Editorials 13. Navani N, Molyneaux PL, Breen RA, Connell DW, Jepson A, Nankivell M, Brown JM, Morris-Jones S, Ng B, Wickremasinghe M, et al. Utility of endobronchial ultrasound-guided transbronchial needle aspiration in patients with tuberculous intrathoracic lymphadenopathy: a multicentre study. Thorax 2011;66:889–893. 14. Du Rand IA, Barber PV, Goldring J, Lewis RA, Mandal S, Munavvar M, Rintoul RC, Shah PL, Singh S, Slade MG, et al.; British Thoracic Society Interventional Bronchoscopy Guideline Group. British Thoracic Society guideline for advanced diagnostic and therapeutic flexible bronchoscopy in adults. Thorax 2011;66:iii1–iii21. 15. Moonim MT, Breen R, Fields PA, Santis G. Diagnosis and subtyping of de novo and relapsed mediastinal lymphomas by endobronchial ultrasound needle aspiration. Am J Respir Crit Care Med 2013;188:1216–1223. 16. Navani N, Nankivell M, Nadarajan P, Pereira SP, Kocjan G, Janes SM. The learning curve for EBUS-TBNA. Thorax 2011;66:352–353. 17. Iqbal S, DePew ZS, Kurtin PJ, Sykes AM, Johnson GB, Edell ES, Habermann TM, Maldonado F. Endobronchial ultrasound and lymphoproliferative disorders: a retrospective study. Ann Thorac Surg 2012;94:1830–1834.
1185 18. Kennedy MP, Jimenez CA, Bruzzi JF, Mhatre AD, Lei X, Giles FJ, Fanning T, Morice RC, Eapen GA. Endobronchial ultrasound-guided transbronchial needle aspiration in the diagnosis of lymphoma. Thorax 2008;63:360–365. 19. Ko HM, da Cunha Santos G, Darling G, Pierre A, Yasufuku K, Boerner SL, Geddie WR. Diagnosis and subclassification of lymphomas and nonneoplastic lesions involving mediastinal lymph nodes using endobronchial ultrasound-guided transbronchial needle aspiration. Diagn Cytopathol [online ahead of print] 31 May 2011; DOI: 10.1002/dc.21741. 20. Marshall CB, Jacob B, Patel S, Sneige N, Jimenez CA, Morice RC, Caraway N. The utility of endobronchial ultrasound-guided transbronchial needle aspiration biopsy in the diagnosis of mediastinal lymphoproliferative disorders. Cancer Cytopathol 2011;119:118–126. 21. Steinfort DP, Conron M, Tsui A, Pasricha SR, Renwick WE, Antippa P, Irving LB. Endobronchial ultrasound-guided transbronchial needle aspiration for the evaluation of suspected lymphoma. J Thorac Oncol 2010;5:804–809. Copyright ª 2013 by the American Thoracic Society DOI: 10.1164/rccm.201309-1701ED
The Lung Microbiome and Viral-induced Exacerbations of Chronic Obstructive Pulmonary Disease: New Observations, Novel Approaches Chronic obstructive pulmonary disease (COPD) is responsible for an enormous global burden of morbidity, mortality, and healthcare expense, and is the only leading cause of death with a rising prevalence (1). The course of COPD is complicated by acute exacerbations (AEs), which are associated with high mortality, accelerated loss of lung function, and a large fraction of the disease’s direct cost (2). The majority of AEs are associated with isolation of bacterial or viral pathogens from respiratory specimens (3), and the concurrent isolation of both is associated with increased inflammation and exacerbation severity (4). The mechanisms of interaction between viruses, bacteria, and the host in the pathogenesis of AEs have not been elucidated, as prior investigations have been hampered by inadequate animal models (5) and by dependence on limited culture-based techniques of microbial identification (6). In this issue of the Journal, Molyneaux and coworkers (pp. 1224–1231) demonstrate how novel investigative approaches may be integrated to bypass these difficulties and elucidate the contributions of microbes to the pathogenesis of AEs (7). They employed a previously described human model of AEs in which rhinovirus is instilled into the nares of subjects with mild COPD, provoking the clinical, physiologic, and inflammatory features associated with AEs (8). The authors collected induced sputum from subjects with and without COPD at multiple time points before and after rhinovirus instillation and characterized the bacterial microbiota of the respiratory tract using a modern culture-independent technique (pyrosequencing of bacterial 16S ribosomal RNA gene libraries). They observed no significant difference in the microbiota of COPD subjects and control subjects at baseline, but significant changes detectable at 15 days after instillation. Unlike that of control subjects, the postexposure sputum of COPD subjects exhibited an increase in the amount of bacterial DNA and a significant change in bacterial community composition, driven by a phylum shift toward Proteobacteria and away from Firmicutes and Bacteroidetes. Several studies in recent years have applied similar molecular techniques to respiratory specimens from patients with
COPD (9–12), though only one to date has included patients experiencing an AE (13). Most reports have found a distinct microbiome in COPD lungs when compared with those of controls, often also reporting a relative increase in Proteobacteria over other prominent phyla (6). In the current study, the apparent lack of difference observed between the preintervention sputum microbiota of COPD subjects and control subjects likely reflects the relatively mild severity of disease in this study’s participants, an unavoidable limitation of this model and the necessities of safe and ethical study of human subjects. Yet the significant differences detected in sputum microbiota 15 days after rhinovirus exposure in the same subjects illustrate the importance of treating the lung microbiome in COPD as a dynamic and context-dependent phenomenon. Importantly, the authors observe that the specific members of Proteobacteria that exhibited the most dramatic postexposure increase in abundance (Haemophilus sp. and Neisseria sp.) were present in lower numbers among the microbiota of the same COPD subjects before rhinovirus exposure. This key observation illustrates the plausibility of the hypothesis that bacteria in AEs are not newly introduced pathogens, but instead reflect selective outgrowth of newly favored species from a previously homeostatic microbial ecosystem. Important as this observation may be, the most significant impact of this study should be what it illustrates about the future and potential of lung microbiome studies. With few exceptions, studies of lung microbiota to date have been cross-sectional, observational, and descriptive (6). With their experimental design, Molyneaux and colleagues have demonstrated a means of testing mechanistic hypotheses in human subjects to a degree previously unapproached in the field. Rather than relying on comparisons of pooled averages of populations, the serial analysis of subject specimens at predefined time points after a controlled exposure permits comparison of subjects with themselves, a more rigorous and informative analysis. The use of changes in lung microbiota as the primary endpoint of an experimental intervention is novel, and will be a necessary approach as future studies attempt to
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unfold the dynamism and complexity of lung microbiota. The use of a viral exposure to influence the bacterial composition of lung microbiota is an important first step in the integration of the various “-omes” of the lung (the microbiome, the virome, the genome, the metabolome, etc.), which will be critical in understanding the complex interface between the host and its microbial exposures. Finally, the authors used induced sputum specimens to detect meaningful bacterial signal that both changed with exposure and correlated tightly with the presence of inflammatory cells (see Molyneaux and colleagues’ Figures 1B and 1D); this confirms that induced sputum is a reasonable noninvasive modality to use in assessing the microbiota of the respiratory tract. A critical limitation of this study is its small number of subjects and, importantly, the heterogeneous response of subjects with COPD to inhaled rhinovirus. The changes in total bacterial DNA and shifts in prominent phyla were driven by the responses of only two subjects (see Molyneaux and colleagues’ Figures 1B and 5B), to whom the reported increase in the number of Proteobacteria sequences at Day 15 can be attributed. This limits the generalizability of the study’s findings and raises the question of why there is no evident change in sputum bacterial community composition among the larger group of other COPD subjects. Possibilities include variable susceptibility to the effects of viral infection and the relatively low resolution of bacterial community profiling achieved (only 927 sequences analyzed per subject). The heterogeneity of responses to rhinovirus infection also underscores the heterogeneity of COPD and the need for refined disease phenotyping (14). The “frequent-exacerbator” phenotype has been proposed as an important and independent subset of the COPD population (15) and has become a target population for recent clinical trials (16). The findings of the current study demonstrate that even among subjects with COPD with similar lung function and baseline lung microbiota, the dynamic response to viral exposure can be profoundly varied. The lung microbiome and its response to perturbation should be incorporated into future attempts to phenotype patients with COPD. Finally, the stark discordance between the authors’ cultureindependent findings and the results of clinical microbial culture is yet further evidence of the limitations of past approaches to understanding the contribution of bacterial infection to the pathogenesis of COPD. Everything we think we know about this topic derives from a half-century of experimentation and observation using culture-based techniques (3). The current study by Molyneaux and colleagues is an important first step in the needed task of revisiting old conclusions and assumptions with fresh approaches. Author disclosures are available with the text of this article at www.atsjournals.org.
Robert P. Dickson, M.D. Department of Internal Medicine University of Michigan Medical School Ann Arbor, Michigan Yvonne J. Huang, M.D. Department of Medicine University of California San Francisco San Francisco, California Fernando J. Martinez, M.D. Department of Internal Medicine University of Michigan Medical School Ann Arbor, Michigan
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Gary B. Huffnagle, Ph.D. Department of Internal Medicine University of Michigan Medical School Ann Arbor, Michigan and Department of Microbiology and Immunology University of Michigan Medical School Ann Arbor, Michigan References 1. Hurd S. The impact of COPD on lung health worldwide: epidemiology and incidence. Chest 2000; 117(2 Suppl)1S–4S. 2. Anzueto A, Sethi S, Martinez FJ. Exacerbations of chronic obstructive pulmonary disease. Proc Am Thorac Soc 2007;4:554–564. 3. Sethi S, Murphy TF. Infection in the pathogenesis and course of chronic obstructive pulmonary disease. N Engl J Med 2008;359:2355–2365. 4. Wilkinson TM, Hurst JR, Perera WR, Wilks M, Donaldson GC, Wedzicha JA. Effect of interactions between lower airway bacterial and rhinoviral infection in exacerbations of COPD. Chest 2006;129:317–324. 5. Wright JL, Cosio M, Churg A. Animal models of chronic obstructive pulmonary disease. Am J Physiol Lung Cell Mol Physiol 2008;295:L1–L15. 6. Dickson RP, Erb-Downward JR, Huffnagle GB. The role of the bacterial microbiome in lung disease. Expert Rev Respir Med 2013;7:245–257. 7. Molyneaux PL, Mallia P, Cox MJ, Footitt J, Willis-Owen SA, Homola D, Trujillo-Torralbo MB, Elkin S, Kon OM, Cookson WO, et al. Outgrowth of the bacterial airway microbiome following rhinovirus exacerbation of chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2013;188:1224–1231. 8. Mallia P, Message SD, Gielen V, Contoli M, Gray K, Kebadze T, Aniscenko J, Laza-Stanca V, Edwards MR, Slater L, et al. Experimental rhinovirus infection as a human model of chronic obstructive pulmonary disease exacerbation. Am J Respir Crit Care Med 2011;183: 734–742. 9. Sze MA, Dimitriu PA, Hayashi S, Elliott WM, McDonough JE, Gosselink JV, Cooper J, Sin DD, Mohn WW, Hogg JC. The lung tissue microbiome in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2012;185:1073–1080. 10. Erb-Downward JR, Thompson DL, Han MK, Freeman CM, McCloskey L, Schmidt LA, Young VB, Toews GB, Curtis JL, Sundaram B, et al. Analysis of the lung microbiome in the “healthy” smoker and in COPD. PLoS ONE 2011;6:e16384. 11. Cabrera-Rubio R, Garcia-Nún˜ez M, Setó L, Antó JM, Moya A, Monsó E, Mira A. Microbiome diversity in the bronchial tracts of patients with chronic obstructive pulmonary disease. J Clin Microbiol 2012;50: 3562–3568. 12. Pragman AA, Kim HB, Reilly CS, Wendt C, Isaacson RE. The lung microbiome in moderate and severe chronic obstructive pulmonary disease. PLoS ONE 2012;7:e47305. 13. Huang YJ, Kim E, Cox MJ, Brodie EL, Brown R, Wiener-Kronish JP, Lynch SV. A persistent and diverse airway microbiota present during chronic obstructive pulmonary disease exacerbations. OMICS 2010; 14:9–59. 14. Han MK, Agusti A, Calverley PM, Celli BR, Criner G, Curtis JL, Fabbri LM, Goldin JG, Jones PW, Macnee W, et al. Chronic obstructive pulmonary disease phenotypes: the future of COPD. Am J Respir Crit Care Med 2010;182:598–604. 15. Hurst JR, Vestbo J, Anzueto A, Locantore N, Müllerova H, Tal-Singer R, Miller B, Lomas DA, Agusti A, Macnee W, et al.; Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE) Investigators. Susceptibility to exacerbation in chronic obstructive pulmonary disease. N Engl J Med 2010;363:1128–1138. 16. Albert RK, Connett J, Bailey WC, Casaburi R, Cooper JA Jr, Criner GJ, Curtis JL, Dransfield MT, Han MK, Lazarus SC, et al.; COPD Clinical Research Network. Azithromycin for prevention of exacerbations of COPD. N Engl J Med 2011;365:689–698. Copyright ª 2013 by the American Thoracic Society DOI: 10.1164/rccm.201309-1573ED
1185 18. Kennedy MP, Jimenez CA, Bruzzi JF, Mhatre AD, Lei X, Giles FJ, Fanning T, Morice RC, Eapen GA. Endobronchial ultrasound-guided transbronchial needle aspiration in the diagnosis of lymphoma. Thorax 2008;63:360–365. 19. Ko HM, da Cunha Santos G, Darling G, Pierre A, Yasufuku K, Boerner SL, Geddie WR. Diagnosis and subclassification of lymphomas and nonneoplastic lesions involving mediastinal lymph nodes using endobronchial ultrasound-guided transbronchial needle aspiration. Diagn Cytopathol [online ahead of print] 31 May 2011; DOI: 10.1002/dc.21741. 20. Marshall CB, Jacob B, Patel S, Sneige N, Jimenez CA, Morice RC, Caraway N. The utility of endobronchial ultrasound-guided transbronchial needle aspiration biopsy in the diagnosis of mediastinal lymphoproliferative disorders. Cancer Cytopathol 2011;119:118–126. 21. Steinfort DP, Conron M, Tsui A, Pasricha SR, Renwick WE, Antippa P, Irving LB. Endobronchial ultrasound-guided transbronchial needle aspiration for the evaluation of suspected lymphoma. J Thorac Oncol 2010;5:804–809. Copyright ª 2013 by the American Thoracic Society DOI: 10.1164/rccm.201309-1701ED
The Lung Microbiome and Viral-induced Exacerbations of Chronic Obstructive Pulmonary Disease: New Observations, Novel Approaches Chronic obstructive pulmonary disease (COPD) is responsible for an enormous global burden of morbidity, mortality, and healthcare expense, and is the only leading cause of death with a rising prevalence (1). The course of COPD is complicated by acute exacerbations (AEs), which are associated with high mortality, accelerated loss of lung function, and a large fraction of the disease’s direct cost (2). The majority of AEs are associated with isolation of bacterial or viral pathogens from respiratory specimens (3), and the concurrent isolation of both is associated with increased inflammation and exacerbation severity (4). The mechanisms of interaction between viruses, bacteria, and the host in the pathogenesis of AEs have not been elucidated, as prior investigations have been hampered by inadequate animal models (5) and by dependence on limited culture-based techniques of microbial identification (6). In this issue of the Journal, Molyneaux and coworkers (pp. 1224–1231) demonstrate how novel investigative approaches may be integrated to bypass these difficulties and elucidate the contributions of microbes to the pathogenesis of AEs (7). They employed a previously described human model of AEs in which rhinovirus is instilled into the nares of subjects with mild COPD, provoking the clinical, physiologic, and inflammatory features associated with AEs (8). The authors collected induced sputum from subjects with and without COPD at multiple time points before and after rhinovirus instillation and characterized the bacterial microbiota of the respiratory tract using a modern culture-independent technique (pyrosequencing of bacterial 16S ribosomal RNA gene libraries). They observed no significant difference in the microbiota of COPD subjects and control subjects at baseline, but significant changes detectable at 15 days after instillation. Unlike that of control subjects, the postexposure sputum of COPD subjects exhibited an increase in the amount of bacterial DNA and a significant change in bacterial community composition, driven by a phylum shift toward Proteobacteria and away from Firmicutes and Bacteroidetes. Several studies in recent years have applied similar molecular techniques to respiratory specimens from patients with
COPD (9–12), though only one to date has included patients experiencing an AE (13). Most reports have found a distinct microbiome in COPD lungs when compared with those of controls, often also reporting a relative increase in Proteobacteria over other prominent phyla (6). In the current study, the apparent lack of difference observed between the preintervention sputum microbiota of COPD subjects and control subjects likely reflects the relatively mild severity of disease in this study’s participants, an unavoidable limitation of this model and the necessities of safe and ethical study of human subjects. Yet the significant differences detected in sputum microbiota 15 days after rhinovirus exposure in the same subjects illustrate the importance of treating the lung microbiome in COPD as a dynamic and context-dependent phenomenon. Importantly, the authors observe that the specific members of Proteobacteria that exhibited the most dramatic postexposure increase in abundance (Haemophilus sp. and Neisseria sp.) were present in lower numbers among the microbiota of the same COPD subjects before rhinovirus exposure. This key observation illustrates the plausibility of the hypothesis that bacteria in AEs are not newly introduced pathogens, but instead reflect selective outgrowth of newly favored species from a previously homeostatic microbial ecosystem. Important as this observation may be, the most significant impact of this study should be what it illustrates about the future and potential of lung microbiome studies. With few exceptions, studies of lung microbiota to date have been cross-sectional, observational, and descriptive (6). With their experimental design, Molyneaux and colleagues have demonstrated a means of testing mechanistic hypotheses in human subjects to a degree previously unapproached in the field. Rather than relying on comparisons of pooled averages of populations, the serial analysis of subject specimens at predefined time points after a controlled exposure permits comparison of subjects with themselves, a more rigorous and informative analysis. The use of changes in lung microbiota as the primary endpoint of an experimental intervention is novel, and will be a necessary approach as future studies attempt to
1186
AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE
unfold the dynamism and complexity of lung microbiota. The use of a viral exposure to influence the bacterial composition of lung microbiota is an important first step in the integration of the various “-omes” of the lung (the microbiome, the virome, the genome, the metabolome, etc.), which will be critical in understanding the complex interface between the host and its microbial exposures. Finally, the authors used induced sputum specimens to detect meaningful bacterial signal that both changed with exposure and correlated tightly with the presence of inflammatory cells (see Molyneaux and colleagues’ Figures 1B and 1D); this confirms that induced sputum is a reasonable noninvasive modality to use in assessing the microbiota of the respiratory tract. A critical limitation of this study is its small number of subjects and, importantly, the heterogeneous response of subjects with COPD to inhaled rhinovirus. The changes in total bacterial DNA and shifts in prominent phyla were driven by the responses of only two subjects (see Molyneaux and colleagues’ Figures 1B and 5B), to whom the reported increase in the number of Proteobacteria sequences at Day 15 can be attributed. This limits the generalizability of the study’s findings and raises the question of why there is no evident change in sputum bacterial community composition among the larger group of other COPD subjects. Possibilities include variable susceptibility to the effects of viral infection and the relatively low resolution of bacterial community profiling achieved (only 927 sequences analyzed per subject). The heterogeneity of responses to rhinovirus infection also underscores the heterogeneity of COPD and the need for refined disease phenotyping (14). The “frequent-exacerbator” phenotype has been proposed as an important and independent subset of the COPD population (15) and has become a target population for recent clinical trials (16). The findings of the current study demonstrate that even among subjects with COPD with similar lung function and baseline lung microbiota, the dynamic response to viral exposure can be profoundly varied. The lung microbiome and its response to perturbation should be incorporated into future attempts to phenotype patients with COPD. Finally, the stark discordance between the authors’ cultureindependent findings and the results of clinical microbial culture is yet further evidence of the limitations of past approaches to understanding the contribution of bacterial infection to the pathogenesis of COPD. Everything we think we know about this topic derives from a half-century of experimentation and observation using culture-based techniques (3). The current study by Molyneaux and colleagues is an important first step in the needed task of revisiting old conclusions and assumptions with fresh approaches. Author disclosures are available with the text of this article at www.atsjournals.org.
Robert P. Dickson, M.D. Department of Internal Medicine University of Michigan Medical School Ann Arbor, Michigan Yvonne J. Huang, M.D. Department of Medicine University of California San Francisco San Francisco, California Fernando J. Martinez, M.D. Department of Internal Medicine University of Michigan Medical School Ann Arbor, Michigan
VOL 188
2013
Gary B. Huffnagle, Ph.D. Department of Internal Medicine University of Michigan Medical School Ann Arbor, Michigan and Department of Microbiology and Immunology University of Michigan Medical School Ann Arbor, Michigan References 1. Hurd S. The impact of COPD on lung health worldwide: epidemiology and incidence. Chest 2000; 117(2 Suppl)1S–4S. 2. Anzueto A, Sethi S, Martinez FJ. Exacerbations of chronic obstructive pulmonary disease. Proc Am Thorac Soc 2007;4:554–564. 3. Sethi S, Murphy TF. Infection in the pathogenesis and course of chronic obstructive pulmonary disease. N Engl J Med 2008;359:2355–2365. 4. Wilkinson TM, Hurst JR, Perera WR, Wilks M, Donaldson GC, Wedzicha JA. Effect of interactions between lower airway bacterial and rhinoviral infection in exacerbations of COPD. Chest 2006;129:317–324. 5. Wright JL, Cosio M, Churg A. Animal models of chronic obstructive pulmonary disease. Am J Physiol Lung Cell Mol Physiol 2008;295:L1–L15. 6. Dickson RP, Erb-Downward JR, Huffnagle GB. The role of the bacterial microbiome in lung disease. Expert Rev Respir Med 2013;7:245–257. 7. Molyneaux PL, Mallia P, Cox MJ, Footitt J, Willis-Owen SA, Homola D, Trujillo-Torralbo MB, Elkin S, Kon OM, Cookson WO, et al. Outgrowth of the bacterial airway microbiome following rhinovirus exacerbation of chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2013;188:1224–1231. 8. Mallia P, Message SD, Gielen V, Contoli M, Gray K, Kebadze T, Aniscenko J, Laza-Stanca V, Edwards MR, Slater L, et al. Experimental rhinovirus infection as a human model of chronic obstructive pulmonary disease exacerbation. Am J Respir Crit Care Med 2011;183: 734–742. 9. Sze MA, Dimitriu PA, Hayashi S, Elliott WM, McDonough JE, Gosselink JV, Cooper J, Sin DD, Mohn WW, Hogg JC. The lung tissue microbiome in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2012;185:1073–1080. 10. Erb-Downward JR, Thompson DL, Han MK, Freeman CM, McCloskey L, Schmidt LA, Young VB, Toews GB, Curtis JL, Sundaram B, et al. Analysis of the lung microbiome in the “healthy” smoker and in COPD. PLoS ONE 2011;6:e16384. 11. Cabrera-Rubio R, Garcia-Nún˜ez M, Setó L, Antó JM, Moya A, Monsó E, Mira A. Microbiome diversity in the bronchial tracts of patients with chronic obstructive pulmonary disease. J Clin Microbiol 2012;50: 3562–3568. 12. Pragman AA, Kim HB, Reilly CS, Wendt C, Isaacson RE. The lung microbiome in moderate and severe chronic obstructive pulmonary disease. PLoS ONE 2012;7:e47305. 13. Huang YJ, Kim E, Cox MJ, Brodie EL, Brown R, Wiener-Kronish JP, Lynch SV. A persistent and diverse airway microbiota present during chronic obstructive pulmonary disease exacerbations. OMICS 2010; 14:9–59. 14. Han MK, Agusti A, Calverley PM, Celli BR, Criner G, Curtis JL, Fabbri LM, Goldin JG, Jones PW, Macnee W, et al. Chronic obstructive pulmonary disease phenotypes: the future of COPD. Am J Respir Crit Care Med 2010;182:598–604. 15. Hurst JR, Vestbo J, Anzueto A, Locantore N, Müllerova H, Tal-Singer R, Miller B, Lomas DA, Agusti A, Macnee W, et al.; Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE) Investigators. Susceptibility to exacerbation in chronic obstructive pulmonary disease. N Engl J Med 2010;363:1128–1138. 16. Albert RK, Connett J, Bailey WC, Casaburi R, Cooper JA Jr, Criner GJ, Curtis JL, Dransfield MT, Han MK, Lazarus SC, et al.; COPD Clinical Research Network. Azithromycin for prevention of exacerbations of COPD. N Engl J Med 2011;365:689–698. Copyright ª 2013 by the American Thoracic Society DOI: 10.1164/rccm.201309-1573ED
