables are derived from linear and logistic generalized estimating equations (GEE) models to account for the repeated exacerbations in the same subject. Non-normally distributed data was transformed to the natural logarithm or using log10 before statistical analysis. FEV1: forced expiratory volume in 1 second BMI: body mass index CFRD: cystic fibrosis related diabetes ABPA: allergic bronchopulmonary aspergillosis Δ: change N/A: not applicable
placebo-controlled trials of inhaled recombinant human alpha-1 antitrypsin in adult CF patients failed to demonstrate a statistically significant reduction in neutrophil elastase activity [25], phase II studies of this drug, using amore clinically relevant primary outcome of pulmonary exacerbations, are
ongoing. Corticosteroids are another potent anti-inflammatory class of drugs known to inhibit neutrophilic accumulation at sites of inflammation [26]. They have been studied mostly as chronic suppressive medications in CF, however, long-term use has been limited by concerns of side effects [27]. A randomized
Table 3 Univariable logistic regression analysis for recovery of lung function at day 14 of antibiotic treatment.
Blood measures Δ WBC (109/L) Δ ESR(mm/h) Δ CRP (mg/L) Sputum measures n Δ Density(log10 CFU/ml) Δ IL-8 (ng/ml) Δ NE (μg/ml)
Responders n = 36 Median (range)
Non-Responders n = 34 Median (range)
Odds Ratio ⁎ (95% CI)
P value
-2.5 (-10.4; 14.1) -4.0 (-62; 18) -4.8 (-135.4; 5.3)
-1.9 (-14.7; 7.6) -2 (-70; 25) -15.3 (-155.8; 6.7)
0.59 (0.19, 1.85) 1.14 (0.72, 1.82) 1.02 (0.72, 1.45)
0.37 0.57 0.90
26 -2.17 (-8.30; 1.5) -66.5 (-411; 647.4) -140.9 (-1106.7; 158.1)
23 -1.42 (-6.3; 2.9) -6.0 (-299; 287.0) -30.1(-826.9; 421.45)
N/A 1.12 (0.94, 1.25) 3.85 (0.95, 16.67) 4.0 (1.26, 12.50)
N/A 0.21 0.06 0.02
P-values were derived from logistic generalized estimating equations (GEE) models to account for the repeated exacerbations in the same subject. The changes in WBC, ESR, CRP, IL-8 and NE were transformed as described in the method section prior to statistical analysis. CI: confidence intervals Δ: change from day 0 to day 14 of antibiotic treatment CFU: colony forming units WBC: white blood cell count ESR: erythrocyte sedimentation rate CRP: C reactive protein NE: neutrophil elastase N/A : not applicable ⁎ Odds Ratio indicates the odds for responding with greater decrease in values. Please cite this article as: Waters VJ, et al, Factors associated with response to treatment of pulmonary exacerbations in cystic fibrosis patients, J Cyst Fibros (2015), http://dx.doi.org/10.1016/j.jcf.2015.01.007
V.J. Waters et al. / Journal of Cystic Fibrosis xx (2015) xxx–xxx
controlled trial examining the short-term use of oral prednisone in all CF patients presenting with pulmonary exacerbations demonstrated a modest improvement in lung function [28]. Focusing this anti-inflammatory treatment on CF patients with exacerbations who are not responding to antibiotic treatment may maximize its potential benefit while minimizing harmful side effects. Additional anti-inflammatory agents, such as ibuprofen and azithromycin [29,30], have also been used in individuals with CF but their role in modifying the response during an acute pulmonary exacerbation has not yet been defined. This study had several limitations including the relatively small sample size (70 pulmonary exacerbations) which decreased the power to detect smaller effect sizes. Therefore, absence of statistically significant differences between patient groups did not necessarily mean that they were similar. In addition, incomplete long-term pulmonary function testing prohibited meaningful analysis of the four groups identified at follow up. Thus, although the majority of non-responders were treated with longer antibiotic courses and appeared to have recovered lung function at follow up, the small group sizes precluded any such conclusions. We also included repeated pulmonary exacerbations from the same patient which could bias the results but controlled for this in the analysis using generalized estimating equations accounting for repeated measures from the same individual. Finally, measures of bacterial density were limited to P. aeruginosa in this study population of CF patients with chronic P. aeruginosa infection; it is possible that changes in the composition of other members of the pulmonary microbiome may have influenced the response to antimicrobial treatment. However, none of the patients included in this analysis had B. cepacia complex or MRSA infection, previously identified as risk factors for failing to recover FEV1 following a pulmonary exacerbation. In summary, although limited in power due to a small sample size, this study demonstrates that inadequate reduction of pulmonary inflammation during an exacerbation is associated with failure to recover lung function and increased risk of subsequent re-exacerbation in CF patients. To improve our success in recovering lung function following exacerbations, we may need to target the underlying inflammatory process in the CF lung with adjuvant therapies in individuals not responding to antimicrobial treatment.
Authors’ contributions V Waters, YCW Yau, F Ratjen and E Tullis designed the study, participated in the data collection, analysis and interpretation, and wrote the paper. S Stanojevic, M Klingel and N Sonneveld did the statistical analysis and assisted with the writing of the manuscript. H Grasemann supervised the sputum processing and inflammatory marker measurements and assisted with the writing of the manuscript. P Wilcox, A Freitag and M Chilvers undertook the enrolment of patients, supervised treatment, participated in data collection and interpretation, and assisted with the writing of the manuscript.
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