Viral Infections in BAL and Bronchial Biopsies of Stable COPD Patients
Condition: HRV and RSV Presence in the Lungs of Stable COPD Patients.
Study type: Observational

Summary: 
      Background: The presence of low-grade viral infection in the airways of patients with stable
      chronic obstructive pulmonary disease (COPD) could potentially have implications in the
      pathogenesis and progression of the disease, but previous studies have reported very
      different rates of human rhinovirus (HRV) and respiratory syncytial virus (RSV) genome
      detection in nasal and sputum samples. However, no study has investigated the presence of
      these viruses directly in the lungs by bronchoalveolar lavage (BAL) and bronchial biopsies.
      This study aimed to investigate whether HRV and RSV are present in the lungs of stable COPD
      patients by performing BAL and bronchial biopsies, and relate their presence with disease
      severity.

      Methods: Consecutive patients with stable COPD and control subjects, who underwent diagnostic
      (e.g., lung cancer) and/or therapeutic (e.g., hemoptysis) fibreoptic bronchoscopy in a
      university hospital in Athens, Greece, were enrolled. The collected BAL and bronchial
      biopsies during bronchoscopy were subsequently processed for HRV and RSV RNA detection with
      real-time polymerase chain reaction (PCR). More specifically, the nucleocapsid gene and 5΄
      non-coding region were searched for RSV and HRV detection, respectively.
    

Detailed description: 
      Patients and control subjects The population of the study consisted of consecutive patients
      with stable COPD and control subjects, who were referred to the investigators between October
      2012 and November 2014 in order to undergo fibreoptic bronchoscopy for diagnostic (e.g., lung
      cancer) or therapeutic (e.g., hemoptysis) purposes. At study time, the clinical and
      functional status of COPD patients had been stable for at least 4 weeks, and none had
      received antibiotic treatment during the last 4 weeks. Exclusion criteria included atopic
      history, asthma, extensive pleural effusions, bronchiectasis, immunosuppression due to
      chemotherapy or systemic corticosteroids and all the contraindications of the bronchoscopic
      procedures. COPD severity was classified using postbronchodilator spirometric values
      according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines.
      Also, the patients were evaluated according to the combined COPD assessment. In this
      classification the impact of the disease on an individual patient is determined from the
      combination of the symptomatic assessment (CAT or mMRC scores), the patient's spirometric
      classification and/or the risk of exacerbations (number of exacerbations and history of
      hospitalization due to exacerbation in the preceding year).The control group was consisted of
      non-smokers or current/ex-smokers without COPD. The ex-smoking condition was defined as the
      cessation of smoking habit at least 1 year previously.

      All the participants completed a questionnaire which included demographic characteristics and
      cigarette smoking status (pack/year). Τhe clinical status was evaluated according to the
      modified Medical Research Council (mMRC) scale of breathlessness and the Body mass , airflow
      Obstruction, Dyspnea, Exercise (BODE) index. Pulmonary function testing was performed and
      arterial blood gases and blood oxygen saturation by pulse oximetry in the sitting position
      were measured. Patients were asked about the number and the characteristics of the preceding
      exacerbations, the number and duration of preceding hospitalizations for respiratory
      disorders, their past medical history (asthma, allergy, sinusitis or nasal polyps), the
      presence of comorbidities (heart disease, malignancies, e.t.c.), and all the medication they
      used.

      Both the COPD patients and control subjects were appropriately prepared (local anesthesia and
      intravenous sedation) in order to undergo transnasal fibreoptic bronchoscopy. After the
      routine bronchoscopic inspection of the tracheobronchial tree, the investigators performed
      BAL from subsegmental bronchi and bronchial biopsies (3-4 per patient) from non infiltrated
      carina.

      All the samples were aliquoted, frozen and stored within 1 hour after sampling. HRV and RSV
      detection RNA samples were extracted from BAL and lung biopsies using the Maxwell 16 viral
      total nucleic acid (Promega). A Nucleocapsid (N) gene partial sequence, as well as a 5΄
      non-coding region (5΄NCR) partial sequence were amplified for RSV and HRV, respectively.

      Real-time PCR studies were performed. The primers/probes mix, that were used, have broad
      homology with a large range of HRV serotypes. Also, the primers/probes for RSV are able to
      distinguish RSV subtypes A and B whilst excluding the closely related human
      Metapneumoviruses. The primer and probe mix provided exploited the so-called TaqMan
      principle. A fluorogenic probe was included in the same reaction mixture which consisted of a
      DNA probe labeled with a 5'-dye and a 3'-quencher. Besides this, the probe was cleaved and
      the reporter dye and quencher were separated. The resulting increase in fluorescence could be
      detected on a range of real-time PCR platforms. Each time the kits were used, a positive and
      a negative control reaction were included in the run.
    

Intervention name(s): BRONCHOSCOPIC PROCEDURE
Intervention type(s): Procedure
Intervention description(s): After the routine bronchoscopic inspection of the tracheobronchial tree, the investigators performed BAL from subsegmental bronchi and bronchial biopsies (3-4 per patient) from non infiltrated carina
Observational model: Case-Control
Time perspective: Prospective
Sampling method: Probability Sample
Study population: 
        We enrolled 31 patients with COPD and 22 control subjects. The most common symptoms were
        dyspnea (32%), cough (19%) and hemoptysis (13%). Whereas 47% of participants did not
        mention any symptom, but referred to our department in order to investigate
        bronchoscopically abnormal radiographic findings revealed in chest computed tomography. The
        commonest comorbidities of COPD patients were arterial hypertension (47%), hyperlipidemia
        (20.7%) and coronary artery disease (17%), and of control subjects arterial hypertension
        (50%), hyperlipidemia (22,7%) and diabetes mellitus type 2 (13,6%).
      
Biospecimen description: 
      BAL and bronchial biopsies
    
Keywords: stable COPD; bronchoalveolar lavage; biopsy; HRV; RSV