Absence of human rhinovirus and respiratory syncytial virus from bronchoalveolar lavage and bronchial biopsies of selected patients with stable chronic obstructive pulmonary disease

Previous studies have reported very different rates of human rhinovirus (HRV) and respiratory syncytial virus (RSV) genome detection in nasal and sputum samples, but not in bronchoalveolar lavage (BAL) and bronchial biopsy samples. Our study aimed to investigate the presence of HRV and RSV in the lungs of 31 consecutive patients with stable COPD (11 GOLD stage I, 11 II, and 9 III) and 22 control subjects (12 current or past smokers, and 10 non-smokers), who underwent diagnostic (e.g., lung cancer) and/or therapeutic (e.g., hemoptysis) fibreoptic bronchoscopy in a university hospital in Athens, Greece. Viral RNA of HRV and RSV were not detected in any of the samples of COPD patients or control subjects after being processed with real-time PCR.


Background
The potential role of persistent low-grade, latent viral infection in disease progression in stable COPD is a matter of great importance [1,2] and controversy partly due to differences in the reported occurrence of asymptomatic virus infection in COPD. Indeed, in the study by Seemungal et al. [3] HRV and RSV were detected in blood samples/nasal aspirates in 7,4 and 23,5 % of patients with stable COPD, respectively, whereas in the study by Falsey et al [4] nasal and sputum samples were positive for RSV in only 0 and 0,9 %, respectively. Importantly, none of the previous studies evaluated samples coming directly from the lower airways, i.e., bronchoalveolar lavage (BAL) fluid or bronchial biopsies, which are fully representative of the lower respiratory tract. Nevertheless, it is constantly stressed out that further studies across the spectrum of disease as well as in smoking and non smoking control subjects are required to better understand the role of asymptomatic viral infection in stable COPD [1,2]. Therefore, the aim of this study was 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
The population of the study consisted of consecutive patients with stable COPD and control subjects (nonsmokers or current/ex-smokers), who were referred to the Sismanoglion Hospital, Athens, Greece, 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 [5], 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.
All the participants completed a questionnaire which included demographic characteristics, cigarette smoking status (pack/year) and their past medical history. Τheir 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. We performed BAL from subsegmental bronchi and bronchial biopsies (3-4 per patient) from non infiltrated carina. RNA samples were extracted from BAL and lung biopsies and a nucleocapsid (N) gene partial sequence, as well as a 5' noncoding region (5΄NCR) partial sequence were amplified for RSV and HRV, respectively. Real-time PCR studies were performed in Stratagene Mx3005 Real-Time PCR Instrument (Agilent Technologies, Santa Clara, CA, USA). The primer and probe mix provided exploited the so-called Taq-Man principle. Each time the kits were used, a positive and a negative control reaction were included in the run.
The study was approved by the local Medical Ethics Committee of Sismanoglion Hospital and all subjects gave their informed consent.
Anthropometric characteristics and lung function data of both COPD patients and control subjects are shown in Table 1. Controls and COPD patients had comparable age, FVC, D LCO and PaCO 2 . As expected, COPD patients had lower spirometric values, arterial blood gases measurements and higher rate of smoking habits, mMRC scale and BODE index scores compared to control subjects.
All BAL and bronchial biopsy samples (53 and 181, respectively) were processed for the detection of HRV and RSV genome with real-time PCR. In Fig.1a and b we can see representative amplification plots for HRV and RSV, respectively.
In all amplification plots for HRV and RSV, samples' and negative controls' fluorescence signals were at the same level indicating that these two viruses were not detected in any of the samples of COPD patients and control subjects.

Discussion
There are sparse publications and controversial reports concerning viral infection of the respiratory tract in stable COPD [1,3,4,6]. Our study had several limitations that  probably influenced the results. More specifically, we included patients from 3 of the 4 GOLD stages as it was rather difficult to perform bronchoscopy in patients with very severe COPD. This could have an impact on our results as the relative importance of airway bacterial infection in COPD has been shown to increase with disease severity [7]. Also, most of the COPD patients did not experience any exacerbations during the year before recruitment, while there is a tendency for patients in whom HRV and RSV are detected in stable COPD to give a history of more frequent exacerbations in the previous year [3]. Additionally, the number of participants in the study was relatively small, and the enrollment of patients was performed in only one center. On the other hand, the detection rates for HRV and RSV among the two groups of normal control subjects and the results were zero, which is similar to previous reports [8,9]. In addition to this, the majority of our participants did not use inhaled and oral corticosteroids on a chronic basis. This is very important as prolonged shedding of RSV has been described in a number of immunosuppressive conditions such as corticosteroid use and HIV infection [10]. The PCR kits that we used are designed to have the broadest detection profile possible whilst remaining specific to the HRVsp and to the RSV_spp genome. We ensured that the samples were suitable in terms of purity, concentration, and RNA/ DNA integrity and we always run at least one negative control with the samples. An alternative explanation for the discrepancy between this study and previous reports is the possibility of PCR contamination which leads to high rates of HRV and RSV RNA identification in subjects with stable COPD.
In conclusion, we have shown for the first time using bronchoscopy that both HRV and RSV RNA were not detected in the BAL and bronchial biopsy samples collected from patients with stable COPD and from control subjects. The possibility of chronic HRV and RSV infection as a mechanism of disease progression could have enormous impact on the management of the disease but our study does not support this hypothesis.