Abstract
Background
Bronchiectasis is increasingly recognized as an inflammatory disease in which “disease activity” is not fully captured by radiology or composite severity scores. Although sputum neutrophil elastase (NE) activity is a validated biomarker, the prognostic value of segment-specific NE activity in bronchial lavage fluid (BLF) remains limited.
Objectives
To evaluate NE activity in BLF collected from affected bronchi and determine its association with one-year bronchiectasis outcomes.
Design
Multicenter prospective cohort study.
Methods
Adults with stable bronchiectasis undergoing bronchoscopy at two hospitals in Ho Chi Minh City were enrolled between April 2022 and January 2024. BLF was collected from the most affected bronchus (ABLF) and a reference bronchus (NBLF). NE activity was quantified using a fluorogenic assay. Participants were followed for 12 months to assess exacerbations and time to first exacerbation.
Results
Of 168 screened patients, 139 were enrolled and 121 completed follow-up. Post-tuberculosis bronchiectasis was the most common etiology. NE activity was detectable in all BLF samples and was higher in ABLF than NBLF (median 2.0 vs. 0.4 μg/mL). Exacerbations occurred in 42.1% of patients. ABLF NE activity was higher in patients with chronic bacterial infection than in those without (median 3.6 vs. 1.8 μg/mL; p=0.001), and in patients who developed exacerbations compared with those who did not (median 3.0 vs. 1.2 μg/mL; p<0.001). After adjustment for baseline severity indices, higher ABLF NE activity remained associated with increased exacerbation risk. Kaplan–Meier analysis showed shorter exacerbation-free survival with increasing ABLF NE activity, with estimated mean exacerbation-free survival times of 10.5, 8.5, and 6.9 months in the mild, moderate, and high NE groups, respectively (log-rank p=0.001).
Conclusion
Segment-specific NE activity in BLF is elevated in affected bronchi and is an independent predictor of exacerbations in this TB-endemic Asian bronchiectasis cohort, supporting BLF NE as a clinically meaningful biomarker of disease activity.
Keywords: bronchial lavage fluid, bronchiectasis, bronchoscopy, exacerbation, neutrophil elastase
Summary
Neutrophil elastase activity measured in bronchial lavage fluid from radiologically affected bronchi was a strong and independent predictor of bronchiectasis exacerbations over one year, regardless of baseline disease severity. These findings support the association between airway neutrophil elastase activity and exacerbation risk in an Asian bronchiectasis population.
Introduction
Bronchiectasis (BE) is a chronic airway disease characterized by irreversible bronchial dilatation, persistent infection, and predominantly neutrophilic airway inflammation. Patients experience recurrent exacerbations, progressive decline in lung function, and reduced quality of life, with a growing economic and healthcare burden worldwide. 1 Recent studies have emphasized bronchiectasis as an inflammatory airway disease, in which “disease activity” reflects ongoing airway inflammation that is not fully captured by radiological extent or conventional severity indices.2,3 Prognostic tools such as the Bronchiectasis Severity Index and FACED score are useful for estimating the risk of exacerbations, hospitalization, and mortality; however, these instruments do not directly quantify airway inflammatory activity and may perform variably across populations.4–6 There is therefore a need for biomarkers that more directly quantify airway inflammation and help refine risk stratification, particularly in Asian and tuberculosis (TB)-endemic settings.
Neutrophil elastase (NE), a serine protease released by activated neutrophils, contributes to airway injury, mucus dysfunction, impaired host defense, and disease progression in bronchiectasis. Its activity reflects ongoing neutrophilic inflammation and has emerged as a clinically relevant treatable trait. This concept has gained further importance following the U.S. Food and Drug Administration approval of brensocatib, a dipeptidyl peptidase 1 inhibitor that reduces activation of neutrophil serine proteases, as the first approved disease-specific treatment for non-cystic fibrosis bronchiectasis. Sputum NE activity is a well-validated biomarker associated with exacerbation frequency, lung-function decline, and disease severity.7,8 However, evidence regarding NE activity in bronchial lavage fluid (BLF) remains limited.
Sputum sampling has practical and biological limitations. Some patients, particularly those with early-stage disease, mild disease, or dry cough, are unable to produce adequate sputum.9,10 In addition, expectorated sputum may contain secretions from both affected and unaffected airways, as well as contamination from the upper airway. Bronchoscopy with BLF sampling is more invasive and is not intended to replace sputum-based assessment in routine practice. Nevertheless, computed tomography-targeted BLF collection may provide a lesion-specific assessment of local airway inflammatory and protease activity in selected patients, especially when non-invasive samples are unavailable or when infection requires further evaluation. In tuberculosis-endemic regions, bronchoscopy may also assist in assessing active tuberculosis, nontuberculous mycobacterial disease, fungal infection, or obstructive/endobronchial lesions when clinically suspected.
Despite the growing interest in NE as a biomarker and therapeutic target, few studies have evaluated BLF-derived NE activity in relation to longitudinal clinical outcomes in bronchiectasis. This gap is particularly relevant in Asian populations, where post-tuberculosis sequelae contribute substantially to disease etiology.11–13 Therefore, this multicenter prospective cohort study aimed to investigate NE activity in BLF collected from diseased segments in stable BE patients and evaluate its association with one-year clinical outcomes, specifically exacerbation frequency and time to first exacerbation. These findings may support the utility of BLF NE activity as a prognostic biomarker and help inform future biomarker-guided strategies in bronchiectasis.
Methods
We conducted a prospective cohort study involving adult patients with stable BE at Cho Ray Hospital and University Medical Center Ho Chi Minh City between April 2022 and January 2024. All adult patients undergoing bronchoscopy who met the British Thoracic Society 2019 criteria for chest computed tomography (CT)-confirmed BE were recruited. We excluded patients with traction bronchiectasis, active pulmonary tuberculosis, or those who had used antibiotics within the preceding four weeks. Patients receiving long-term therapy with macrolides, immunosuppressants, oral corticosteroids, or NE inhibitors were also excluded. The reporting of this study conforms to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement. 14
Data and sample collection, neutrophil elastase activity measurement
Demographic and clinical data were recorded, including age, sex, body mass index (BMI), symptoms (sputum, dyspnea), history of hemoptysis (documented recently or within two years prior to enrolment), prior exacerbations, smoking status, complete blood count, pulmonary function, specifically Forced Expiratory Volume in 1 second (FEV1) and Forced Vital Capacity (FVC), chest CT features, modified Reiff score, Bronchiectasis Severity Index (BSI), and St. George’s Respiratory Questionnaire (SGRQ) score (calculated using the SGRQ software developed by Marco Gelpi, Jonathan Argentiero, and Andreas Ronit at https://sgrq.github.io/).
BLF was collected from the most radiologically affected bronchus and from a reference bronchus, designated as affected BLF (ABLF) and reference/least-affected BLF (NBLF), respectively. The affected bronchus was selected as a priority based on the most recent chest CT as the segment with the greatest extent and severity of bronchiectasis. When BE involved multiple lobes with similar severity, the target site was the bronchus with the most prominent endobronchial inflammatory features at inspection (e.g., purulent secretions). For the reference bronchus, we preferentially selected a bronchus in a lobe without bronchiectasis on CT. In participants with diffuse bronchiectasis and no clearly normal lobe, NBLF was obtained from the least affected segment (minimal radiologic abnormality).
To minimize cross-contamination, the reference-site lavage was performed first, followed by the affected-site lavage. For the ABLF collection, 60 mL of sterile normal saline was instilled into the radiologically most affected bronchus and then aspirated into a sterile container. For NBLF collection, 20 mL of sterile normal saline was instilled into the reference bronchus and then aspirated into a separate sterile container.
After collection, BLF samples were kept at 2°C-8°C and transferred to the laboratory for processing. Each sample was initially centrifuged at 320 ×g for 10 minutes at 4°C to separate the cellular pellet from the supernatant. The resulting supernatant was subsequently centrifuged at 6000 ×g for 5 minutes at 4°C to remove residual mucus and debris. The clarified supernatant was aliquoted and stored at −80°C until neutrophil elastase activity measurement. No mucolytic agent was added before NE measurement, and visible mucus burden in BLF was not quantitatively assessed. NE activity was measured in recovered cell-free BLF supernatant using a fluorogenic substrate-based neutrophil elastase activity assay kit (Neutrophil Elastase Activity Assay Kit, Fluorometric; MAK246, Sigma-Aldrich, Burlington, MA, USA), according to the manufacturer’s protocol with minor modifications for BLF samples, and reported as µg/mL. A 5 ng/µL NE working standard was prepared to generate a standard curve of 0–25 ng/well. For each reaction, 50 µL of BLF supernatant, standard, or assay buffer blank was added to a 96-well plate, followed by 50 µL of NE substrate reaction mix. Fluorescence was measured kinetically at 37°C using excitation/emission wavelengths of 380/500 nm. Samples above the upper standard were diluted and reanalyzed within the linear range. Final NE-equivalent activity concentrations were calculated using the sample volume and corresponding dilution factor and reported as µg/mL. 15
For microbiology testing, bacterial culture, fungal culture, and cell differentials from ABLF were performed according to the physician’s discretion. Details of BLF microbiological sampling and culture procedures have been reported in our earlier related publication. 16
Follow-up procedure and outcomes
Patients were followed for 12 months after the initial bronchoscopy. Follow-up visits were scheduled at intervals of every three months. For patients without in-person visits, telephone interviews were conducted to monitor clinical status. Patients who missed scheduled visits and could not be contacted by telephone after two attempts were classified as lost to follow-up.
Clinical data collected during follow-up included the results of bacterial cultures from respiratory specimens (performed at the physician’s discretion), lung function, which was typically assessed at three time points: enrolment, six months, and study completion (12 months). The primary outcome recorded was the incidence of exacerbations. Exacerbations were defined according to the European Multicentre Bronchiectasis Audit and Research Collaboration (EMBARC) consensus criteria as worsening of at least three key symptoms for at least 48 hours, including cough, sputum volume and/or consistency, sputum purulence, breathlessness or exercise tolerance, fatigue/malaise, and hemoptysis, requiring a change in clinical intervention. 17 In this study, prescription of a new course of antibiotics was required to confirm an exacerbation event. Pulmonary function testing was performed during stable follow-up visits, but was not routinely performed at the onset of exacerbation. Chronic bacterial infection (CBI) was defined as the isolation of the same pathogen from respiratory samples on at least two occasions, spaced three or more months apart, within a one-year period. 18
Statistical analysis
To assess the association between NE activity and the development of BE exacerbations [risk ratio (RR)=1.61, as previously reported 7 ], and considering the one-year exacerbation rate in BE patients (p0=0.5), the estimated required sample size was 109 patients, with α=0.01, β=0.2, and an additional 10% to account for dropouts. Continuous variables were tested for normality using the Kolmogorov–Smirnov test. For binary variables (bacterial isolation, chronic bacterial infection, exacerbation development), differences in NE activity between the two groups were analyzed using the Mann-Whitney U test. Time to first exacerbation was evaluated using Kaplan–Meier analysis, and differences among NE activity tertile groups were compared using the log-rank test. Estimated mean exacerbation-free survival times and 95% confidence intervals were reported. A two-sided p-value <0.05 was considered statistically significant, with 95% confidence intervals (CI). Data analyses were performed using IBM SPSS Statistics version 22.0 (IBM Corp., Armonk, NY, USA). Data visualization and figures were generated using JASP version 0.18.3.0 (University of Amsterdam, Amsterdam, Netherlands) and Stata version 17 (StataCorp, College Station, TX, USA).
Results
Baseline characteristics
Of 168 patients screened, 139 met the inclusion criteria and were included in the baseline analysis. Among them, 121 completed the one-year follow-up and were included in the longitudinal outcome analyses (Figure 1). Baseline demographic and clinical characteristics of the 139 included patients are summarized in Table 1. The mean age was 60 ± 13 years, 82 patients were female (59.0%), and 73 patients (52.5%) were aged ≥60 years.
Figure 1.
Study flowchart of patient recruitment and one-year follow-up. Abbreviations: AFB, acid-fast bacilli; BLF, bronchial lavage fluid; NTM, nontuberculous mycobacteria; PCR, polymerase chain reaction.
Table 1.
Characteristics of bronchiectasis patients from each participating center.
| Characteristics | Total (n = 139) | University Medical Center HCMC (n = 50) | Cho Ray’s Hospital (n = 89) | P-value |
|---|---|---|---|---|
| Age (years) | 60 ± 13 | 57 ± 14 | 61 ± 12 | 0.091 * |
| Female sex | 82 (59.0) | 41 (82.0) | 41 (46.1) | < 0.001 $ |
| Productive cough | 100 (71.9) | 35 (70.0) | 65 (73.0) | 0.702 $ |
| Dyspnea | 83 (59.7) | 26 (52.0) | 57 (64.0) | 0.165 $ |
| History of hemoptysis | 58 (41.7) | 16 (32.0) | 42 (47.2) | 0.081 $ |
| Exacerbation in the previous year | 31 (22.3) | 7 (14.0) | 24 (27.0) | 0.078 $ |
| BMI (kg/m2) | 21.1 ± 3.0 | 21.3 ± 3.1 | 20.9 ± 2.9 | 0.441 * |
| WBC (K/mm3) | 7.9 [6.7 – 9.9] | 7.9 [5.9 – 9.9] | 7.9 [6.8 – 9.9] | 0.618 † |
| EOS (K/mm3) | 0.2 [0.1 – 0.3] | 0.2 [0.1 – 0.4] | 0.2 [0.1 – 0.3] | 0.461 † |
| NLR | 2.7 [1.8 – 4.0] | 2.4 [1.6 – 3.2] | 2.9 [2.0 – 4.8] | 0.016 † |
| Modified Reiff score | 3.0 [2.0 – 5.0] | 3.0 [2.0 – 5.0] | 3.0 [2.0 – 4.0] | 0.460 † |
| %FEV1 | 70.0 [55.0 – 81.0] | 78.0 [64.0 – 90.8] | 66.0 [48.5 – 76.0] | 0.005 † |
| %FVC | 71.0 [60.0 – 82.0] | 76.0 [67.8 – 89.3] | 66.0 [54.0 – 79.8] | 0.006 † |
| BSI | 4.0 [3.0 – 7.0] | 3.0 [2.0 – 5.0] | 5.0 [3.0 – 8.0] | 0.001 † |
| • St. George’s Respiratory Questionnaire (SGRQ) | ||||
| Total score | 20.6 [8.1 – 41.7] | 16.3 [8.1 – 31.4] | 24.9 [8.1 – 50.6] | 0.088 † |
| Symptom score | 39.8 [20.5 – 57.7] | 34.9 [18.8 – 52.6] | 48.2 [27.4 – 59.5] | 0.056 † |
| Activity score | 23.5 [5.3 – 47.7] | 20.4 [3.9 – 39.6] | 29.5 [5.9 – 59.5] | 0.234 † |
| Impact score | 10.9 [3.4 – 34.4] | 7.2 [2.9 – 25.6] | 14.8 [3.6 – 44.7] | 0.176 † |
| • Distribution of bronchiectasis lesions | ||||
| Upper lobe | 95 (68.4) | 29 (58.0) | 66 (74.2) | 0.145 $ |
| Middle lobe | 76 (54.7) | 39 (78.0) | 37 (41.6) | < 0.001 $ |
| Lower lobe | 75 (54.0) | 23 (46.0) | 52 (58.4) | 0.192 $ |
| Bilateral bronchiectasis | 80 (57.6) | 28 (56.0) | 52 (58.4) | 0.861 $ |
| • Bronchiectasis pattern | ||||
| Cylindrical | 88 (63.3) | 36 (72.0) | 52 (58.4) | 0.166 $ |
| Varicose | 13 (9.4) | 2 (4.0) | 11 (12.4) | |
| Cystic | 15 (10.8) | 3 (6.0) | 12 (13.5) | |
| Mixed | 23 (16.5) | 9 (18.0) | 14 (15.7) | |
| • Potential etiologies | ||||
| Post-tuberculosis bronchiectasis | 46 (33.1) | 15 (30.0) | 31 (34.8) | 0.561 $ |
| Suspected NTM infection | 26 (18.7) | 9 (18.0) | 17 (19.1) | 0.873 $ |
| Other causes | 24 (17.3) | 7 (14.0) | 17 (19.1) | 0.445 $ |
| Unknown cause | 43 (30.9) | 19 (38.0) | 24 (27.0) | 0.177 $ |
Note. Data are presented as number (percentage), mean ± standard deviation, or median [interquartile range].
Abbreviations: BMI = body mass index; BSI = Bronchiectasis Severity Index; FEV1 = forced expiratory volume in 1 second; FVC = forced vital capacity; NLR = neutrophil-to-lymphocyte ratio; WBC = white blood cell count; EOS = eosinophil count; NTM = nontuberculous mycobacteria.
*Student’s t-test.
†Mann–Whitney U test.
$Chi-square test.
In terms of etiology, post-tuberculosis BE was the most prevalent cause, while 30.9% were classified as idiopathic. Cylindrical bronchiectasis was the most frequent radiological pattern. Bilateral disease was present in 68.4% of patients, with frequent upper-lobe involvement. The median FEV1 was 70.0% predicted. According to the Bronchiectasis Severity Index, 56.8% of patients were classified as having mild disease.
Bronchial lavage fluid analysis
NE activity was detected in all collected specimens. Median NE levels were notably higher in ABLF (2.0 μg/ml; [interquartile range (IQR) 0.8 - 3.4 μg/ml]) compared to NBLF (0.4 μg/ml; [IQR 0.3-0.7 μg/ml]). NE activity in ABLF correlated positively with Bronchiectasis Severity Index score (rho=0.365; p<0.001), modified Reiff score (rho=0.297; p<0.001), and total SGRQ score (rho=0.218; p=0.034).
In the post-tuberculosis bronchiectasis subgroup, paired BLF samples were obtained from the most affected bronchiectatic area and from a reference or least affected area in all 46 patients. NE activity was significantly higher in bronchiectatic areas than in reference areas, 2.04 μg/mL [IQR: 0.80–3.35] vs. 0.35 μg/mL [IQR: 0.24–0.61], p <0.001.
Cell differentials in ABLF were available for 109 patients (78.4% of the baseline cohort). Analysis revealed a median neutrophil percentage of 65.0% [IQR 24.5-85.0%], a median lymphocyte percentage of 16.0% [IQR 1.0-48.0], and a median eosinophil percentage of 0.7% [IQR 0.0-5.5%].
Bacterial culture of ABLF was performed in 136 patients, 97.8% of the baseline cohort, and was positive in 51 patients, 37.5%. The distribution of all isolated bacteria is detailed in Figure 2. Pseudomonas aeruginosa was the most common pathogen, accounting for 29.6% of isolates. Multidrug-resistant bacteria accounted for 40.7% of isolates, 22/54, including two isolates of methicillin-resistant Staphylococcus aureus.
Figure 2.
Bacterial isolates in bronchial lavage fluid from radiologically affected bronchi.
Fungal cultures were positive in 26.3% (26/99) of indicated cases, primarily Candida spp (n= 20) and Aspergillus spp. (n=6). Fungal positivity correlated with significantly higher radiological severity (Reiff score median 4.5 [IQR 2.8–6.0] vs. 3.0 [2.0–4.0], p=0.010).
Patient treatment and outcomes after one-year follow-up
During the one-year follow-up, the majority of the cohort (82/121, 67.8%) received only symptomatic therapy or antibiotics for bacterial exacerbations. Inhalation therapy (long-acting β2-agonists, long-acting muscarinic antagonists, or inhaled corticosteroids) was prescribed in 15 cases (12.4%). Etiology-directed treatment, including treatment for nontuberculous mycobacterial pulmonary disease, rheumatoid arthritis-associated bronchiectasis, or immunodeficiency-associated bronchiectasis, was given in 9 patients, and 6 patients underwent lobectomy. Long-term macrolide therapy was prescribed in five patients, and an additional four patients (19 in total) were instructed to perform daily airway-clearance therapy. No patients were receiving Dornase alfa or any other DNase therapy at baseline or during the 12-month follow-up period.
A total of 13 cases met the diagnostic criteria for CBI. The pathogens identified were P. aeruginosa, n = 9, Escherichia coli, n = 1, Haemophilus influenzae, n = 1, Proteus mirabilis, n = 1, and S. aureus, n = 1. It should be noted that sputum and BLF cultures were obtained at the attending physician’s discretion rather than by fixed protocol, which may limit the precise estimation of CBI incidence.
One-year spirometric follow-up was available in 78 patients. No significant changes were observed in FEV1, p = 0.309, or FVC, p = 0.202, between baseline and one year.
Exacerbations were reported in 51 patients (42.1%) during the one-year follow-up. The frequencies of total exacerbations, hospitalized exacerbations, and non-hospitalized exacerbations are shown in Figure 3. The median number of exacerbations during the one-year follow-up was 2 (IQR: 1–2).
Figure 3.
Frequency distribution of exacerbations among patients who experienced at least one exacerbation during follow-up. Panel A shows total exacerbations per patient among the 51 patients who experienced at least one exacerbation. Panels B and C show type-specific distributions among patients with at least one non-hospitalized exacerbation (n=45) or hospitalized exacerbation (n=15), respectively. The non-hospitalized and hospitalized groups are not mutually exclusive; 9 patients experienced both types of exacerbation.
Out of 121 cases, three deaths were documented: a 32-year-old female with BE related to common variable immunodeficiency (due to bacterial exacerbation), a 77-year-old male with bronchiectasis-chronic obstructive pulmonary disease (COPD) overlap syndrome (due to severe COPD exacerbation), and a 62-year-old male with idiopathic BE (due to nasopharyngeal cancer).
Association of neutrophil elastase activity with outcomes and development of a prediction model for bronchiectasis exacerbation
Levels of NE activity were significantly higher in BE patients with CBI compared to those without, both in ABLF (3.6 μg/ml [IQR: 2.8–5.1] vs. 1.8 μg/ml [IQR: 0.8–3.2], p = 0.001) and in NBLF (0.8 μg/ml [IQR: 0.7–1.7] vs. 0.4 μg/ml [IQR: 0.3–0.6], p = 0.003).
In an exploratory analysis using baseline ABLF bacterial culture data, patients with positive bacterial isolation had higher ABLF NE activity than those without positive isolation (2.6 [1.1–3.9] vs. 1.8 [0.8–3.0] μg/mL; p=0.017). Positive baseline ABLF bacterial isolation was also more frequent among patients who developed exacerbations during follow-up than among those who did not (45.1% vs. 26.9%; p=0.039).
Patients who developed exacerbations showed more severe disease than those without, as reflected by higher Modified Reiff scores (p<0.001) and BSI values (p=0.002). NE activity was also higher in patients with subsequent exacerbations, both in ABLF (3.0 μg/ml [IQR 1.6–4.2] vs. 1.2 μg/ml [IQR 0.7–2.7], p<0.001) and in NBLF (0.6 μg/ml [IQR 0.3–0.9] vs. 0.4 μg/ml [IQR 0.2–0.6], p=0.006).
Multivariable regression models were constructed to evaluate the association between ABLF NE activity and exacerbation during the one-year follow-up. Based on prior causal reasoning and the use of a causal directed acyclic graph (DAG), the models were adjusted for the following confounding variables: BSI score, female gender, productive cough, and history of hemoptysis. Treatment interventions were considered potential colliders and, therefore, were not included in the models. NE activity in ABLF was significantly associated with the risk of bronchiectasis exacerbations across all three models (Table 2). This was a strong and consistent predictor, with each 1 μg/ml increase in NE activity associated with a 33.9%–40.2% increase in the odds of exacerbations. Model performance demonstrated a good fit to the data, as assessed by the Hosmer–Lemeshow test with p values >0.05.
Table 2.
Multivariable logistic regression models accessing the association between Neutrophil elastase activity in bronchial lavage fluid and risk of bronchiectasis exacerbations.
| Models | Variables | β cofficient | Odds ratio | 95% confident interval | P value |
|---|---|---|---|---|---|
| Model 1 | Constant | -1.799 | 0.165 | | <0.001 |
| Female | 0.070 | 1.073 | 0.483 -2.383 | 0.864 | |
| BSI | 0.151 | 1.163 | 1.019 – 1.327 | 0.023 | |
| NE activity in ABLF | 0.292 | 1.339 | 1.059 – 1.694 | 0.015 | |
| Model 2 | Constant | -3.348 | 0.035 | | <0.001 |
| Female | 0.159 | 1.172 | 0.503 – 2.729 | 0.713 | |
| BSI | 0.141 | 1.152 | 1.005 – 1.320 | 0.043 | |
| NE activity in ABLF | 0.338 | 1.402 | 1.095 – 1.796 | 0.007 | |
| Productive cough | 1.834 | 6.259 | 1.993 – 19.657 | 0.002 | |
| Model 3 | Constant | -2.989 | 0.050 | | <0.001 |
| Female | 0.144 | 1.155 | 0.492 – 2.712 | 0.741 | |
| BSI | 0.166 | 1.180 | 1.024 -1.361 | 0.022 | |
| NE activity in ABLF | 0.315 | 1.371 | 1.069 – 1.758 | 0.013 | |
| Productive cough | 1.712 | 5.542 | 1.762 – 17.429 | 0.003 | |
| History of hemoptysis | -0.624 | 0.536 | 0.227 – 1.266 | 0.155 |
ABLF: Abnormal bronchial lavage fluid; BSI: bronchiectasis severity index; NE: neutrophil elastase.
Bold values indicate statistically significant results (P < 0.05).
Regarding exacerbation frequency, negative binomial regression also confirmed that NE activity in ABLF was independently associated with the number of exacerbations during the one-year follow-up (rate ratio 1.185; 95% CI 1.015–1.384; p=0.032).
Among the 121 patients with one-year follow-up, ABLF NE activity was divided into tertiles: low, 0.145–1.033 μg/mL, n=40; intermediate, >1.033–2.899 μg/mL, n=39; and high, >2.899 μg/mL, n=42. For the entire cohort, the estimated mean exacerbation-free survival time was 8.6 months (95% CI 7.8–9.4). Kaplan–Meier analysis demonstrated significant differences in time to first exacerbation among the three NE activity groups (log-rank p = 0.001; Figure 4). The estimated mean exacerbation-free survival times were 10.5 months (95% CI 9.4–11.6) in the mild NE activity group, 8.5 months (95% CI 7.1–10.0) in the moderate NE activity group, and 6.9 months (95% CI 5.5–8.4) in the high NE activity group, indicating progressively shorter exacerbation-free survival with increasing ABLF NE activity.
Figure 4.
Kaplan–Meier curve for time to first exacerbation according to ABLF NE activity tertiles. The y-axis shows exacerbation-free survival probability. Differences among groups were assessed using the log-rank test.
Discussion
This prospective multicenter study evaluated NE activity in segment-specific BLF and its prognostic value in stable BE. Our cohort was characterized by an elderly, female-predominant population with a high prevalence of post-tuberculosis bronchiectasis (33.1%), reflecting the epidemiological profile of bronchiectasis in Vietnam and other tuberculosis-endemic Asian settings. 13 NE activity was measurable in all BLF specimens but was substantially higher in ABLF than in NBLF. This spatial difference suggests that computed tomography-targeted bronchoscopic sampling can identify airway compartments with greater local inflammatory and protease activity. Importantly, NE activity measured in BLF from radiologically affected bronchi was independently associated with future exacerbations over one-year. After adjustment for established severity indices, higher ABLF NE activity remained associated with greater exacerbation risk and a shorter time to first exacerbation, supporting NE as an activity-linked biomarker that adds prognostic information beyond structural extent or composite scores. 3
The detection of NE activity in all BLF samples should be interpreted in the context of the sampling method and assay platform. Previous sputum-based studies have not detected free NE activity in all patients with bronchiectasis, even in severe disease. 7 In contrast, our study used lobar-targeted bronchoscopic sampling and a quantitative fluorogenic substrate assay, which may detect low-level localized NE activity in bronchial regions that are diluted, variably represented, or below predefined thresholds in expectorated sputum or semi-quantitative point-of-care assays. However, detectability in BLF should not be equated with high sputum NE activity or severe clinical disease. More than half of our cohort had mild bronchiectasis according to the BSI, and NE activity was substantially lower in NBLF than in ABLF. These findings suggest that low-level NE activity may be measurable even in reference or least affected regions, whereas higher localized activity reflects greater airway protease burden and disease activity.
To our knowledge, this is among the first studies to demonstrate the prognostic value of NE activity using segment-specific BLF sampling in bronchiectasis. Sputum NE is already a well-established biomarker associated with exacerbation frequency, lung-function decline, and disease severity.7,19 Our findings extend this evidence by showing that the same prognostic signal is evident when NE activity is measured directly in the lower-airway compartment. This supports the concept that airway protease activity is consistently associated with clinical instability in bronchiectasis, irrespective of sampling method.
Bronchoscopy is more invasive than sputum sampling. Nevertheless, BLF sampling may have value in selected patients because CT-targeted bronchoscopy allows lavage of the radiologically most affected airway segment and may provide a more lesion-specific assessment of local airway inflammatory/protease activity than sputum sampling. BLF may also be useful when patients cannot expectorate sputum or when sputum results are negative or clinically discordant. In addition, bronchoscopy can provide simultaneous microbiological and diagnostic information, including evaluation for NTM, fungal disease, active tuberculosis in TB-endemic settings when clinically suspected, or obstructive/endobronchial causes. Recent real-world evidence from De Angelis et al. supports this selected-use approach, showing high microbiological yield, management changes in more than half of patients, and a low rate of minor adverse events. 20 Together, these data support the potential value of bronchoscopy-based BLF assessment for selected patients and for segment-specific phenotyping in research.
Patients with CBI and those with positive bacterial isolation had higher ABLF NE activity, supporting infection as an important driver of airway protease activity. At the same time, BLF NE activity may still provide information beyond culture positivity alone. Unlike bacterial isolation, NE activity may reflect several overlapping processes, including infection, neutrophil activation, extracellular release of enzymatically active NE, degranulation, protease–antiprotease imbalance, and structural airway damage. Thus, BLF NE activity may capture the downstream inflammatory and proteolytic consequences of airway infection and structural lung damage, rather than simply duplicating culture results.
Importantly, this study adds evidence for the relationship between NE activity and future exacerbations in an Asian bronchiectasis population. This is clinically relevant given the emergence of dipeptidyl peptidase 1 inhibitors, including brensocatib, which reduce activation of neutrophil serine proteases and have recently expanded the therapeutic landscape for bronchiectasis. 21 It has been observed that Asian patients (incorporating predominantly Japan, Korea, and China) appear particularly responsive to NE inhibition with DPP-1 inhibitors (e.g., Brensocatib), achieving lower rates of exacerbations and better therapeutic responses compared to other populations. 22 By establishing a strong relationship between NE activity and future exacerbations in this Vietnamese cohort, our findings further support NE activity as a clinically prognostic marker and strengthen the rationale for biomarker-guided approaches in future bronchiectasis trials.
The substantial proportion of patients with post-tuberculosis bronchiectasis in our cohort is worth noting, since respiratory morbidity and structural damage frequently persist even after successful microbiological cure. In this subgroup, NE activity was significantly higher in bronchiectatic areas than in reference areas, suggesting that localized protease activity continues to be a prominent driver of ongoing airway inflammation in post-tuberculosis lung damage. While we observed low-level NE activity in the reference areas of these patients, whether this represents normal physiological turnover, subclinical airway inflammation, or broader inflammatory effects of previous infection remains uncertain. Future studies incorporating multi-lobar sampling, paired microbiological assessments, and broader inflammatory profiling are needed to fully map the spatial distribution of disease activity in the post-tuberculosis phenotype.
Our study has several limitations. First, the cohort represents patients willing and eligible to undergo bronchoscopy, which may limit generalizability. Second, NE was measured at a single stable time point; repeated measurements could clarify intra-individual variability and whether changes in NE track with treatment response or evolving microbiology. Additionally, differential cell counts were performed only in BLF from affected bronchi. Consequently, we could not compare neutrophil proportions between bronchiectatic and reference areas across the cohort, which limited our ability to fully map the spatial inflammatory gradient, particularly within the post-tuberculosis subgroup. Exacerbation diagnosis relied partly on patient-reported information, which may introduce recall bias. Finally, microbiological surveillance during follow-up was performed at the clinician’s discretion rather than by protocolized longitudinal sampling, and lacked quantitative bacterial burden assessments and NET-specific biomarkers. Consequently, we may have underestimated the true incidence of CBI and could not determine whether bacterial burden acts as a confounder, mediator, or effect modifier. Therefore, the association between NE activity and future exacerbations should be interpreted as reflecting airway protease activity in a clinical context where bacterial infection may also contribute to risk.
Conclusion
In conclusion, segment-specific NE activity in BLF was higher in radiologically affected airways and was independently associated with one-year exacerbation outcomes in patients with stable bronchiectasis. These findings support NE as a clinically meaningful marker of disease activity in TB-endemic populations and strengthen the therapeutic rationale for protease-targeted approaches in Asian patients.
Acknowledgements
We thank Pham Thi Van Thanh, Duong Minh Ngoc, Tran Ngoc Thai Hoa, Le Thi Kim Chi, Nguyen Kim Tuyen, Nguyen Tiet Au, Nguyen Tuan Anh, and Do Duc Quan for their support in conducting bronchoscopy and collecting bronchial lavage fluid. We also thank Kieu-Minh Le and Van Thanh Niem Vo for their support in measuring neutrophil elastase activity.
Appendix.
Abbreviations
- ABLF
Affected Bronchial Lavage Fluid
- BE
Bronchiectasis
- BLF
Bronchial Lavage Fluid
- BMI
Body Mass Index
- BSI
Bronchiectasis Severity Index
- CBI
Chronic Bacterial Infection
- CI
Confidence Interval
- COPD
Chronic Obstructive Pulmonary Disease
- CT
Computed Tomography
- DAG
Directed Acyclic Graph
- DPP-1
Dipeptidyl Peptidase-1
- EMBARC
European Multicentre Bronchiectasis Audit and Research Collaboration
- FACED
FEV1, Age, Colonization, Extension, Dyspnea
- FEV1
Forced Expiratory Volume in 1 second
- FVC
Forced Vital Capacity
- IQR
Interquartile Range
- MDR
Multidrug-Resistant
- NBLF
Normal (Reference) Bronchial Lavage Fluid
- NE
Neutrophil Elastase
- NTM
Nontuberculous Mycobacteria
- RR
Risk Ratio
- SGRQ
St. George’s Respiratory Questionnaire
- TB
Tuberculosis.
Author contributions: Lam Nguyen-Ho: Conceptualization; Data curation; Formal analysis; Investigation; Methodology; Writing – original draft; Writing – review and editing.
Quoc-Khanh Tran-Le: Data curation; Formal analysis; Investigation; Writing – original draft; Writing – review and editing.
Hoang Kim Tu Trinh: Conceptualization; Methodology; Writing – original draft; Writing – review and editing.
Diem My Vu: Conceptualization; Methodology; Writing – original draft; Writing – review and editing.
Vu Le-Thuong: Conceptualization; Investigation; Writing – original draft; Writing – review and editing.
Ngoc Tran-Van: Conceptualization; Investigation; Writing – original draft; Writing – review and editing.
James D. Chalmers: Conceptualization; Methodology; Writing – original draft; Writing – review and editing.
All authors reviewed and approved the final version of the manuscript and agreed to be accountable for the work.
Funding: The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was partly funded by the University of Medicine and Pharmacy at Ho Chi Minh City (NO. 112/2021/HÐ-ÐHYD).
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Artificial intelligence disclosure: Gemini (Google) was used solely to assist with grammar checking and English-language proofreading. It was not used for data generation or analysis, interpretation of the results, generation of references, or creation of images. All suggested edits were critically reviewed and approved by the authors, who take full responsibility for the accuracy, originality, and integrity of the submitted work.
Conference presentation: Part of the results were previously presented as a poster at the ERS Congress 2025. This study forms part of the PhD thesis of Lam Nguyen-Ho at the University of Medicine and Pharmacy at Ho Chi Minh City.
ORCID iDs
Lam Nguyen-Ho https://orcid.org/0000-0001-7171-2257
Quoc-Khanh Tran-Le https://orcid.org/0000-0002-7788-3767
Ethical considerations
The study protocol was approved by the Ethics Committee in Biomedical Research of the University of Medicine and Pharmacy at Ho Chi Minh City, Vietnam (approval No. 528/HĐĐĐ-ĐHYD; approved on 9 November 2021).
Consent to participate
Written informed consent to participate was obtained from all participants before enrollment.
Consent for publication
The manuscript does not contain identifiable personal information, images, videos, or other materials requiring specific participant consent for publication.
Data Availability Statement
The data supporting the findings of this study are available from the corresponding author upon reasonable request. Access to the data is subject to applicable institutional requirements, ethical approval, and protection of participant confidentiality.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The data supporting the findings of this study are available from the corresponding author upon reasonable request. Access to the data is subject to applicable institutional requirements, ethical approval, and protection of participant confidentiality.