In this observational single-center prospective study, we recruited bronchiectasis patients aged 18–75 years from out-patient clinics of The First Affiliated Hospital of Guangzhou Medical University between March 2017 and November 2018. Bronchiectasis was diagnosed according to chest high-resolution computed tomography (reviewed by an experienced radiologist) with compatible clinical symptoms (e.g. chronic cough, sputum production). Eligible patients remained clinically stable (respiratory symptoms not exceeding normal daily variations), and had no use of antibiotics (except for low-dose macrolides) for four weeks. Active tuberculosis, malignancy, acute respiratory tract infections within four weeks and asthma or COPD as the primary diagnosis were excluded. The study protocol was approved by The Ethics Committee of The First Affiliated Hospital of Guangzhou Medical University (Medical Ethics 2012, the 29th). All patients signed informed consent.

At initial visits, clinical evaluations included demography, clinical history, spirometry and exacerbation rate within the preceding 12 months. Blood and sputum were collected. Spirometry was performed according to international guidelines.20 Radiologic severity was assessed with modified Reiff score.21 Disease severity was calculated with bronchiectasis severity index (BSI)8 and E-FACED score.22 Patients were followed up at 3–6-month intervals until November 2018 (multiple visits), and were requested to contact investigators upon significant worsening of symptoms for an additional visit, scheduled within 48h (antibiotic use, if any, did not exceed 24h). The upper limit of duration from symptom onset was 7 days (5 days after confirming symptom onset) for AE visits. Symptom questionnaire (see Online Supplement) which queried upper and lower airway symptoms [rating the severity with visual analog scale (VAS, range: 0–10)], spirometry, sputum and blood specimens were obtained during each follow-up, including stable visits and AE visits.

AEs were defined as significant deterioration (>48h) of ≥3 symptoms, including cough frequency, sputum volume and/or consistency, sputum purulence, breathlessness and/or exercise tolerance, fatigue and/or malaise, hemoptysis, which required immediate changes in treatment.23 Treatment decisions were made before all testing results became available.

Details are shown in Online Supplement. Briefly, patients thoroughly rinsed their mouth, followed by deep cough for collecting spontaneous sputum. Sputum plugs (the most purulent portion) were selected from eligible samples (leukocytes/epithelial cells >2.5:1).10,11 No uniform techniques of chest physiotherapy was employed. Sputum was immediately split for bacterial culture, viral detection with multiplex quantitative polymerase chain reaction (qPCR), and ultracentrifugation (20,000×g) for 2h at 4°C for sputum sol preparation and storage at −80°C for inflammatory biomarkers (interleukin-1β, CXC motif chemokine-8, tumor necrosis factor-α and interferon-γ) multiplex assays as described previously.10,11

We did bacterial culture by homogenizing fresh sputum with SPUTASOL (Oxoid SR089A, UK), followed by inoculation in blood and chocolate agar plates (Biomeurix Inc., France) for overnight incubation.11 Pathogenic bacteria included, but not limited to, Pseudomonas aeruginosa, Haemophilus influenzae, Haemophilus parainfluenzae, Klebsiella pneumoniae, Streptococcus pneumoniae, Streptococcus aureus and Escherichia coli.10 Isolation of new bacteria denoted sputum culture findings switching from negative to positive, or from any pathogenic bacterium to another pathogenic bacterium.

We extracted viral nucleic acids using extraction kit (TaKaRa MiniBEST Viral RNA/DNA Extraction Kit Ver. 5.0). We conducted qPCR based on TaqMan™ probes to identify sixteen common respiratory viruses: rhinovirus, influenza virus A/B, parainfluenza virus 1–4, human coronavirus (HCoV-229E, OC43, NL63 and HKU1), respiratory syncytial virus, adenovirus, enterovirus, bocavirus and human metapneumovirus. Validated viral detection kits were purchased from Guangzhou HuYanSuo Medical Technology Co., Ltd., Guangzhou, China.11,24 The cycle threshold (Ct) of <40 was considered positive. Lower Ct indicated higher viral loads.

No data exist regarding the proportion of patients with bacterial plus viral isolations in bronchiectasis. Assuming an equivalent proportion of patients with bacterial isolation during stable-states and AEs, and the difference of 20% in virus detection rate between AEs and stable-states,11 107 bronchiectasis patients would be needed based on the two-sided significance of 0.05 and power of 80%, taking into account a 25% drop-out rate.

Data were expressed as mean±standard deviation or median (interquartile range, IQR) for continuous variables, and count (percentage) for categorical variables. Generalized estimating equations with logit link were used to explore the association between pathogen isolation and the odds of AEs compared with stable visits, taking into account repeated observations in individual participants. Continuous variables were analyzed with t-test, analysis-of-variance, Mann–Whitney or Kruskal–Wallis test depending on the variable distribution. Categorical variables were compared with Chi-square or Fisher's exact test. Missing values were not imputed. Statistical analysis was performed using SPSS 18.0 (SPSS Inc., Chicago, USA) and Graphpad Prism version 5.0 (Graphpad Inc., USA).

Of 130 patients screened, 108 patients were enrolled and 98 were followed-up (Fig. 1). The median follow-up duration was 13.0 months. The 108 patients provided 375 sputum samples (299 for stable-visits; 76 for AEs), with a median (IQR) of 3.0 (2.0–4.0) sputum specimens per patient. Seventy-three patients (74.5%) experienced at least one AE, and reported 169 AEs during follow-up (76 AEs sampled because 63.2% contacted too late, 21.1% declined due to no availability, 10.5% administered antibiotics for >2 days, and 5.2% yielded no sputum). Sputum was mostly sampled before antibiotic administration during AEs except that 2 samples were sampled within 24h of antibiotic administration. The clinical characteristics did not differ between patients who did and did not provide sputum during AEs (Table E1).

Fig. 1.

Flow chart of patient recruitment.

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Patient characteristics of the full and AE cohort are shown in Table 1. The mean age was 46.8 years. The median BSI was 7.0 (IQR: 4–9) and the E-FACED score was 2.5 (IQR: 1.0–4.0). The most common etiologies were post-infective and tuberculosis. Asthma was the primary etiology in eight (7.4%) patients.

The percentage of no pathogen detection, bacterial isolation, viral detection and bacterial plus viral detection was 35.8%, 52.8%, 4.4% and 7.0%, respectively during stable-visits, while the corresponding percentage was 23.7%, 47.3%, 14.5% and 14.5%, respectively during AEs (P=.001, Fig. 2A). 59.8% of stable-visits samples and 61.9% of AEs samples tested positive to bacteria (P=.753). The three prevalent species isolated in stable-visits and AEs samples were Pseudomonas aeruginosa (44.4% vs. 32.1%), Haemophilus influenzae (9.8% vs. 15.4%) and Escherichia coli (2.0% vs. 3.8%). However, we noted a higher detection rate of Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae during AEs. The overall bacterial compositions that took into account all bacterial species did not differ remarkably between stable-visits and AEs (P=.070, Fig. 2B).

Fig. 2.

Percentage and composition of pathogens in sputum samples at AEs and stable-visits. Percentage of pathogens in sputum samples at AEs and stable visits. Bacterial and viral composition in sputum samples at AEs and stable visits. AE: acute exacerbation of bronchiectasis. Other bacteria consisted of Proteus mirabilis (n=4), Acinetobacter baumannii (n=2), Moraxella catarrhalis (n=2), Pseudomonas ozanae (n=1), Staphylococcus aureus (n=1), Haemophilus haemolyticus (n=1), Haemophilus parahaemolyticus (n=1), Streptococcus pneumoniae (n=1), Shewanella algae (n=1), Actinomyces ureae (n=1), Pasteurella multocida (n=1), Enterobacter aerogen (n=1) and Serratia marcescens (n=1). There were more patients isolated with two bacteria when clinically stable. Hence, the overall percentage of patients isolated with pathogenic bacteria appeared higher when clinically stable compared with AE onset.

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The proportion of patients tested positive to virus increased from 11.4% at stable-visits to 29.0% at AEs (P=.003). Prevalent viruses included coronavirus (3.4%), herpes simplex virus (2.4%) and influenza A (2.0%) at stable-visits, and influenza B (7.7%), coronavirus (7.7%) and rhinovirus (6.4%) at AEs. The viral spectrum alone did not differ between stable-visits and AEs (P=.396). Dual viral species were detected during 4 (5.3%) AE episodes (Table E2).

Of the 49 patients, 17 (34.7%) provided ≥2 AE sputum samples, for which no identical virus was detected whereas an identical (colonized) bacteria was isolated in 8 patients.

Among 375 sputum samples, isolation of bacteria alone did not correlate with AEs (P>.05, Fig. 3). Nonetheless, isolation of new bacteria occurred more frequently during AEs than stable-visits (OR=2.52, 95%CI: 1.35–4.71), in which culture switching from negative to positive accounted for 66.7% (12/18) of episodes [from culture negative to Haemophilus influenzae (38.9%) and Moraxella catarrhalis (11.1%)], whereas bacterial class-switch accounted for 33.3% (6/18) of episodes [from Pseudomonas aeruginosa to Haemophilus influenzae (16.7%) and other bacteria (11.1%); Pseudomonas aeruginosa was not isolated at most AEs].

Fig. 3.

Association between the detection of different pathogens and the risks of AEs. Notes: OR= Odds Ratio, PA= Pseudomonas aeruginosa, HI= Haemophilus influenzae; AEs: acute exacerbations of bronchiectasis. Any bacteria denotes bacterial culture positive for any bacteria; Isolation of new bacteria denotes sputum culture switching from negative to positive, or sputum culture positive switching from one pathogenic bacterium to other pathogenic bacterium.

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Viral isolations occurred more frequently during AEs than stable-visits (OR=3.28, 95%CI: 1.76–6.12). The odds was highest for rhinovirus (OR=8.14, 95%CI: 1.90–34.81), followed by influenza A/B (OR=4.81, 95%CI: 1.64–14.13). However, isolation of coronavirus did not correlate with AEs (OR=2.80, 95%CI: 0.96–8.21). Moreover, bacterial plus viral isolations occurred more frequently during AEs than stable-visits (OR=2.24, 95%CI: 1.11–4.55).

At baseline, pathogen (including Haemophilus influenzae) isolation status failed to predict future risks of AEs during follow-up (Table E3). Neither bacterial nor viral isolations alone at baseline predicted a shorter time to the next AEs during follow-up (Fig. E1).

We collected 52 and 24 samples from warmer (May-October) and colder seasons (November-April), between which the detection rate of viruses differed significantly (36.5% vs. 12.5%, P=.032). However, we noted no significant difference in the rate of bacterial isolation (61.5% vs. 62.5%, P=.936), nor did the rate of isolating new bacteria (19.2% vs. 33.3%, P=.179) (Fig. E2).

Next, we stratified patients at AEs as: (1) New bacterial AE (50.0%): isolation of new bacteria; (2) Viral AE (21.1%): detection of any virus; (3) Unexplained AE (26.3%): AE without isolation of new bacteria or detection of viruses. Two AEs were not analyzed because of simultaneous detection of virus and new bacteria. Demographic and clinical characteristics did not differ among these subgroups (Table E4).

Symptom questionnaires were obtained from 58 (78.4%) AEs. Viral AEs tended to yield more coryza symptoms than unexplained AEs and new bacterial AEs (P=.053, Table 2). Neither the number nor the VAS of lower airway symptoms differed among the three groups during stable-visits (Fig. 4A). However, increased sputum purulence (mean difference: 2–4 for VAS) and breathlessness (mean difference: 1.6 for VAS) deteriorated most notably during new bacterial AE (Fig. 4B and C).

Fig. 4.

The severity of lower airway symptoms assessed with the visual analog scale. (A) The VAS during stable visits. (B) The VAS during AEs. (C) The difference in VAS between AEs and stable visits. Notes: AE: acute exacerbation of bronchiectasis; VAS: visual analog scale.

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Spirometry was assessed in 46 (62.2%) AEs. Lung function decline did not differ among viral, new bacterial and unexplained AEs (Figure E3). Sputum cytokines and total leukocyte count were detected in 53 (71.6%) and 39 (51.3%) AEs, respectively. Median interleukin-1β levels increased significantly in new bacterial AEs than in unexplained AEs (P=.006) and viral AEs (P=.005), as did tumor necrosis factor-α levels except for the comparison with viral AEs (P=.138). New bacterial AEs trended toward higher blood neutrophil counts, while viral AEs yielded higher monocyte counts (Fig. 5).

Fig. 5.

The airway and systemic inflammations in different groups. (A) The level of sputum cytokines during AEs. (B) White blood cell count during AEs. (C) The level of C-reactive protein during AEs. (D) The difference in inflammatory cell count between AEs and stable visits. Notes: AE: acute exacerbation of bronchiectasis.

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To further investigate the characteristics of bacterial plus viral isolations, AEs were divided into: no bacteria/viruses detected (B−V−, n=18); Bacteria detected alone (B+V−, n=36); Viruses detected alone (B−V+, n=11); both bacteria and viruses detected (B+V+, n=11).

There were more coryza symptoms in B+V+ group than in B−V− group (median: 4.6 vs. 1.6, P=.019). Ocular itching appeared more frequent in B+V+ group (42.9%) than in B−V− (0%) and B+V− group (7.1%, Table 3). The VAS for cough, sputum and sputum purulence tended to be higher in B+V+ and B+V− groups (Ptrend<0.05, Fig. E4). The greatest lung function decline (Ptrend=0.043 for forced vital capacity) was noted in B−V− group (Fig. E5). However, there was no notable among-group difference in sputum inflammatory biomarker levels (all P>.05) (Fig. E6).