Bronchiectasis

Chapter 1

Bronchiectasis: epidemiology and causes D. Bilton*,#," and A.L. Jones*,#,"

Summary

Keywords: Aetiology, bronchiectasis, epidemiology, non-cystic fibrosis

*Dept of Cystic Fibrosis, Royal Brompton Hospital, # NIHR Biomedical Research Unit into Advanced Lung Disease, Royal Brompton Hospital, and " Dept of Cystic Fibrosis, National Heart and Lung Institute, Imperial College London, London, UK. Correspondence: D. Bilton, Royal Brompton Hospital, Sydney Street, London, SW3 6NP, UK, Email [email protected]

D. BILTON AND A.L. JONES

Bronchiectasis remains a significant cause of morbidity and mortality in the developed world. The true prevalence of the condition remains elusive, in part, because of the innate difficulty in determining causation, when more than one respiratory condition exists in the same patient, but also due to the increasing rate of diagnosis by radiological means where no clinical symptoms are present. The wide ranging aetiology of bronchiectasis will be discussed in this chapter; however, some aspects will be discussed in greater detail throughout this Monograph. The diagnosis of bronchiectasis should be the beginning of a targeted search for causation, which may lead to directed treatment, thereby limiting the disease progression. Over the next 5 years a reduction in the number of cases labelled as idiopathic bronchiectasis should be expected, as the continual expanding knowledge of immunology and immunogenetics, with respect to large studies of patients with bronchiectasis, can be applied.

Eur Respir Mon 2011. 52, 1–10. Printed in UK – all rights reserved. Copyright ERS 2011. European Respiratory Monograph; ISSN: 1025-448x. DOI: 10.1183/1025448x.10003110

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ronchiectasis was first described by Laennec [1] in 1819 as part of a wider work describing the use of his novel invention, the stethoscope. In his book ‘‘De l’Auscultation Mediate ou Traite du Diagnostic des Maladies des Poumons et du Coeur’’ [1], he described the condition through the use of case reports, detailing clinical examination and correlating this with post mortem findings. He identified that any illness characterised by chronic sputum production could lead to bronchiectasis with tuberculosis and pertussis infection identified as the most likely causative conditions.

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A century later, in 1919, A. Jex-Blake delivered a lecture at the Hospital for Consumption (London, UK) on the condition of bronchiectasis [2]. He examined the case records for the hospital over a 20-year period and gave a detailed account of the condition and its causes. He identified that bronchiectasis itself was a secondary condition to a preceding disorder of the lung and,

as such, its frequency was likely to be underestimated as the preceding condition was of such severity that the presence of bronchiectasis was overlooked. He also identified that the condition was apparent in 2% of the hospital’s admissions over the same 20-year period, but estimated that the true figure could be as high as 5%. Perhaps, unsurprisingly, in this pre-antibiotic era a third of patients were identified as having bronchiectasis secondary to an episode of pneumonia or pleurisy, a third due to chronic bronchitis and a further third due to bronchial obstruction, the majority of which were malignant tumours. Since the introduction of antibiotic therapy the incidence of bronchiectasis due to tuberculosis or other infections decreased markedly from the beginning of the 20th century. Perhaps the most striking evidence for the effect of antibiotic introduction was a report in 1969 by FIELD [3] into childhood admissions for the condition. The author reported a reduction from 24–99 per 10,000 hospital admissions to 6–13 per 10,000 admissions for five large children’s hospitals between 1952 and 1960. Despite this decline in cases in the antibiotic era of medicine, non-cystic fibrosis (CF) bronchiectasis remains a significant cause of morbidity.

EPIDEMIOLOGY AND CAUSES

Epidemiology Remarkably the current knowledge of the true incidence of bronchiectasis has changed very little from when A. Jex-Blake gave his lecture almost a century ago. In part, the reason for this remains similar to what was perceived in 1919. Bronchiectasis is often noted as a secondary phenomenon to a more severe pulmonary pathology, as is the case of asthma or chronic obstructive pulmonary disease (COPD), and as such goes unreported. Conversely, the widespread use of computer tomography (CT) as a diagnostic tool in respiratory medicine has resulted in the identification of an increased number of radiological bronchiectasis cases in patients who showed no symptoms and who would have otherwise not been classified as having it. Future studies of the prevalence of bronchiectasis should not be confined to radiological evidence alone but should include the assessment of clinical symptoms. One of the first large-scale studies to determine the incidence of bronchiectasis was performed in 1953 and examined the population of Bedford, a town in the UK [4]. The authors identified an incidence of bronchiectasis as 1.3 per 1,000 people. The relevance of this data, collected prior to the widespread use of antibiotics and where the authors excluded patients with bronchiectasis as a consequence of other pulmonary pathology, is perhaps limiting. However, more recent data has been collected from cohorts in Finland, New Zealand and the USA [5–7]. The data from Finland suggested an incidence of 2.7 per 100,000 people, while in New Zealand an overall incidence in children of 3.7 per 100,000 was noted but showed wide variations with regards ethnicity. For example, children from a Pacific Island descent had an incidence of 17.8 per 100,000 compared with an incident of 1.5 per 100,000 for those of a Northern European descent. Unsurprisingly, given the often chronic nature of its development, the prevalence of bronchiectasis and hospital admission related to bronchiectasis increased with age. Studies from the USA estimate a prevalence of 4.2 per 100,000 people in those aged 18–34 years, increasing to 271.8 per 100,000 in people aged .75 years [7].

Aetiology There are a wide range of conditions that can cause bronchiectasis and there are a number of ways in which one could classify these aetiological factors; however, an approach based on pathological processes appears to be the most logical and is described in table 1.

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Bronchial dilatation can be caused by a structural defect in the wall itself, an effect of abnormal airway pressure on the bronchial wall or by damage to the airway elastic tissue and cartilage as a result of bronchial wall inflammation.

The lungs are continuously exposed to inhaled pathogens and have developed an advanced mechanism for trapping and removing them. The human airways are lined with ciliated epithelium with submucosal goblet cells secreting mucus that makes up the top layer of the airway surface liquid, the lower layer being the periciliary fluid that bathes the cilia and ensures they function appropriately. In healthy individuals the mucus traps inhaled pathogens and the continuously motile cilia transport the mucus and its contents out of the lung. Any defect in this mucociliary clearance mechanism can lead to the retention of pathogens resulting in the progression of airway infection, inflammation and ultimately bronchiectasis.

Structural lung conditions The effect of obstructions within the bronchus itself was identified by LAENNEC [1] as a significant cause of bronchiectasis. Obstruction of the bronchi with foreign objects or tumours is now a relatively rare cause of bronchiectasis. Unsurprisingly most patients with bronchiectasis secondary to retained objects are young children. Congenital disorders affecting the structure of the bronchial tree can lead to bronchiectasis through a direct effect on the bronchial wall itself, although impaired clearance of sputum through the abnormally dilated structures can further compound the condition.

Table 1. Aetiology of bronchiectasis Structural lung conditions Williams–Campbell syndrome Mounier–Kuhn syndrome Ehlers–Danlos syndrome Toxic damage to airways Inhalational injury Aspiration secondary to neuromuscular disease GERD Obstruction of single bronchus Tumour Foreign body Obstructive airways disease Asthma COPD AAT deficiency Defects of mucociliary clearance Ciliary dyskinesia Primary ciliary dyskinesia Secondary ciliary dyskinesia Channelopathies CFTR dysfunction ENaC dysfunction ABPA Immunodeficiency CVID XLA CGD Antibody deficiency with normal Ig Secondary immunodeficiency Haematological malignancy Post-allogeneic bone marrow transplant Drug-induced immunosuppression Infections Childhood infections Tuberculosis Pneumonia Measles Whooping cough Nontuberculous mycobacteria Bronchiectasis in systemic diseases Inflammatory bowel disease Connective tissue diseases Yellow nail syndrome Idiopathic bronchiectasis

D. BILTON AND A.L. JONES

Inflammation within the bronchial wall can be the result of an infection within the airway, inhalation of injurious agents or an endogenous condition such as an autoimmune disease.

GERD: gastro-oesophageal reflux disease; COPD: chronic obstructive pulmonary disease; AAT: a1- antitrypsin deficiency; CFTR: cystic fibrosis transmembrane conductance regulator; ENaC: epithelial sodium channel; ABPA: allergic bronchopulmonary aspergillosis; CVID: common variable immunodeficiency; XLA: X-linked agammaglobulinaemia; CGD: chronic granulomatous disease; Ig: immunoglobulin.

Williams–Campbell syndrome was first described in 1960 after the case reports of five children were studied by WILLIAMS and CAMPBELL [8]. Histological examination of the bronchial wall revealed a deficiency or absence of cartilage, mostly from the third division of the bronchi down. WILLIAMS and CAMPBELL [8] went on to describe a further 11 children with the same clinical findings of bronchiectasis and cartilage deficiency.

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Mounier–Kuhn syndrome (tracheobronchomegaly) is characterised by dilatation of the trachea and large bronchi, usually presenting in young adults. Its underlying pathology is not clearly understood but histological examination has shown atrophy of airway cartilage and smooth muscle.

Case reports suggesting an association with Ehlers–Danlos syndrome, and the appearance of the condition in siblings, could point to an unidentified genetic cause for the condition.

Obstructive airways disease To carry on the theme of defects in the gross airway structure itself, perhaps a continuation of this is to consider whether obstructive airways diseases, namely asthma and COPD, could lead to bronchiectasis. It is natural to assume that these conditions would lead to bronchiectasis as both have clearly been shown to cause airway inflammation and structural blockage of airways, either through bronchospasm or fixed airways obstruction, in the case of COPD.

Asthma A number of studies have highlighted the presence of airway remodelling in chronic asthma patients using high-resolution CT (HRCT) scanning techniques. The airway remodelling can vary from mild airway wall thickening to blatant bronchiectasis. Bronchial wall thickening has been found in up to 82% of asthmatic patients in a cohort [9] and in patients with mild asthma [10]. As bronchial wall thickening is indicative of airway inflammation this suggests that a significant number of patients with asthma are at risk of developing bronchiectasis.

EPIDEMIOLOGY AND CAUSES

The prevalence of bronchiectasis in these studies is estimated at 17.5–40% [9–11]. In the largest of these studies, which comprised of 463 patients with severe asthma, 40% of patients were shown to have evidence of bronchiectasis on HRCT scans [11]. However, study participants were selected for HRCT on the basis of clinical indication, the most common being a suspicion of bronchiectasis. The studies suggest that bronchiectasis is associated with a more severe obstruction and is more apparent in patients who present with a longer history of asthma symptoms, consequently a subgroup of severe asthma patients appear to be at risk of developing bronchiectasis [9–11].

COPD COPD is a term encompassing a number of pathological processes including chronic bronchitis, asthma, emphysema and bronchiectasis. Therefore, it is difficult to fully attribute COPD as the cause of bronchiectasis as in some cases bronchiectasis may be the primary diagnosis. Certainly it is probable that bronchiectasis in COPD is common. A study of moderate-to-severe COPD patients demonstrated the prevalence of bronchiectasis to be 50% [12]. The COPD patients with bronchiectasis were found to have more severe exacerbations and increased sputum inflammatory markers. Further studies are required to elucidate the mechanisms that predispose COPD patients to developing bronchiectasis; severity of airflow obstruction may be a key driver in this mechanism.

a1-antitrypsin deficiency

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a1-antitrypsin (AAT) deficiency is classically associated with predominantly lower lobe emphysema. Bronchiectasis has also been associated with the enzyme deficiency, whether this is a direct consequence of the deficiency or secondary to the emphysema-associated airways obstruction is less clear. In a study of patients with severe AAT deficiency the vast majority of subjects had some evidence of bronchiectasis on a HRCT scan (70 out of 74 subjects), with 27% having clinically significant bronchiectasis with a correlation between forced expiratory volume in 1 second (FEV1) and bronchial wall thickness [13]. In a study of the distribution of AAT alleles in a population of bronchiectasis patients, there was no difference in AAT allele distribution between healthy controls and bronchiectasis patients [14]. However, there was an over representation of hetero- and homozygote AAT deficiency alleles in those patients with bronchiectasis and coexistent asthma. Therefore, the evidence would suggest that AAT deficiency is related to airway obstruction rather than a direct effect of the enzyme deficiency on the bronchial wall structure.

Defects of mucociliary clearance Ciliary dyskinesia Abnormalities of cilia structure and/or motility cause a decreased mucus clearance from the lungs. These abnormalities can be due to a primary defect in the structure or function of the cilia or secondary damage to the cilia from external agents, such as bacteria or inhaled noxious agents.

Primary ciliary dyskinesia Airway cilia are complicated structures containing more than 250 proteins. The ciliary structures are composed of microtubules which are mobilised by structures known as dynein arms, these are divided into two groups the outer and inner dynein arms. This complicated polypeptide structure can be affected by numerous genetic mutations and, as such, primary ciliary dyskinesia (PCD) is a genetically heterogenous disorder. Among the most commonly identified mutations are those of the genes DNAI1 and DNAH5, which code for proteins responsible for the assembly of outer dynein arms. As cilia are present throughout the body, patients with PCD will often present with multiple symptoms such as sinusitis, recurrent otitis media, infertility and defects of organ lateralisation with situs inversus or situs ambiguus. The triad of bronchiectasis, chronic sinusitis and situs inversus is also known as Kartagener’s syndrome.

A number of noxious agents, both organic and inorganic, have been shown to affect the function of cilia in human airway epithelia. Certain bacteria, such as Pseudomonas aeruginosa and Haemophilus influenzae, have been shown to disable mucociliary clearance by releasing products that inhibit ciliary beat frequency, allowing them to persist and propagate infection [15, 16]. Inhaled inorganic substances such as diesel particles [17] and cigarette smoke [18] have also been shown to have a direct effect on ciliary function, inhibiting ciliary beat frequency. It is important to note here that no causal role for tobacco smoking and the development of bronchiectasis has been made, indeed outside of COPD bronchiectasis appears to be a disease of the nonsmoker. Aspiration of gastric contents is a well recognised, but perhaps under diagnosed, cause of bronchiectasis. Whilst aspiration of both acid and nonacid stomach contents leads to direct inflammation of the bronchial wall, ciliary function may also be affected by these agents.

D. BILTON AND A.L. JONES

Secondary ciliary dyskinesia

Channelopathies As previously mentioned, the epithelial lining of the airway is coated in a liquid known as the airway surface liquid. It contains two layers, the outer mucus layer and an inner periciliary layer. Ion channels within the apical surface of the epithelial levels regulate the fluid content of this layer to ensure adequate hydration. This enables the cilia to move in a liquid layer but also prevents the desiccation of the mucus into a thick, sticky substance that is difficult to mobilise.

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Defects in the ion channels of the epithelial layer can lead to dehydration of the airway surfaces, thereby affecting the depth of the periciliary layer and bringing the cilia into contact with the viscous mucus layer, further impeding its function. The most widely recognised of these defects is that found in CF. Here, the loss of a chloride channel known as the CF transmembrane regulator (CFTR) protein leads to the inability of the epithelial cells to excrete chloride. The dysregulation of the ion transport is further compounded by the effect of CFTR on another ion channel, that of the epithelial sodium channel (ENaC). CFTR is an inhibitor of the ENaC channel and therefore the loss of CFTR is postulated to lead to hyperactivity of the sodium channel, resulting in a large

increase in the transport of sodium into the epithelial cell with a corresponding movement of water out of the airway liquid. In theory, genetic defects of the ENaC channel could lead to bronchiectasis if such a mutation led to over activity of the channel. Whilst mutations of ENaC genes have been identified in patients with idiopathic bronchiectasis [19], a significant number of these were also carriers of a CFTR mutation. Furthermore, a single CFTR mutation is frequently observed in patients with diffuse bronchiectasis. A study comparing patients with either none, one or two CFTR mutations suggested a continuum of CFTR dysfunction (as measured by nasal potential differences) existed and that this may lead to the development of bronchiectasis in some patients who are CFTR heterozygotes [20].

Allergic bronchopulmonary aspergillosis

EPIDEMIOLOGY AND CAUSES

Allergic bronchopulmonary aspergillosis (ABPA) is a pulmonary condition caused by a hypersensitivity reaction to the ubiquitous environmental fungus Aspergillus fumigatus. It is most commonly seen in patients with pre-existing asthma or CF and is clinically characterised by recurrent wheeze, pulmonary infiltrates and the development of bronchiectasis. The hypersensitivity reaction has mixed features of immediate hypersensitivity (type I), antigen–antibody complexes (type III) and inflammatory cell responses (type IV) [21]. The inflammatory cell response seen in ABPA shows a predominance of T-helper cell type 2 (Th2) cells leading to a release of cytokines mediating allergic inflammation (as opposed to the Th1, cytotoxic pathway) [22]. The type I hypersensitivity reaction causes local degranulation of mast cells and histamine release leading to bronchoconstriction. The combination of airway inflammation, which leads to viscous, eosinophil-laden mucus, plugging and airway obstruction, and bronchospasm leads to a reduction in mucociliary clearance and the development of bronchiectasis. As such bronchiectasis in ABPA is common. In three large case studies it was found that central bronchiectasis was present in 69–76% of patients with ABPA [23–25].

Immunodeficiency Defects in the immune system leave the lungs vulnerable to infection and in some cases the development of bronchiectasis can be the first indication of immunodeficiency. The most common forms of primary immune deficiencies observed in patients with bronchiectasis are common variable immune deficiency (CVID), X-linked agammaglobulinaemia (XLA) and chronic granulomatous disease (CGD).

Common variable immune deficiency CVID is characterised by reduced levels of immunoglobulins (Igs) with associated recurrent bacterial infections. An increased risk of autoimmune conditions and malignancy has also been identified. The majority of patients present with recurrent pulmonary infections at a mean age 29 years [26]. CVID is the most common primary immune deficiency to cause bronchiectasis. A case series undertaken in a UK population identified 68% of the patients with CVID as having evidence of bronchiectasis [27]. The most likely cause of this high rate of incidence could be the delay in the diagnosis of CVID, with a mean duration of 4 years between reporting of symptoms and diagnosis [27].

X-linked agammaglobulinaemia

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XLA is caused by a mutation of a tyrosine kinase gene that is involved in the development of Blymphocytes, leading to an absence of circulating B-lymphocytes and the absence of Igs. Given the severity of the immune deficiency it usually presents much earlier than CVID, usually being diagnosed in early childhood [28]. Despite treatment with replacement Igs, chronic lung disease can still develop with the risk of developing bronchiectasis increasing with age [29].

Chronic granulomatous disease CGD is a group of disorders characterised by a loss of phagocytic NADPH oxidase, without which phagocytes are unable to produce the reactive oxygen species required to kill ingested bacteria. Infections are mainly due to Staphylococcus aureus, Serratia marcescens, Salmonella sp., Klebsiella sp. and Burkholderia cepacia.

Antibody deficiency with normal Igs In a study of patients with bronchiectasis and normal IgG levels, 11% were shown to have specific antibody production deficiencies with an inability to respond to pneumococcal and H. influenzae vaccines [30].

Secondary immunodeficiency The development of bronchiectasis in HIV-infected patients has been noted in a number of case series. While recurrent pulmonary infection is likely to be the major factor in the development of bronchiectasis in these patients, the development of lymphocytic interstitial pneumonia may also be implicated [31].

Infections

A number of childhood respiratory infections have been implicated in the pathogenesis of bronchiectasis. The most widely recognised infectious causes of bronchiectasis are measles and pertussis infection in the West [32], with tuberculosis being a major cause elsewhere.

Nontuberculous mycobacterial infection Globally, Mycobacterium tuberculosis infection remains a major cause of morbidity and mortality and a significant cause of bronchiectasis. In developed countries with screening programmes and adequate access to treatment, the incidence of new infections remains low. However, the incidence of nontuberculous mycobacterial (NTM) pulmonary infections is increasing. These mycobacteria vary in pathogenecity with Mycobacterium avium complex (MAC) being the most pathogenic whilst other organisms, such as Mycobacterium gordonae and Mycobacterium abscessus, act as opportunistic pathogens and are only found in patients with underlying lung diseases. NTM is commonly present in one of three clinical forms; 1) a tuberculosis-like pattern with a predominant upper lobe fibrocavitatory disease, mostly found in older males with COPD; 2) nodular bronchiectasis, most commonly seen in middle-aged females; and 3) hypersensitivity pneumonitis [33].

D. BILTON AND A.L. JONES

Childhood infections

The second of these clinical forms is also known as ‘‘Lady Windermere syndrome’’, and was first described in 1992 in a case series of 29 predominately elderly, female patients [34]. The patients had MAC infection with bronchiectasis predominantly affecting the middle lobe and lingula. The authors postulated that persistent voluntary cough suppression could lead to chronic inflammatory processes in these poorly draining lung regions which are susceptible to MAC infection [34].

Bronchiectasis in systemic diseases Inflammatory bowel disease

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The development of bronchiectasis in patients with ulcerative colitis is a well recognised phenomenon and the subject of a number of case series [35]. Classically, bronchiectasis develops after resection of the large bowel, suggesting a common immune system response that becomes

concentrated on the bronchial wall after the bowel is removed. The common embryonic origin and similar structures of bowel and bronchial wall (columnar epithelial and submucosal glands) add weight to this theory. The link between Crohn’s disease and bronchiectasis is less clear with only a small number of case reports detailing their coexistence [36], perhaps too few to determine a definite association.

Connective tissue diseases A number of connective tissue diseases have been noted to be associated with bronchiectasis, largely based on case series reviews of small numbers of patients. The clearest association is that between rheumatoid arthritis and bronchiectasis. Studies have estimated the incidence of bronchiectasis in rheumatoid arthritis patients to be as high as 41% with a significant number of them being asymptomatic [37]. Again no clear pathological process has been identified as the cause of this association, although studies have suggested common genetic predisposition with an association between human leukocyte antigen sub-groups [38]. An effect of the immunosuppressive agents used in rheumatoid arthritis treatment has also been postulated, although a significant number of patients develop bronchiectasis prior to the onset of arthropathy. Associations between bronchiectasis and Sjo¨gren’s syndrome [39], systemic sclerosis [40], systemic lupus erythematosus [41], ankylosing spondylitis [42, 43] and relapsing polychondritis [44] have all been made in small case series reviews.

EPIDEMIOLOGY AND CAUSES

Yellow nail syndrome Yellow nail syndrome is a rare syndrome that was first described in 1964 by SAMMAN and WHITE [45] and is characterised by bronchiectasis, lymphoedema and a characteristic appearance of the nails. The underlying pathological defect is not clear, although a recent study revealing an association with chronic rhinosinusitis suggests a possible defect in an inflammatory pathway or mucociliary clearance rather than a structural defect within the lung itself [46].

Idiopathic bronchiectasis In two large studies [47, 48], which identified the cause of bronchiectasis in adults, a significant proportion of patients (26% and 53%, respectively) were found to have no identifiable cause and were labelled as having idiopathic bronchiectasis, the majority of whom were found to be female and nonsmokers. As all the patients studied had undergone rigorous clinical testing and their history had been reported, leading to the exclusion of all known causes, including genetic disorders, it is unlikely under recognition of known causes of bronchiectasis could have occurred. Even in paediatric studies, with much shorter follow-up periods and clear exposure histories, no cause could be found for bronchiectasis in 25% of the patients [32]. It is clear, therefore, that there is still much to learn about bronchiectasis and its underlying pathogenesis.

Statement of interest None declared.

References

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39. Uffmann M, Kiener HP, Bankier AA, et al. Lung manifestation in asymptomatic patients with primary Sjo¨gren syndrome: assessment with high resolution CT and pulmonary function tests. J Thorac Imaging 2001; 16: 282–289. 40. Andonopoulos AP, Yarmenitis S, Georgiou P, et al. Bronchiectasis in systemic sclerosis. A study using high resolution computed tomography. Clin Exp Rheumatol 2001; 19: 187–190. 41. Fenlon HM, Doran M, Sant SM, et al. High-resolution chest CT in systemic lupus erythematosus. AJR Am J Roentgenol 1996; 166: 301–307. 42. Souza AS Jr, Muller NL, Marchiori E, et al. Pulmonary abnormalities in ankylosing spondylitis: inspiratory and expiratory high-resolution CT findings in 17 patients. J Thorac Imaging 2004; 19: 259–263. 43. Casserly IP, Fenlon HM, Breatnach E, et al. Lung findings on high-resolution computed tomography in idiopathic ankylosing spondylitis – correlation with clinical findings, pulmonary function testing and plain radiography. Br J Rheumatol 1997; 36: 677–682. 44. Davis SD, Berkmen YM, King T. Peripheral bronchial involvement in relapsing polychondritis: demonstration by thin-section CT. AJR Am J Roentgenol 1989; 153: 953–954. 45. Samman PD, White WF. The "Yellow Nail" syndrome. Br J Dermatol 1964; 76: 153–157. 46. Nisbet M, Deveraj A, Meister M, et al. Yellow nail syndrome and bronchiectasis. Am J Respir Crit Care Med 2009; 179: A3216. Available from: http://ajrccm.atsjournals.org/cgi/reprint/179/1_MeetingAbstracts/A3216.pdf. 47. Shoemark A, Ozerovitch L, Wilson R. Aetiology in adult patients with bronchiectasis. Respir Med 2007; 101: 1163–1170. 48. Pasteur MC, Helliwell SM, Houghton SJ, et al. An investigation into causative factors in patients with bronchiectasis. Am J Respir Crit Care Med 2000; 162: 1277–1284.

Chapter 2

Pulmonary defence mechanisms and inflammatory pathways in bronchiectasis B.N. Lambrecht*,#, K. Neyt* and C.H. GeurtsvanKessel*,"

Over recent years there has been a tremendous increase in the understanding of pulmonary immunity, mostly driven by large research efforts in understanding the basis of asthma and chronic obstructive pulmonary disease. Bronchiectasis is well understood. In this article, an overview of pulmonary defence mechanisms as well as inflammatory mechanisms is given as a basis to understand the pathogenesis of bronchiectasis.

Keywords: Bronchiectasis, inflammatory mechanisms, immunity, pulmonary defence

*Dept of Pulmonary Medicine, Laboratory of Immunoregulation and Mucosal Immunology, Ghent University, Ghent, Belgium. # Dept of Pulmonary Medicine, and " Dept of Virology, Erasmus University Medical Center, Rotterdam, The Netherlands. Correspondence: B.N. Lambrecht, Dept of Pulmonary Medicine, Laboratory of Immunoregulation and Mucosal Immunology, Ghent University, De Pintelaan 185, B-9000 Ghent, Belgium, Email [email protected]

B.N. LAMBRECHT ET AL.

Summary

Eur Respir Mon 2011. 52, 11–21. Printed in UK – all rights reserved. Copyright ERS 2011. European Respiratory Monograph; ISSN: 1025-448x. DOI: 10.1183/1025448x.10003210

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ronchiectasis is a chronic disorder characterised by permanent dilatation of the bronchi accompanied by inflammatory changes in their walls and in the adjacent lung parenchyma. The pathogenesis is related to recurrent inflammation of the bronchial walls combined with fibrosis in the surrounding parenchyma. The resultant traction on weakened walls leads to eventual irreversible dilatation [1]. Bronchiectasis can result from defective pulmonary defence mechanisms that lead to recurrent, severe and tissue-damaging microbial insults or chronic bacterial colonisation with persistent inflammation leading to structural changes to the airway wall. Given the fact that restoration of inflammation and return to immune homeostasis is crucial in the lung to protect the delicate gas exchange machinery, it is also possible that bronchiectasis results from defective anti-inflammatory pathways that serve to dampen chronic inflammation. Therefore, in this chapter we provide a brief overview of lung defence mechanisms and how these immune defence mechanisms can contribute to chronic inflammation and structural changes to the airway wall if not properly counter-regulated by anti-inflammatory pathways. The major inflammatory cell types found in bronchiectasis are neutrophils in the airway lumen causing purulent sputum and macrophages, dendritic cells (DCs) and lymphocytes in the airway wall [2, 3].

The latter cells often occur in lymphoid aggregates or so-called tertiary lymphoid follicles, and are typically seen in patients with tubular bronchiectasis and are a major cause of small airway obstruction [4].

Mechanical and physical pulmonary defence mechanisms

PULMONARY DEFENCE AND IMMUNITY

The inspired air is contaminated with toxic gases, particulates and microbes. The first line of defence of the lung is made up of the complex physical shape of the conducting upper and lower airways, causing a highly turbulent airflow that facilitates the impaction, sedimentation and deposition of particulate matter and microorganisms on the mucosa, followed by the removal of these deposited particles by the mucociliairy blanket and/or the physical expulsion from the respiratory tract by sneezing, coughing or swallowing. Reductions in the cough reflex are associated with increased frequency of respiratory infections, but it is not known at present whether this would also predispose to development of bronchiectasis [5]. The presence of isolated middle lobe bronchiectasis and colonisation with nontuberculous mycobacteria (the so-called Lady Windermere syndrome) has been proposed to be caused by cough suppression [6]. The action of the mucociliary blanket is a dynamic and complexly regulated escalator for bringing inhaled particles to the throat so that they can be swallowed. Defects in the function of the mucociliary blanket can cause bronchiectasis. The conducting airways are lined with ciliated epithelium and the structure and function of the cilia in propulsing mucus has been extensively studied [7–9]. Genetic defects in the structure of the outer dynein arm proteins that connect microtubules in cilia are the cause of primary ciliary dyskinesia [10]. Other mutations involve the ktu gene, which is involved in the assembly of both the outer dynein and the inner dynein arm [11]. Defects in radial spoke head proteins are associated with abnormalities of the central microtubule pair of the cilium (presence of only one microtubulus rather than two) [10]. Ciliary disturbances (sometimes associated with situs inversus; Kartagener syndrome) almost always lead to bronchiectasis and are often also associated with chronic rhinosinusitis. The correct movement of cilia and function of the mucociliary escalator also depend on the low viscosity of the periciliary fluid layer, physically a hydrated sol layer, allowing sufficient separation between the apical side of the epithelium and the viscous mucous blanket covering the cilia. If the periciliary fluid layer is concentrated (i.e. like in cystic fibrosis (CF)), the periciliary fluid layer becomes thinner and the cilia become entangled in the mucus layer, thus impeding normal ciliary propulsion of the mucus [12, 13].

Humoral innate immune mechanisms in the lung Innate immune defences are evolutionary conserved pathways of defence that kill microbes in a generic pathway, often relying on the recognition and antagonism of common motifs in microbial proteins or lectins, the so-called pathogen-associated molecular patterns (PAMPs), which are so crucial for the function of the microbe that their antagonism leads to loss of pathogenicity. Just like acquired or adaptive immunity, innate immunity consists of a humoral and a cellular part.

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Humoral innate defence mechanisms are elaborate in the lung and consist of lactoferrin, lyzozyme, defensins, complement, cathelicidins and collectins [14]. These molecules can be produced by airway structural cells or by recruited innate immune cells such as neutrophils and macrophages (see later). Lactoferrin chelates Fe2+ molecules that are crucial for the growth of some bacteria but also stimulates the function of neutrophils. Lyzozyme degrades Gram-positive cell walls. Defensins are made by neutrophils (a-defensins) and epithelial cells (b-defensins). They serve to make pores in bacterial cell walls, and thus are truly antibacterial peptides but also neutralise viruses and fungi and recruit DCs via activation of the CCR6 chemokine receptor on these cells [15]. The proper function of defensins depends on the correct salt concentration in the airway surface liquid [16]. Thus, in CF patients defensin function against Staphylococcus aureus is defective, possibly explaining the susceptibility to colonisation, although this theory has also been questioned. LL37 is a well-known airway cathelicidin that is also salt sensitive and has broad antimicrobial activity but

also has effects on innate and adaptive immune cells [17]. Surfactant protein A and D are collectins that opsonise bacteria and viruses such as influenza. A closely related collectin family member is mannose binding lectin (MBL), it is not secreted into the lung lining fluid but is an important circulating factor that can activate the complement cascade. Deficiency of MBL is a cause of recurrent bacterial infections and could be a cause of bronchiectasis. Low MBL levels in CF patients and other forms of bronchiectasis are also associated with a more rapid decline in lung function [18].

Cellular innate immune mechanisms in the lung The cellular arm of innate immunity in the lung is primarily made up of alveolar macrophages and recruited neutrophils (fig. 1). Alveolar macrophages serve an important function in the phagocytosis, killing and/or neutralisation of inhaled particulate antigens. Resident alveolar macrophages continuously encounter inhaled substances due to their exposed position in the alveolar lumen. These cells are packed with enzymes, metabolic products and cytokines that are vital to defence of the alveolar space but can potentially damage the alveolocapillary membrane. To avoid collateral damage to type I and type II alveolar epithelial cells (AEC) in response to harmless antigens, they are kept in a quiescent state, producing few inflammatory cytokines [19]. It has been estimated previously, that the pool of alveolar macrophages can handle up to 109

B.N. LAMBRECHT ET AL.

Stimulus

Ingestion by alveolar macrophages Direct triggering of epithelial cells Activation of dendritic cells

Secondary neutrophil influx

TNF-α, IL-1, G-CSF, GM-CSF, chemokines (IL-8)

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Figure 1. When a pathogen enters the lung, it triggers both epithelial cells, macrophages and dendritic cells. The epithelial cells make chemokines that subsequently attract neutrophils that help in phacocytosing the pathogens. All recruited cells together with epithelial cells then make cytokines and growth factors that further enforce innate immune responses to the pathogen by further recruitment of inflammatory cells. TNF: tumour necrosis factor; IL: interleukin; G-CSF: granulocyte colony-stimulating factor; GM-CSF: granulocyte-macrophage colony-stimulating factor.

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PULMONARY DEFENCE AND IMMUNITY

intratracheally injected bacteria before there is spill-over of bacteria to DCs and before adaptive immunity is induced [20]. Elegant studies have demonstrated that in vivo elimination of alveolar macrophages using clodronate filled liposomes lead to overt inflammatory reactions to otherwise harmless particulate and soluble antigens [21], but also to an increased sensitivity to bacterial, fungal and viral infection. In their exposed position, alveolar macrophages serve as the first line of defence against inhaled pathogens not only by directly acting as the main phagocytes, but also as an important producer of pro-inflammatory chemokines, cytokines and lipid mediators; bioactive mediators that recruit other cell types to the lung. In contrast to alveolar macrophages that reside in the lung and serve as an immediate line of innate defence against inhaled pathogens, neutrophils are recruited within minutes following inoculation of microbes into the lung. The main function of neutrophils is phagocytosis and killing of microbes, particularly fungi such as Aspergillus sp. and Pneumocystis jeroveci. They can also kill microorganisms through the release of a-defensins and lyzozyme. Neutrophil killing function depends on oxidative enzymes such as those of the NADPH oxidase system and myeloperoxidase. Chronic granulomatous disease is caused by missense, nonsense, frameshift, splice or deletion mutations in the genes for p22(phox), p40(phox), p47(phox), p67(phox) (autosomal chronic granulomatous disease) or gp91(phox) (X-linked chronic granulomatous disease), which result in variable production of neutrophil-derived reactive oxygen species [22]. Neutrophil extravasation is also a highly organised process requiring the rolling, arrest and diapedesis of cells on the vessel wall. Defects in certain integrins, selectins or their activator can cause defective neutrophil recruitment and cause recurrent pulmonary infections [23]. Once recruited, neutrophils can also further enhance more neutrophil recruitment through production of cytokines (interleukin (IL)-1, tumour necrosis factor (TNF)-a and IL-6) as well as through release of calcium binding proteins of the S100 family (S100A8, A9 and A12) that act on the RAGE (receptor for advanced glycation end products) receptor.

Induction of innate immune responses in the lung The above mechanisms of innate defence act in a coordinated fashion. Although a single aspect of the innate defence system can be triggered directly through recognition of foreign PAMPs, the innate defence mechanisms are often induced simultaneously via triggering of common receptors on both phagocytes (for cellular defences) and epithelial cells (for inducing the production of humoral innate defence mechanisms). The most famous pattern recognition receptors belong to the family of Toll-like receptors (TLR)1-11, NOD-like receptors, RIG-I-like receptors and C-type lectin receptors [24]. These receptors recognise particular conserved PAMPs on specific groups of microbes. The archetypical TLR4 is expressed at the cell surface and recognises the Gram-negative cell wall component lipopolysaccharide, whereas TLR2 recognises peptidoglycan and TLR5 recognises bacterial flagellin. The endosomal TLR receptors TLR3 recognise double-stranded RNA, TLR7 and TLR8 single-stranded RNA and TLR9 unmethylated CpG motifs [24]. The exact cellular localisation and downstream signalling mechanisms of these pathways have been studied extensively over the past few years and several clinical primary immunodeficiency syndromes have been brought back to deficiencies in one of the signalling intermediates of these pathways. Deficiency of IRAK4, a critical intermediary in TLR4 signalling causes recurrent bacterial infections, particularly at a young age [25]. Deficiency of the C-type lectin receptor dectin-1 or the downstream signalling intermediate molecule CARD9 causes immunodeficiency to candida and P. jeroveci, most probably due to reduced induction of T-helper cell (Th)17 responses [26]. Conversely, over activity of these signalling cascades, for example caused by small polymorphisms in or mutations of negative regulators of these pathways are associated with auto-immunity and overzealous inflammatory pathways. As one example, polymorphisms in the ubiquitin editing enzyme TNF-a-induced protein 3 (TNFAIP3, also known as A20), cause hypersensitivity of TLR and cytokine receptors and are often found in patients with systemic lupus erythematosus [27]. Our own unpublished data also show that genetic deficiency of A20 in epithelial cells causes severe mucosal inflammation in response to inhalation of intrinsically harmless proteins, but it is

unknown at present how this could be implicated in the regulation of inflammatory pathways relevant to bronchiectasis.

Adaptive cellular immunity Like innate immunity, adaptive or acquired immunity consists of a cellular and a humoral arm. Cellular adaptive immunity is made up of different types of T-lymphocytes, whereas humoral immunity is made up of B-lymphocytes and plasma cells and their secreted product; immunoglobulins (Ig).

DCs are potent antigen presenting cells that have emerged as key regulators of adaptive immunity (see [28] for a more detailed review on the biology of lung DC function). The general function of lung DCs is to recognise and pick up foreign antigens at the periphery of the body, and subsequently migrate to the draining mediastinal lymph nodes where the antigen is processed into immunogenic peptides and displayed on major histocompatibility complex (MHC)I and MHCII molecules for presentation to naı¨ve T-cells. In fact, these cells should be seen as specialised cells of the mononuclear phagocyte system, which have evolved from the cells of the innate immune system to control adaptive immunity that came later in evolution [29]. DCs express all the pattern recognitions receptors shared with phagocytes of the innate immune system, yet at the same time also have the machinery to talk to T-cells and B-cells and relay information about the type of antigen to these cells, so that a tailor-made adaptive response is induced and long-term memory is initiated. As these cells respond to many noxious stimuli from both the outside world (PAMPs) and from within (danger-associated molecular patterns) and at the same time closely communicate with lung structural cells such as alveolar epithelial cells, endothelial cells and fibroblasts, it has been proposed that they could be crucial players in many lung diseases, particularly where T-cell responses are involved in initiation of maintenance of the disease [30]. Very recently the first case reports of patients presenting with defects in the DC system have been reported. These DC-deficient patients are at risk of severe viral skin infections and pulmonary infections with atypical mycobacteria, which also leads to bronchiectasis [31, 32]. Our own experiments employing DC-deficient mice have elucidated a crucial role for these cells in the induction of antiviral immunity to influenza virus, via induction of both CD4 and CD8 T-cell responses [33]. Similar conclusions have been reached in models of tuberculosis and bacterial lung infections [34]. Conversely, DCs are also heavily involved in maintaining immunopathology in which T-cells play a predominant role, the best example being the mucosal inflammation seen in asthma and chronic obstructive pulmonary disease (COPD) [35]. In humans with bronchiectasis, as well as in a rat model of bronchiectasis, there is an increased infiltration of the airway wall with DCs [2, 3]. The airways of patients with diffuse panbronchiolitis, a disorder of the small bronchioles that can also lead to bronchiectasis, contain increased numbers of DCs that have a clearly activated phenotype, while treatment with neomacrolides reduces the antigen presenting capacities of these DCs [36, 37].

B.N. LAMBRECHT ET AL.

Induction of adaptive cellular immunity by DCs

Constituents of adaptive cellular immunity

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Adaptive cellular immunity consists of defined subsets of CD4+ Th cells and CD8+ cytotoxic Tcells. Once DCs transport their antigenic cargo to the draining lymph nodes, they induce the proliferation and differentiation of naı¨ve T-cells into particular types of T-cell responses (fig. 2). Discrete types of Th cells provide crucial help for different parts of the innate and adaptive immune response [38]. Th1 cells make interferon (IFN)-c and mainly provide help to monocytic cells, including macrophages and DCs, thus enforcing killing of intracelullar pathogens, and at the same time enforcing opsonisation of these through provision of B-cell help. Conversely, Th2 cells make IL-4, IL-5 and IL-13 providing help to eosinophils, mast cells and basophils to eliminate

IL-4

Th2 Gata-3 c-maf STAT6

IL-10 TGF-β Th0

Treg

IL-4 IL-5 IL-13 TNF-α

IL-10 TGF-β

Foxp3 IL-6 TGF-β IL-1 IL-23

IL-12

Th17

IL-17 IL-22

RORγ STAT3

Th1 T-bet STAT4 STAT1

IFN-γ TNF-α

complex helminths, and at the same time induce IgG1 and IgE from Bcells to arm the basophils and mast cells with effector potential. For a long time since the original description of the Th1/Th2 concept, it has been unclear which subtype of TAnti-inflammatory Suppresses lymphocytes cell help was important for induProfibrotic? cing neutrophilic responses and protection from extracellular pathogens such as fungi. This gap has Antifungal been breached recently by the disStimulates neutrophils covery of the cytokines IL-17 and Autoimmunity IL-22 which are produced by Th17 cells that induce neutrophilic inflammation and production of defensins Intracellular pathogens Stimulates macrophages by epithelial cells and are important Stimulates lgG2a for clearance of fungi and extracelDelayed hypersensitivity lular bacteria [39].

Antihelminthic Stimulates eosinophils Stimulates lgE, lgG1 Allergy

The precise signals that induce different types of Th lineage-comtheir secreted cytokines. When a T-helper cell (Th) type 0 encounters mitment of naı¨ve T-cells has been antigen on a DC, it will be induced to differentiate into various intensely studied [38]. Antigen premutually exclusive cell fates. Each T-cell differentiation programme is senting cells can provide different controlled by transcription factors such as Gata-3, forkhead box P3 levels and quality of signal one (Foxp3), RAR-related orphan receptor gamma (RORc) or T-bet, which enforce Th cell lineage choice. Eventually Th cells emerge (peptide-MHC), signal two (cothat are specialised for performing various antimicrobial tasks. stimulatory molecules) and signal IL: interleukin; Treg: T-regulatory cell; TGF: transforming growth three (instructive cytokines) to factor; TNF: tumour necrosis factor; IFN: interferon; Ig: immunoglonaı¨ve T-lymphocytes upon antigen bulin; STAT: signal transducer and activator of transcription. encounter and triggering of their pattern recognition receptors [29]. When stimulated through the unique T-cell receptor (TCR), naı¨ve CD4+ T-cells differentiate into Th1 cells in the presence of high amounts of IL-12. IL-12 instructs Th1 development via activation of signal transducer and activator of transcription (STAT)4 and the lineage instructing transcription factor T-bet. IL-17 producing cells are induced when exposed to a cocktail of cytokines including transforming growth factor (TGF)-b, IL-6, and IL1a/b, while IL-23 further enhances the proliferation of these cells. The Th17 lineage specific transcription factor RAR-related orphan receptor ct enforces Th17 characteristics in naı¨ve T-cells, and is induced by the cocktail of cytokines instructive to their development. The mechanisms leading to Th2 cell differentiation in vivo are still poorly understood, but in most instances require a source of IL-4 to activate the transcription factors STAT6 and GATA-3, and a source of IL-2, IL-7 or thymic stromal lymphopoietin to activate the transcription factor STAT5 [40–44]. Despite the overwhelming evidence that IL-4 is necessary for most Th2 responses, DCs were, however, never found to produce IL-4 and it was therefore assumed that Th2 responses would occur by default, in the absence of strong Th1 or Th17 instructive cytokines in the immunological DC T-cell synapse, or when the strength of the MHCII-TCR interaction or the degree of co-stimulation offered to naı¨ve T-cells was weak [45–48]. In this model, naı¨ve CD4 T-cells were the source of instructive IL-4. In an alternative view, IL-4 is secreted by an accessory innate immune cell type, such as natural killer T-cells, eosinophils, mast cells or basophils, that provide IL-4 in trans to activate the Th2-differentiation programme [49]. In the lung allergic response to house dust mite allergen, we have recently found that basophils help DCs to induce Th2 immunity by providing an important, but not essential source of IL-4 [50].

PULMONARY DEFENCE AND IMMUNITY

Figure 2. T-cell polarisation induced by dendritic cells (DCs) and

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Lung DCs are also essential in instructing the selection and expansion of CD8 cytotoxic T-cells that recognise virus-infected cells, cells infected with intracellular bacteria and tumourally

transformed cells via presentation of endogenous cellular antigen on the MHCI complex [33]. An important conceptual point is that DCs do not have to be infected themselves to perform this task, but can phagocytose virally infected or transformed cells and use the process of cross-presentation to present the exogenous antigen into their MHCI loading machinery. Once activated by DCs and CD4 T-cell help, cytotoxic T-cells can lyse and kill infected cells in a process requiring granzyme and/or perforin, or kill target cells in a FasL- and/or TNF receptor-like apoptosis inducing liganddependent manner, causing apoptotic cell death in targets [51].

Humoral immune mechanisms in the lung Humoral immunity plays a predominant role in protection from severe infections with encapsulated bacterial strains. Antibodies are well known for their neutralising effects on secondary infections and this is the principle of most vaccinations against childhood infections. During a primary infection, however, antibodies, some of which have broad-spectrum specificity (so-called natural antibodies), also have the capacity to activate complement and opsonise bacterial cell walls and capsules, thus facilitating clearance of the pathogens. Antibodies of the IgA and IgG class are actively secreted into the airway lumen via the action of the polymeric Ig receptor. Airway luminal IgA is an important defence against viral entry. Maybe the most prevalent cause of bronchiectasis is deficiencies in humoral immunity, such as common variable immunodeficiency (CVID), a group of disorders characterised by low to absent Ig and various degrees of T-lymphocyte abnormalities [18, 58]. CVID can be caused by mutations in the proteins involved in T–B-cell communication such as ICOS, BAFF, TACI and APRIL [59, 60]. This is a rapidly evolving field and it is only a matter of time before all these mutations can be diagnosed on a routine basis.

B.N. LAMBRECHT ET AL.

Several defects in adaptive immunity are associated with increased susceptibility to lung infection and can be an important risk factor for later development of bronchiectasis. Defects in the IL-12/ IFNcSTAT1 axis are a well-known risk factor for mycobacterial infections and invasive Salmonellosis [52]. Defects in the IL-23//Th17 axis are associated with increased risk of fungal infections and P. jeroveci infections [53]. Patients with sporadic or autosomal dominant forms of the hyper IgE syndrome (Job’s syndrome when associated with connective tissue abnormalities) have mutations in STAT3, and hence deficient differentiation of Th17 cells [54, 55]. These patients are at risk for severe recurrent Staphyloccal infections, pneumatocoeles and mucocutaneous candidiasis. In recessive forms of the hyper IgE syndrome, mutations in DOCK8 have been described, and these patients are similarly at risk for recurrent sinopulmonary infection and have defects in Th17 generation [56]. The few biopsy studies that have been performed in bronchiectasis have seen increased infiltration of the bronchial wall with CD4 and CD8 T-cells. The neutrophilic inflammation seen in CF and other forms of bronchiectasis is typically associated with the increased presence of Th17 cells [57]. In bronchiectasis associated with allergic bronchopulmonary aspergillosis, one has also observed increased numbers of Th2 cells, thus explaining the association with sputum eosinophilia.

Organised lymphoid structures and bronchiectasis

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The organised accumulation of lymphocytes in lymphoid organs serves to optimise both homeostatic immune surveillance, as well as chronic responses to pathogenic stimuli [61]. During embryonic development, circulating haemopoietic cells gather at predetermined sites throughout the body, where they are subsequently arranged in T- and B-cell specific areas, leading to the formation of secondary lymphoid organs, such as lymph nodes and spleen. In contrast, the body has a limited second set of selected sites that support neo-formation of organised lymphoid aggregates in adult life. However, these are only revealed at times of local, chronic inflammation when so-called tertiary lymphoid organs (TLO) appear. Just like in lymph nodes and spleen, areas of TLO are characterised by formation of specialised high endothelial venules and the organised

production of chemokines leads to cellular organisation of T-cells and B-cells in discrete areas. In humans, TLO has been observed in the joint and lung of rheumatoid arthritis [62], around the airways of COPD patients [63] and in the thyroid [64]. Certain infectious diseases are also accompanied by the formation of TLO. Influenza virus infection of the respiratory tract leads to formation of inducible bronchus-associated lymphoid tissue (iBALT) that supports T- and B-cell proliferation and productive Ig class switching in germinal centres [65, 66]. Tertiary lymphoid follicles or iBALT is frequently seen in tubular bronchiectasis, and the close association with bronchi might explain the obstruction of small bronchioles and airway obstruction that is often seen. This is certainly the case in rheumatoid arthritis-associated bronchiectasis, in which bronchial obstruction is often caused by strongly enlarged TLOs that impinge on the lumen of the airway, an entity known as follicular bronchiolitis by pathologists and reflecting the presence of B-cell follicles inside TLO structures [62]. Formation of TLO could be the result of chronic colonisation of bronchiectatic airways by microbes, and indeed it has been proposed that latent adenoviral infection is a cause of follicular bronchiectasis [4]. However, in one school of thought, TLO formation can also be seen as a source of self-specific autoantibodies and a reflection of an underlying auto-immune component of the disease. In TLO associated with rheumatoid arthritisbronchiectasis, one has indeed seen the production of pathogenic antibodies to citrullinated proteins [62].

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Anti-inflammatory pathways With its large surface area, the lung is a portal of entry for many pathogens as inhaled air is contaminated with infectious agents, toxic gases and (fine) particulate matter. At the same time, inhaled microbes and toxic substances can gain easy access to the bloodstream across the delicate alveolar–capillary membrane. Innate and adaptive immune defence of this vulnerable barrier is not easy and needs to be tightly controlled as too much oedema, inflammation and cellular recruitment will lead to thickening of the alveolar wall and will jeopardise the diffusion of oxygen vital to life. Considering the large surface area of the respiratory epithelium and the volume of air inspired on a daily basis it is remarkable that there is so little inflammation under normal conditions, suggesting the presence of regulatory mechanisms that act to protect the gas-exchange mechanism. Even following severe bacterial or viral infection, a return to homeostasis is the usual outcome. Understanding the conditions by which lung immune homeostasis is regulated might be crucial to advance our insight into the pathogenesis of inflammatory lung diseases such as bronchiectasis. One type of cell that has received particular attention in suppressing immune responses in the lung is the alveolar macrophage. Alveolar macrophages adhere closely to AECs at the alveolar wall and are separated by only 0.2–0.5 mm from interstitial DCs. In macrophagedepleted mice, the DCs have a clearly enhanced antigen presenting function [67]. When mixed with DCs in vitro, alveolar macrophages suppress T-cell activation through the release of nitric oxide (mainly in rodents), prostaglandins, IL-10 and TGF-b. Alveolar macrophages also express CD200R, an inhibitory receptor that regulates the strength of innate immunity to inhaled pathogens. Another cell type that has received a lot of attention is the regulatory T-cell (Treg). Natural Tregs express high levels of CD25 and express the lineage specific transcription factor Foxp3 [68]. These cells are generated in the thymus and have a natural reactivity for self antigens as well as some foreign antigens, and mainly suppress autoimmunity [69]. Induced Tregs are generated when DCs encounter self antigen in the periphery or upon chronic immune stimulation. It is assumed that these induced Tregs serve to dampen overt immune activation to stimuli that cannot be fully eliminated, a typical example being chronic helminth infections or mycobacterial infections [70]. As bronchiectasis is a disorder of chronic inflammation accompanied by microbial colonisation, it is very likely that increased Tregs are found inside lesions, although this has not been formally addressed. It is also possible that failure of Treg function at a certain stage of the disease contributes to ongoing inflammation, which might ultimately progress to fibrosis. In this regard it is a striking observation that Tregs also make TGF-b as part of their suppressive programme. TGF-b might be at the crossroads of immunoregulation and fibrosis initiation.

Immune regulation might also stem from changes in stromal cells of the airways, such as epithelial cells. Airway epithelial cells play a predominant role in deciding whether or not an acute or chronic stimulus like endotoxin is recognised or not [71]. Epithelial cells express many pattern recognition receptors and the sensitivity of these can be regulated through negative regulators of signalling. Finally, some epithelial derived cytokines, such as IL-37, have an intrinsically antiinflammatory effect on innate immunity in the lung [72]. It is currently unknown if defects in these counter-regulatory mechanisms are involved in the maintenance of inflammation in patients with bronchiectasis.

Conclusion There has been great progress in our knowledge of innate and adaptive immune responses in the lung. Immune defects in innate and adaptive cellular and humoral immunity can all lead to bronchiectasis. In contrast to other obstructive airway diseases, such as asthma and COPD, we have not yet fully grasped the immunopathogenesis of chronic inflammation in this disorder.

Support statement

None declared.

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Statement of interest

B.N. LAMBRECHT ET AL.

B.N. Lambrecht is supported by grants from Fonds voor Wetenschappelijk Onderzoek Flanders (Odysseus Program), European Research Council (ERC) starting grant and Multidisciplinary Research Platform (GROUP-ID consortium) of University of Ghent, Ghent, Belgium. K. Neyt is supported by a fellowship of FWO Flanders.

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Chapter 3

Histopathology of bronchiectasis M. Goddard

HISTOPATHOLOGY

Summary The clinical presentation of bronchiectasis occurs after initial irreversible damage to the airway has occurred. The clinician then has to control symptoms and limit the progression of the disease. A clearer understanding of the pathogenesis of this disease will enable the development of better treatment strategies. Bronchiectasis is a multi-factorial disease process in which there are a number of key steps, although they are not always clinically identifiable. There is often an initiator or damaging event such as a viral infection which, in an individual with a predisposing risk such as a degree of immune dysfunction or an impaired mucociliary clearance system, leads to persistent and damaging bacterial infections. These infections go on to provoke an inappropriate and self-damaging inflammatory response in which neutrophil activity leads to progressive tissue damage and a relentless cycle of infection, inflammation and bronchial wall injury. Persistent infection and chronic inflammatory cell infiltration further amplify the local inflammatory milieu and may lead to systemic complications. Keywords: Aetiology, bronchiectasis, histopathology, inflammation, neutrophils, pathogenesis

Correspondence: M. Goddard, Dept of Pathology, Papworh Hospital NHS Foundation Trust, Papworth Everard, Cambridge, CB23 3RE, UK, Email [email protected]

Eur Respir Mon 2011. 52, 22–31. Printed in UK – all rights reserved. Copyright ERS 2011. European Respiratory Monograph; ISSN: 1025-448x. DOI: 10.1183/1025448x.10003310

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ronchiectasis was first described at the beginning of the 19th century by LAENNEC [1]. He ascribed the term to the pooling of secretions in the airways leading to wall weakening and dilatation. Whilst the term is often used loosely by radiologists to describe any airway dilatation, pathologically the term is used to describe an irreversible dilatation of the airway often associated with chronic suppuration.

Aetiology and classification Aetiologically, bronchiectasis may be divided into obstructive and nonobstructive types (table 1).

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In the obstructive type of bronchiectasis airway dilation may develop from obstruction due to any cause. The disease is confined to the airways distal to the obstruction. Causes of obstruction may be luminal, such as the inhalation of foreign bodies. This is most common in children and shows a

The pathogenesis of obstructive bronchiectasis is relatively straightforward, as bronchial secretions accumulate distal to the obstruction and become infected producing inflammation and damage to the bronchial wall which becomes weakened and dilated. This was recognised by LAENNEC [1].

Table 1. Causes of bronchiectasis Bronchial obstruction Foreign bodies Tumours Carcinoid tumours Endobronchial chondromas and lipomas Mucous plugs ABPA Nonobstructive Post-infective Measles Adenovirus Pertussis Tuberculosis Mucociliary abnormalities Cystic fibrosis Ciliary dysmotility syndromes Primary ciliary dyskinesia Immunological abnormalities Hypogammaglobulinaemia IgA and IgG sub-class deficiencies Neutrophil function abnormalities Ataxia telangiectasia Associated with systemic diseases Rheumatoid arthritis Sjo¨gren’s syndrome Ankylosing spondylitis a1-antitrypsin deficiency Pulmonary fibrosis ABPA: allergic bronchopulmonary aspergillosis;

There have been several attempts to classify nonIg: immunoglobulin. obstructive bronchiectasis, which are variably based on a mixture of historic bronchographic and, more recently, high-resolution computed tomography (HRCT), appearances (saccular or cystic, fusiform or cylindric) [10] and histological appearances (follicular) with lymphoid follicles in the wall, as defined by WHITWELL [11]. Nonobstructive bronchiectasis is typically more widespread, affecting more than one lobe and most commonly affecting the basal segments of the lower lobes [11–13]. The left lung is more frequently affected than the right and the disease process typically involves the middle-order bronchi (fourth to ninth generations).

M. GODDARD

predilection for the right lower lobe and posterior segment of the right upper lobe [2–5]. The risk of developing bronchiectasis following foreign body inhalation has been assessed as 3–16% [6]. However, other causes of obstruction include mucus inspissation in allergic bronchopulmonary aspergillosis (ABPA) [7], with central or upper lobe bronchiectasis, and distal to broncholitis. Tumours may also cause obstruction and bronchiectasis but this is more prevalent in the slower growing often polypoid tumours, such as carcinoid tumours, endobronchial lipomas and chondromas [8]. Extrinsic compression of the bronchus may also lead to obstruction, and typically hilar tuberculous lymphadenopathy can lead to bronchiectasis, particularly of the right middle and lower lobes. The middle lobe, because of its relatively narrow lumen, is at particular risk of compression or obstruction, a condition sometimes referred to as middle lobe syndrome [9].

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This classification is now probably unsatisfactory and of little pathological significance; although the distribution of changes may provide a clue to the underlying aetiology. Post-infective bronchiectasis is traditionally the most common underlying cause, is most often basal and may be confined to a single lobe. However, nonobstructive bronchiectasis may occur in association with a number of other conditions. In diseases where mucociliary clearance is impaired, bronchiectasis frequently, if not inevitably, develops. In cystic fibrosis [14] and ciliary dysmotility syndromes, such as primary ciliary dyskinesia arising from a defect in the dynein arms [15], bronchiectais is more widespread, sometimes with upper lobe predominance. The association of bronchiectasis with disturbances in mucociliary clearance mechanisms highlights the importance of local defence mechanisms within the airways in aetiological terms and in terms of the pathogenesis of the disease. There may be primary defects in the immune system with abnormalities of neutrophil function, hypogammaglobulinaemia [16], immunoglobulin (Ig)A and IgG sub-class deficiencies [17], and in ataxia telangiectasia. Bronchiectasis may also be associated with some autoimmune conditions including ulcerative colitis [18], rheumatoid disease [19], Sjo¨gren’s syndrome [20] and ankylosing spondylitis. Bronchiectasis is also associated with several noninflammatory conditions within the lung, such as a1-antitrypsin deficiency, and is reported in some cases of pulmonary fibrosis but in these cases may be due to traction effects of the surrounding fibrosis.

Of the known causes or associations of bronchiectasis, childhood infection is probably the most common accounting for up to 30% of cases, with immunodeficiencies present in up to 18%. However, in some studies, no underlying abnormality can be detected in .50% of cases.

Histopathological appearances Histopathologically, bronchiectatic airways appear dilated and on examination have a crosssectional area that is much larger than the accompanying pulmonary artery (fig. 1). The airway lumen is often filled with a mucopurulent exudate with neutrophils and macrophages (figs 2 and 3). The respiratory epithelium lining shows variable changes from a reserve cell hyperplasia to squamous metaplasia (fig. 4) with active inflammation shown by epithelial and mucosal infiltration by neutrophils and in severe exacerbations, ulceration (figs 5 and 6). The bronchial wall is often destroyed due to loss of fibromuscular tissues and the elastic framework, and may show erosion and loss of cartilage [21]. There is usually a reduction in submucosal glands. The wall may be thin but is more often greatly thickened with extensive peribronchial fibrosis extending into the adjacent lung parenchyma (fig. 7) [22]. There is an associated chronic inflammatory cell infiltrate within the wall, predominantly lymphocytes and plasma cells, and in some cases lymphoid follicles with germinal centres may be prominent [23]. The presence of B-cell immune activation through the presence of germinal centres and plasma cells in the walls of bronchiectatic airways, would support the role of antibodies in the immune response to persistent infection. However, bronchiectasis is associated with some autoimmune connective tissue diseases, in particular rheumatoid arthritis [19, 24, 25], and a role for autoimmunity in the destruction of the airway has also been suggested.

HISTOPATHOLOGY

Eosinophils may be seen as part of the infiltrate as with any chronic airways inflammation. Whilst nonspecific, they raise the possibility of an associated fungal infection such as Aspergillus. Although eosinophils are commonly seen in the mucus plugs of ABPA, their presence within the airway wall inflammation is nonspecific. Granulomas and multinucleate giant cells may be seen in the wall and might be a reaction to the inspissated luminal material but the possibility of concomitant fungal or mycobacterial infection should always be considered. In established bronchiectasis, the histological pattern of chronic inflammation within the airway wall with superimposed active inflammation, most likely reacting to concomitant infection, has a fairly uniform appearance and provides little insight into the underlying aetiology or pathogenesis.

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Figure 1. Low-power view of a bronchiectatic airway; note the airway lumen is much larger than that of the accompanying pulmonary artery. Magnification64.

The surrounding lung parenchyma may show a number of changes. Where there is distal luminal obliteration of bronchi and bronchioles, the lung parenchyma may

The airways are supplied by the bronchial arteries and the inflammatory destruction and healing processes result in the formation of bronchopulmonary anastamoses, probably due to a mixture of new vessel formation and the re-opening of pre-existing, pre-capillary bronchopulmonary connections (fig. 8). Ulceration of the airways can lead to severe haemorrhage and haemoptysis. The formation of anastamoses and the loss of some of the alveolar capillary bed leads to the development of pulmonary hypertension [27].

Figure 2. Bronchiectatic airway wall with dense chronic inflammatory cell infiltrate, which includes lymphocytes, plasma cells and eosinophils. Magnification620.

M. GODDARD

show atelectasis due to absorption and collapse. Obliterative changes in small airways are important in contributing to airflow obstruction in bronchiectasis [10, 11]. Destructive inflammation may lead to the formation of an abscess cavity, although this may be difficult to distinguish from a distended, ulcerated airway. There may be accompanying interstitial pneumonitis, particularly in cases of follicular bronchiectasis, and also changes of an organising pneumonia. Small airway changes, such as bronchiolectasis, may be seen as part of the whole disease process or may be part of an underlying disease leading to more proximal dilatation, as has been seen with small airways disease, such as bronchiolitis [18, 26].

Focal proliferations of neuroendocrine cells are also seen and may lead to the formation of multiple tumourlets, small aggregates of neuroendocrine cells in the walls of small airways. These are not specific to bronchiectasis and may be seen in a number of chronic lung conditions [28].

Figure 3. Bronchiectatic airway wall with luminal pus, neutrophil infiltration of the airway epithelium and a dense chronic inflammatory cell infiltrate. Magnification640.

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It has also been recognised that the persistent chronic activation of the immune system in the wall of the airway may lead to the development of bronchus-associated lymphoid tissue (BALT), especially in bronchiectasis associated with Sjo¨gren’s syndrome [29]. Increased incidence of BALTomas,

low-grade, B-cell lymphomas, is associated with Sjo¨gren’s syndrome but not specifically related to bronchiectasis [20]. In chronic disease, further complications may arise. Locally, the lung may develop abscesses and even empyema, although this is less common as the pleural space is often obliterated by fibrous adhesions. Bronchiectatic spaces may become colonised by saprophytic fungi, most commonly Aspergillus sp.

HISTOPATHOLOGY

Systemic dissemination of infection may lead to abscesses in other organs, notably the brain, and chronic suppuration may be complicated by systemic amyloidosis (type AA). The incidence in bronchiectasis is unclear but in one study of patients with systemic amyloidosis requiring haemodialysis, 40% had underlying bronchiectasis [30]. Figure 4. Squamous metaplasia of the epithelium lining in bronchiectasis. Magnification640.

Pathogenesis

The pathogenesis of bronchiectasis is complex and a number of different mechanisms contribute to the development of a similar morphological appearance and different factors act together to set up a cycle of inflammation and destruction that leads to damage and destruction of the bronchial wall [31]. The initiator to this sequence is usually damage to the bronchial epithelium. This may be due to an external insult or to an intrinsic deficiency within the patient. The most common predisposing factor to the development of bronchiectasis is a severe childhood respiratory infection, which may be viral, such as measles or adenovirus, or bacterial, such as Bordetella pertussis [32–34]. The resultant permanent dilatation of the airways is thought to be due not only to inflammation and destruction of the bronchial wall but also, in part, to a traction effect produced by collapse of the surrounding lung parenchyma. However, the persistence of infection and inflammation are of paramount importance in the progression of the disease.

Figure 5. Severely inflamed ulcerated bronchiectatic airway with

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no epithelium and surface granulation tissue. Magnification620.

Whilst an underlying cause is not established in all cases, the number and type of associations for bronchiectasis gives us some indication of what the important underlying pathogenetic mechanisms may be.

In post-infectious causes, the initiating viral infection appears to be transitory and, in the case of adenoviruises, it has not been possible to demonstrate the persistence of the virus within bronchiectasis by in situ hybridisation [35]. However, some respiratory viruses have been shown to lead to abnormalities in ciliary function, which may persist for several weeks [36].

In the early stages of bronchiectasis, the most common bacterial isolate is Haemophilus influenzae, which has the capacity to directly damage the airway epithelium and induce the production of inflammatory mediators [37]. The typical immune response to H. influenzae is a T-helper (Th)1 response. However, some bronchiectasis patients with persistent infection have been found to have a Th2 response with a cytokine profile of interleukin (IL)-4 and IL-10. The release of cytokines contributes to the inflammatory response within the airway and at the same time may also result in a failure of the response to satisfactorily remove the organism [38].

Figure 7. A bronchiectatic airway showing an attenuated inflamed epithelium with surrounding inflammation and fibrosis extending into the peribronchial lung parenchyma. Magnification620.

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Over time, a number of other organisms have been found to be established within the airways, particularly Streptococcus pneumoniae and Pseudomonas aeruginosa. The initial damage to the epithelium lining allows this secondary bacterial colonisation to occur, which further inhibits ciliary clearance and promotes the persistence of infection and damaging inflammation [39]. The importance of this persistence in bacterial colonisation may be related to the production of heatlabile products by the bacteria, which further damage ciliated cells and inhibit ciliary activity. P. aeruginosa can be a particular problem as it is protected from cellular and humoral attack because it survives in a biofilm on the mucosal surface [40]. Pseudomonas has been shown

M. GODDARD

Clinically, it is the recurrence and persistence of bacterial infections in the airways with which most patients present that are of most importance and are linked to the progression of the disease. There is a Figure 6. Ulcerated airway with surrounding fibrosis of the wall. prevailing view that bacterial infecMagnification610. tion in the lower respiratory tract provokes an exaggerated and uncontrolled neutrophilic response and that the complex interplay between bacterial infection and airway inflammation, along with the release of tissue damaging substances, leads to the progressive damage which typifies bronchiectasis.

to produce phenazine pigments that can inhibit ciliary action through a mechanism which leads to a reduction in cellular cAMP and ATP. Furthermore, pseudomonal pyocyanin can lead to epithelial disruption and rhamnolipids have a ciliostatic effect [41, 42]. Alveolar macrophages are an important mediator of defence against Pseudomonas and stimulated macrophages secrete cytokines that both recruit and activate neutrophils, thus potentially amplifying both the inflammatory response and the potential for further tissue damage [43, 44]. The resultant inflammatory reaction is an important pathogenic mechanwall. Magnification620. ism in the weakening of the bronchial wall. Much of the damage appears to relate to the release of proteolytic enzymes and oxygen free radicals from neutrophils. The severity of an inflammatory response is dependent on the interplay of several cytokines, which may be both pro- and anti-inflammatory [45]. In a well-regulated system, the inflammatory cascade is proportionate to the triggering bacterial stimulation and is switched off. There is evidence that in bronchiectasis the inflammatory response is disproportionate to the infective burden and that the inflammatory response persists [43, 46]. Indeed, in the early phases of bronchiectasis, active airway inflammation has even been reported in the absence of identifiable microbial infection, suggesting a dysregulation of the cytokine network independent of infection [47].

HISTOPATHOLOGY

Figure 8. Thick-walled bronchial artery in a bronchiecatic airway

Neutrophils are potent effectors in inflammatory responses and secrete anti-microbial substances, as well as reactive oxygen free radicals [48]. Bronchoalveolar lavage (BAL) studies have demonstrated that neutrophils are consistently present in patients with bronchiectasis, even when sterile and clinically stable, but increase in the presence of potential pathogens [49, 50]. Recruitment and migration of neutrophils in airways is facilitated by the activation of neutrophils and the upregulation of adhesion molecules on endothelial cells [51–53]. These changes are regulated by cytokines, particularly IL-1 and tumour necrosis factor (TNF)-a, as well as lipopolysaccharide (LPS), which have been shown to be increased in the airways of patients with bronchiectasis [54, 55]. Activated neutrophils secrete potentially tissue damaging enzymes such as neutrophil elastase, proteinase 3 and metalloproteinases. Levels of these enzymes in BAL samples have been shown to correlate with neutrophil numbers and markers of disease activity such as 24hour sputum production [56]. These enzymes can directly damage the structural integrity of the airway via damage to the basement membrane and elastin framework [57–60]. Neutrophils are also an important source of oxygen free radicals. Release of oxygen free radicals are an important part of the defence against infection and are regulated by a protective anti-oxidant system. However, the excessive release of these oxidants can overwhelm the defence mechanisms and cause tissue damage via lipid peroxidation. Furthermore, reactive oxygen species may amplify the inflammatory response through the induction of cytokine and chemokine production by the stimulation of genes regulated by nuclear factor-kB. Studies in bronchiectatic patients have shown increased levels of exhaled H2O2 correlating with neutrophil counts and disease activity [61, 62].

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Macrophages also play a role in the disease progression as they secrete TNF-a, which promotes neutrophil recruitment, as well as other inflammatory mediators including IL-8, monocyte chemotactic protein-1 and chemokines [23, 63]. Lymphocytes are also typically present in

bronchial biopsies within the lamina propria and may also infiltrate the overlying epithelium. Studies assessing the relative proportions of CD4+ and CD8+ have produced mixed and, at times, conflicting results. Nonetheless, their presence indicates a cell-mediated immune response contributing to the overall inflammatory process [22]. Whilst the epithelial layer may be seen as a protective barrier through mucociliary clearance and generation of anti-bacterial substances, it also contributes to the inflammatory process through the direct generation of pro-inflammatory cytokines [64]. Exposure to LPS leads to the generation of IL-8 and TNF-a which, as stated previously, are important in neutrophil recruitment. Bronchial epithelial cells are also able to upregulate surface adhesion molecules, such as intracellular adhesion molecule-1, aiding the migration of neutrophils [65, 66]. Thus, a number of pathways lead to the activation and recruitment of neutrophils into the airways which, if not adequately regulated and controlled, results in the destruction of local tissue and the persistence and progression of bronchiectasis. Individual variability in this innate response may help to explain why not all individuals exposed to predisposing triggers will go on to develop bronchiectasis and offers potential targets for therapeutic intervention.

Statement of interest None declared.

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1. Laennec RTH. De l’Auscultation Mediatle ou Traite du Diagnostic des Maladies des Poumons et du Couer. [On Mediate Auscultation or Treatise on the Diagnosis of the Lungs and Heart.] Paris, Brosson and Claude, 1819. 2. Weissberg D, Schwartz T. Foreign bodies in the tracheobronchial tree. Chest 1987; 91: 730–733. 3. Limper AH, Prakash UB. Tracheobronchial foreign bodies in adults. Ann Intern Med 1990; 112: 604–609. 4. Scala R, Aronne D, Palumbo U, et al. Prevalence, age, distribution and aetiology of bronchiectasis: a retrospective study on 144 symptomatic patients. Monaldi Arch Chest Dis 2000; 55: 101–105. 5. Tsao PC, Lin CY. Clinical spectrum of bronchiectasis in children. Act Paeditr Taiwan 2002; 43: 271–275. 6. Barker AF, Bardana EJ. Bronchiectasis: update of an orphan disease. Am Rev Respir Dis 1988; 137: 969–978. 7. Bosten C, Myer J, Greenberger P, et al. Pathologic features of allergic bronchopulmonary aspergillosis. Am J Surg Pathol 1988; 12: 216–222. 8. Box K, Kerr KM, Jefferey RR, et al. Endobronchial lipoma associated with lobar bronchiectasis. Repsir Med 1991; 85: 71–72. 9. Kwon KY, Myers JL, Swensen SJ, et al. Middle lobe syndrome: a clinicopathological study of 21 patients. Hum Pathol 1995; 26: 302–307. 10. Reid LM. Reductions in bronchial subdivisions in bronchiectasis. Thorax 1950; 5: 223–247. 11. Whitwell F. A study of the pathology and pathogenesis of bronchiectasis. Thorax 1952; 7: 213–239. 12. Ogilvie AG. The natural history of bronchiectasis. A clinical, roentgenologic and pathologic study. Arch Intern Med 1941; 68: 395–465. 13. Percy KMA, King DC. Bronchiectasis: A study of prognosis based on a follow-up of 400 cases. Am Rev Tuber 1940; 41: 531–548. 14. Ogninc G, Kampalath B, Tomashefski JF. Destruction and loss of bronchial cartilage in cystic fibrosis. Hum Pathol 19888, 29: 65–73. 15. Rossman CM, Forrest JB, Lee RM, et al. The dyskinetic cilia syndrome. Ciliary motility in immotile cilia syndrome. Chest 1980; 78: 580–582. 16. Watts WJ, Watts MD, Dai W, et al. Respiratory dysfunction in patients with common variable hypogammaglobulinaemia. Am Rev Respir Dis 1986; 134: 699–703. 17. Gracia JD, Rodrigo MJ, Morrell F, et al. IgG subclass deficiencies associated with bronchiectasis. Am J Respir Crit Care Med 1996; 153: 650–655. 18. Butland RJ, Cole P, Citron KM, et al. Chronic bronchial suppuration and inflammatory bowel disease. Q J Med 1981; 50: 63–75. 19. Tahanami I, Imamuma T, Yamamoto Y, et al. Bronchiectasis complicating rheumatoid arthritis. Respir Med 1995; 89: 453–454. 20. Strimlan CV, Rosenow EC, Diverite MB, et al. Pulmonary manifestations of Sjogren’s syndrome. Chest 1976; 70: 354–361. 21. Hayward J, Reid ML. The cartilage of intra-pulmonary bronchi in normal lungs, in bronchiectasis and in massive collapse. Thorax 1952; 7: 98–110.

M. GODDARD

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22. Silva JR, Jones JA, Cole PJ, et al. The immunological component of the cellular inflammatory infiltrate in bronchiecatsis. Thorax 1989; 44: 668–673. 23. Gaga M, Bentley AM, Humbert M, et al. Increases in CD4+ T lymphocytes, macrophages, neutrophils and interleukin 8 positive cells in the airways of patients with bronchiectasis. Thorax 1998; 53: 685–691. 24. Solanki E, Neville E. Bronchiectasis and rheumatoid disease. Is there an association? Br J Rheumatol 1992; 31: 691–693. 25. Mahon MJ, Swinson DR, Shettar S, et al. Bronchiectasis and rheumatoid arthritis: a clinical study. Ann Rheum Dis 1993; 52: 776–779. 26. Moles KM, VArjhess G, Hayes JR. Pulmonary involvement in ulcerative colitis. Br J Dis Chest 1988; 82: 79–83. 27. Liebow AA, Hales MR, Linskog GE. Enlargement of the bronchial arteries and their anastomoses with the pulmonary arteries in bronchiectasis. Am J Pathol 1949; 25: 211–231. 28. Whitwell F. Tumourlets of lung. J Pathol Bact 1955; 70: 529–541. 29. Yousem SA, Colby TV, Carrington CB. Follicular bronchitis/bronchiolitis. Hum Pathol 1985; 16: 700–706. 30. Akcay S, Akman B, Ozdemir H, et al. Bronchiectasis related amyloidosis as a cause of chronic renal failure. Ren Fail 2002; 24: 815–823. 31. Cole PJ. Inflammation: a two edged sword – the model of bronchiectasis. Eur J Respir Dis Suppl 1986; 147: 6–15. 32. Warne WP. Factors causing bronchiectasis. JAMA 1935; 105: 1666–1670. 33. Glauser EM, Cook CD, Harris GBC. Bronchiectasis: a review of 187 cases. Acta Pediatr Scand 1966; 165: 1–15. 34. Whooping cough in the United States and Britain. N Engl J Med 1983; 309: 108–109. 35. Hogg JC, Irving WL, Porter H, et al. In situ hybridisation studies of adenoviral infections of the lung and their relationship to follicular bronchiectasis. Am Rev Respir Dis 1989; 139: 1531–1535. 36. Rayner CFJ, Rutman A, Dewar A, et al. Ciliary disorientation in patients with chronic reparatory tract inflammation. Am J Respir Crit Care Med 1995; 151: 800–804. 37. Starner TD, Zhang N, Kim G, et al. Haemophilus influenzae forms biofilms on airways epithelia: implications in cystic fibrosis. Am J Respir Crit Care Med 2006; 174: 213–230. 38. King PT, Hutchinson PE, Johnson PD, et al. Adaptive immunity to non typeable Haemophilus influenzae. Am J Respir Crit Care Med 2003; 167: 587–592. 39. Wilson R, Dowlin RB, Jackson AD. The biology of bacterial colonisation and invasion of the respiratory mucosa. Eur Respir J 1996; 9: 1523–1530. 40. Mathee K, Ciofu O, Sternberg C, et al. Mucoid conversion of Pseudomonas aeruginosa by hydrogen peroxide: a mechanism for virulence activation in the cystic fibrosis lung. Microbiology 1999; 145: 1349–1357. 41. Lau GW, Hasset DJ, Ran H. The role of pyocyanin in Pseudomonas aeruginosa infection. Trends Mol Med 2004; 10: 599–606. 42. Caldwell C, Chen Y, Goetzmann HS, et al. Pseudomonas aeruginosa exotoxin pyocyanin causes cystic fibrosis airway pathogenesis. Am J Pathol 2009; 175: 2473–2488. 43. Chmiel JF, Davis PB. State of the art: why do the lungs of patients with cystic fibrosis become infected and why can’t they clear infection? Respir Res 2003; 4: 8. 44. Lu W, Hisatsume A, Koga T, et al. Cutting edge: enhanced pulmonary clearance of Pseudomonas aeruginosa by Muc1 knockout mice. J Immunol 2006; 176: 3890–3894. 45. Wouters EFM. Local and systemic inflammation in chronic obstructive pulmonary disease. Proc Am Thorac Soc 2005; 2: 26–33. 46. Aldallal N, Mc Naughton EE, Manzel LJ, et al. Inflammatory response in airway epithelial cells isolated from patients with cystic fibrosis. Am J Respir Crit Care Med 2002; 166: 1248–1256. 47. Angrill J, Agusti C, De Celis R, et al. Bronchial inflammation and colonisation in patients with clinically stable bronchiectasis. Am J Respir Crit Care Med 2001; 164: 1628–1632. 48. Liu Y, Shaw Sk, Ma S, et al. Regulation of leucocyte transmigration: cell surface interactions and signalling events. J Immunol 2004; 172: 7–13. 49. Eller J, Lap e Silva JR, Poulter LW, et al. Cells and cytokines in chronic bronchial infection. Ann NY Acad Sci 1994; 725: 331–345. 50. Loukides S, Bouros D, Papatjheodorou G, et al. Exhaled H2O2 in steady-state bronchiectasis: relationship with cellular composition in induced sputum, spirometry, and extent and severity of disease. Chest 2002; 121: 81–87. 51. Adams DH, Shaw S. Leucocyte-endothelial interactions and regulation of leucocyte migration. Lancet 1994; 343: 831–836. 52. Hogg JC. Leukocyte traffic in the lung. Ann Rev Physiol 1995; 57: 97–114. 53. Zheng L, Tipoe G, Lam WK, et al. Upregulation of circulating adhesion molecules in bronchiectasis. Eur Respir J 2000; 16: 691–696. 54. Carlos T, Kovach N, Schwarz B, et al. Human monocytes bind to two cytokine induced adhesive ligands on cultured human endothelial cells: endothelial-leukocyte adhesion molecule-1 and vascular cell adhesion molecule-1. Blood 1991; 77: 2266–2271. 55. Dustin ML, Rothlein R, Bhan AF, et al. Induction by IL-1 and interferon-c: tissue distribution, biochemistry and function of a nature adherence molecule (ICAM-1). J Immunol 1986; 137: 245–254. 56. Pang JA, Cheng A, Chan HS, et al. The bacteriology of bronchiectasis in Hong Kong investigated by protected catheter brush and bronchoalveolar lavage. Ann Rev Respir Dis 1988; 139: 14–17.

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57. Sepper R, Kontinnen YT, Ding Y, et al. Human neutrophil collagenase (MMP-8) identified in bronchiectasis BAL fluid, correlates with severity of disease. Chest 1995; 107: 1641–1647. 58. Zheng L, Lam WK, Tipoe GL, et al. Overexpression of matrix metalloproteinases-8 and -9 in bronchiectasis airways in vivo. Eur Respir J 2002; 20: 170–176. 59. Sepper R, Kontinnen YT, Sorsa T, et al. Gelatinolytic and type-IV collagenolytic activity in bronchiectasis. Chest 1994; 106: 1129–1133. 60. Doring G. The role of neutrophil elastase in chronic inflammation. Am J Respir Crit Care Med 1994; 150: S114–S117. 61. Lloberes P, Monserrat E, Monserrat JM, et al. Sputum sol phase proteins and elastase activity in patients with clinically stable bronchiectasis. Thorax 1992; 47: 88–92. 62. Loukides S, Horvath I, Wodehouse T, et al. Elevated levels of expired breath hydrogen peroxide in bronchiectasis. Am J Respir Crit Care Med 1998; 158: 991–994. 63. Simpson JI, Grissell TV, Gibson PG. Innate immune activation in bronchiectasis. Eur Respir J 2004; 24: Suppl. 48, 210S. 64. Devalia JL, Davies RJ. Airway epithelial cells and mediators of inflammation. Respir Med 1993; 87: 405–408. 65. Look DC, Rapp SR, Keller BT, et al. Selective induction of intercellular adhesion molecule-1 by interferon-c in airway epithelial cells. Am J Physiol 1992; 263: L79–L87. 66. Humlicek AL, Pang L, Look DC. Modulation of airway inflammation and clearance by epithelial ICAM-1. Am J Physiol Lung Cell Mol Physiol 2004; 287: L598–L607.

Chapter 4

Assessment and investigation of adults with bronchiectasis M. Drain and J.S. Elborn

ASSESSMENT AND INVESTIGATION

Summary The diagnosis of bronchiectasis is made on the basis of highresolution computed tomography (HRCT) scan findings. A diagnosis of bronchiectasis should be considered in all patients with persistent cough productive of sputum, where another clear diagnosis has not been made. This includes patients with an initial diagnosis of chronic obstructive pulmonary disease or severe asthma. Once bronchiectasis has been confirmed by HRCT scanning, patients should undergo a range of investigations to determine whether or not there is an underlying cause. This can usually be determined in approximately 50% of patients with bronchiectasis. The common conditions which should be sought are cystic fibrosis, immunodeficiency syndromes, primary ciliary dyskinesia, and autoimmune diseases, such as rheumatoid arthritis and ulcerative colitis. For many of these conditions, there is specific treatment to improve symptoms and reduce lung injury but, without an accurate diagnosis, appropriate therapy may not be instituted. Keywords: Bronchiectasis, computed tomography scan, cystic fibrosis, primary ciliary dyskinesia, primary immunodeficiency

B

Centre for Infection and Immunity, School of Medicine, Dentistry and Biomedical Sciences, Queen’s University Belfast, Belfast, UK. Correspondence: J.S. Elborn, Centre for Infection and Immunity, School of Medicine, Dentistry and Biomedical Sciences, Queen’s University Belfast, 97 Lisburn Road, Belfast, BT9 7BL, UK, Email [email protected]

Eur Respir Mon 2011. 52, 32–43. Printed in UK – all rights reserved. Copyright ERS 2011. European Respiratory Monograph; ISSN: 1025-448x. DOI: 10.1183/1025448x.10003410

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ronchiectasis is a generic diagnostic term that describes the pathological dilation of the airways found in a number of chronic lung conditions. The aetiology of bronchiectasis is varied (table 1), and, in most series, an underlying cause can only be definitively identified in 50% of cases [1, 2]. The importance of determining a cause lies in facilitating treatment that may improve symptoms, reduce exacerbations and alter the course of the disease by preserving lung function. In one series in children, extensive investigation detected a specific cause in 74% of those investigated, and this led to a change in treatment in 56% [3]. In an adult series from the same geographical area, a diagnosis was again reached in 74%, and the treatment of 37% of these was affected by knowledge of the diagnosis [4].

Congenital

Acquired

Cystic fibrosis# Primary ciliary dyskinesia# a1-Antitrypsin deficiency Congenital anatomical defects Tracheo-oesophageal fistula Bronchotracheomalacia Tracheomegaly Pulmonary sequestration Yellow nail syndrome Marfan’s syndrome Cystic fibrosis# Primary ciliary dyskinesia#

Following infection# Bacterial# Whooping cough Tuberculosis Nontuberculous mycobacteria Viral Measles HIV# Fungal ABPA# Immunodeficiency Primary Common variable immunodeficiency# X-linked agammaglobulinaemia# IgA deficiency MHC class II deficiency B-cell deficiency Hyper-IgE syndrome Secondary Following chemotherapy# Haematological malignancy# Graft-versus-host disease Interstitial lung disease# (traction bronchiectasis) Autoimmune disease Rheumatoid arthritis# Ulcerative colitis Sjo¨gren’s syndrome Sarcoidosis Following surgery Inhaled foreign body Chronic GORD

ABPA: allergic bronchopulmonary aspergillosis; Ig: immunoglobulin; MHC: major histocompatibility complex; GORD: gastro-oesophageal reflux disease. #: more common conditions that should be considered when making an initial diagnosis [2].

Childhood respiratory infection, e.g. whooping cough, measles, tuberculosis (TB) or severe bacterial pneumonia, is cited as being responsible for a large proportion of cases of bronchiectasis, i.e. up to 50% [4–6]. This potential cause, however, is subject to recall bias, particularly since the majority of cases present in the fifth and sixth decades of life. Many people of this age have had measles, whooping cough or other childhood infections associated with respiratory infection, including pneumonia. In addition, the first episode of pulmonary infection could represent the first exacerbation of bronchiectasis. Bronchiectasis is found in association with numerous multisystemic diseases, such as cystic fibrosis (CF) [7], immunodeficiencies [8], a1-antitrypsin (a1-AT) deficiency [9], primary ciliary dyskinesia (PCD) [10], rheumatoid arthritis and inflammatory bowel diseases, especially ulcerative colitis [1, 7, 11].

M. DRAIN AND J.S. ELBORN

Table 1. Causes of bronchiectasis in adults

Prevalence The prevalence of bronchiectasis is almost certainly underestimated. This is because it is a condition that many healthcare practitioners are unfamiliar with, and it is frequently misdiagnosed as asthma or chronic obstructive pulmonary disease (COPD) due to the similarities in clinical findings (table 2).

33

In the USA, the prevalence of bronchiectasis has been estimated at 4.2 per 100,000 population among those aged 18–34 years, rising to 272 per 100,000 population in those aged .75 years [12].

Table 2. Clinical findings in chronic obstructive pulmonary disease (COPD), asthma and bronchiectasis

Symptom Cough Sputum production Dyspnoea Wheeze Haemoptysis Fever Lethargy Recurrent infection Clinical signs Finger clubbing Breath sounds Added sounds Lung function Spirometry Reversibility Lung volumes Transfer factor Hypoxia Radiology Chest radiography

ASSESSMENT AND INVESTIGATION

CT findings

COPD

Bronchiectasis

Asthma

+ + ++ + +/+/+

+ ++ +/+/+ + + ++

+ +/+ ++ +

No Q/wheeze Wheeze

Extensive disease Normal/Q Crackles

No Normal/wheeze Wheeze

FEV1Q, FVCQ FEV1/ FVCQ 15% Q/q Normal Yes

FEV1Q/normal FVCQ/normal FEV1/FVCQ/normal 40% Normal/Q Normal/Q Yes/no

FEV1Q/normal, FVC normal FEV1Q/normal Yes Normal Normal No

Chronic inflammatory changes, hyperinflation Hyperinflation, airtrapping, bullae, may have mildly dilated airways or thickened bronchial wall

Tramlines, ring shadows/normal

Normal/ hyperinflation Normal/air-trapping, may have mildly dilated airways or thickened bronchial wall

Dilated bronchi, thickened bronchial wall, lack of tapering of bronchi, bronchi visible in outer 1–2 cm, air-trapping

CT: computed tomography; FEV1: forced expiratory volume in 1 second; FVC: forced vital capacity; -: uncommon; +/-: occurs sometimes; +: common; ++: very common; Q: decrease; q: increase.

However, these values are derived from a database of claims from 30 different healthcare insurance plans made over a 2-year period. They are likely to underestimate the true prevalence as they exclude not only the uninsured population but also those who use alternative plans. It cannot, therefore, be claimed that they are truly representative of the real population prevalence. The prevalence of non-CF bronchiectasis in Northern Ireland is estimated at around 5,000 in a population of approximately 2 million [13], leading to 300–400 admissions per annum for the treatment of an infective exacerbation. In children, bronchiectasis is less common. It can be extrapolated that the prevalence should be falling following the advent of improved antibiotic therapies and vaccination of children during the first year of life. Only two national studies have been reported with very different rates, 0.5 per 100,000 population in Finland [14] and 3.7 per 100,000 population in New Zealand [15]. In certain indigenous population groups, the prevalence is much higher, e.g. the New Zealand figure doubles among the Maori and Pacific Islander populations [11, 15], Aboriginal rural communities have 14.7 per 1,000 population affected among those aged ,16 years [16] and 16 per 1,000 population in Alaskan natives [17]. This is thought to occur due to an increased rate of severe pulmonary infection in early childhood, due to a combination of socioeconomic factors rather than solely a genetic predisposition.

Approach to diagnosis

34

The diagnostic approach to a patient with bronchiectasis should first establish that there is radiological evidence of airways dilatation and secondarily consider possible underlying conditions [1].

People presenting with a chronic productive cough lasting for .4 weeks or recurrent episodes, with two or more episodes occurring over 8 weeks, should have the diagnosis of bronchiectasis considered [2]. In the scheme outlined in figure 1, all of the first-line investigations should be considered as routine in patients being investigated for bronchiectasis. Most people have already undergone some investigations prior to referral, such as a sputum culture, chest radiography or computed tomography (CT), which may guide further investigations.

Symptoms and physical findings Cough productive of sputum is the most common symptom associated with bronchiectasis [1, 18–21]. In some studies, 25% of patients do not report excessive daily sputum production, but describe a marked increase in volume during an exacerbation [1, 18]. Occasional haemoptysis is a frequent symptom and is reported by half of all patients. This is often associated with a pulmonary exacerbation. Shortness of breath, fever and chest pain are also common complaints among non-CF bronchiectasis patients [18], although they are common symptoms in other chronic inflammatory lung disease that may coexist, e.g. COPD and asthma (table 2). Patients presenting with such symptoms who do not respond as expected to usual therapy should raise the possibility of bronchiectasis and this should be investigated. Some symptoms point to specific diagnoses (table 3).

Physical examination

Diagnostic suspicion of bronchiectasis

Consider other Negative diagnosis

Chest HRCT scan

Sputum culture (including mycobacteria)

Spirometry

Chest radiography

Assessment of functional status and infection in all patients

Positive

Sweat [Cl-] (CF)

Genetics Nasal PD

Igs (CVID)

α1-AT levels and phenotype (α1-AT)

Vaccination studies: Pneumovax and tetanus

Genetics

Nasal NO RF/autoantibodies (PCD) (CTD)

EM studies Genetics Functional studies

Aspergillus IgE and IgG and eosinophilia (ABPA)

M. DRAIN AND J.S. ELBORN

Physical findings are of modest help in the assessment of patients with bronchiectasis. The classic findings of wet crackles and finger clubbing are now uncommon and should trigger investigation for conditions associated with severe bronchiectasis, such as CF. Crackles with some associated

First-line diagnostic investigations to be considered in all patients

Further studies in case of diagnostic suspicion or doubt

Figure 1. Diagnostic approach to bronchiectasis. HRCT: high-resolution computed tomography; [Cl-]: chloride

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ion concentration; CF: cystic fibrosis; Ig: immunoglobulin; a1-AT: a1-antitrypsin; PCD: primary ciliary dyskinesia; NO: nitric oxide; RF: rheumatoid factor; CTD: connective tissue disease; ABPA: allergic bronchopulmonary aspergillosis; PD: potential difference; EM: electron microscopy; CVID: common variable immunodeficiency.

Table 3. Specific historical features suggestive of a particular diagnosis in adults Primary ciliary dyskinesia Cystic fibrosis

Common variable immunodeficiency

Neonatal respiratory distress, middle ear disease, infertility Culture of Staphylococcus aureus, Pseudomonas aeruginosa or Burkholderia cepacia complex, malabsorption symptoms, infertility, recurrent pancreatitis, nasal polyposis Recurrent respiratory, urinary, gastrointestinal and skin infections

wheeze are the most common findings, with finger clubbing now a rare feature, and usually associated with severe disease. Other aspects of examination should focus on clinical signs of associated diseases, such as CF, immune deficiency or a connective tissue disease.

Diagnostic tests Blood tests

ASSESSMENT AND INVESTIGATION

A complete blood count, although nonspecific, is important in monitoring the ongoing condition of each individual patient. Haemoglobin level can be low secondary to anaemia of chronic disease, and, conversely, patients may be polycythaemic secondary to chronic hypoxia. An elevated white cell count may indicate the presence of acute infection. The differential white cell count can reveal lymphopenia, which may prompt further investigation for immunodeficiency syndromes or eosinophilia, which can occur in but is not diagnostic of allergic bronchopulmonary aspergillosis (ABPA). C-reactive protein (CRP) is an acute-phase reactant commonly measured in respiratory patients with acute exacerbations in order to assist in determining whether or not there is a systemic inflammatory response [1, 22, 23]. In bronchiectasis patients in a stable state, it has been shown that CRP levels are elevated from baseline [22]. The CRP level also correlated with decline in lung function and severity of disease on high-resolution CT (HRCT) in the same series [22].

Radiology Although suspected with a history of recurrent lower respiratory tract infection on a background of chronic cough and sputum production, the diagnosis of bronchiectasis can only be confirmed radiologically [2]. The gold-standard investigation is HRCT. This was first described in 1982 [16], and permits a detailed examination of the lung architecture using a noninvasive technique. Historically, the diagnosis was based on bronchography, which involved instillation of a radioopaque dye into the airways and fluoroscopic screening. This technique has been superseded due to the greater detail available in a safer more easily tolerated imaging method and is now obsolete. Volumetric HRCT has some advantages over conventional HRCT as it provides more-detailed images, but it is more prone to image degradation due to motion artefact and requires a higher radiation dose. Standard HRCT is appropriate for the majority of patients. Findings on HRCT are bronchial wall thickening with dilatation of the bronchi to a diameter greater than that of the accompanying arteriole (the signet-ring sign); lack of normal tapering of bronchi/bronchioles on sequential slices; and visualisation of bronchi in the outer 1–2 cm (fig. 2) [1, 23, 24]. The bronchiectatic changes in CF have been quantified using a number of scoring systems, but the value of these in diagnosis or follow-up care has not been established.

36

The histopathological appearance of bronchiectasis has been further subcategorised as cylindrical, saccular and varicose, depending on the shape of the bronchi [25]. The true clinical significance of these subdivisions is unclear. However, cystic bronchiectasis has been associated with an increased frequency of exacerbation and more-clinically significant disease [24]. HRCT appearance can also be used to confirm any other parenchymal or bronchiolar pathology, such as interstitial lung

a)

b)

c)

changes in bronchiectasis. a) Signet-ring sign, i.e. dilatation of the bronchi to a diameter greater than that of the accompanying vessel. b) Visualisation of the bronchi in the outer 1–2 cm. c) Thickened bronchial walls. The circled areas indicate ring shadows.

disease [25, 26]. The distribution of ectatic airways throughout the lung fields can be used to guide investigation of underlying causes, but most changes are nonspecific (table 4) [27, 28]. Although the diagnosis is confirmed radiologically using HRCT, a posteroanterior chest radiograph should be obtained as a baseline with which to compare future films in the event of acute exacerbation. Depending on the distribution of the bronchiectasis and the degree of damage, the chest radiograph may show minimal change from normal or be markedly abnormal. Traditionally, the radiographic changes associated with bronchiectasis are tramlines and ring shadows [18]; these markings correspond to the thickened mucosa of the more-severely inflamed airways in transverse or cross-section.

M. DRAIN AND J.S. ELBORN

Figure 2. High-resolution computed tomography

Table 4. High-resolution computed tomography features of bronchiectasis General features Bronchial dilatation (bronchus diameter greater than that of adjacent vessel) Bronchial wall thickening Bronchial plugging Areas of reduced attenuation (mosaic pattern) Specific features ABPA: upper-zone central bronchiectasis Cystic fibrosis: upper-zone bronchiectasis NTM/MAC: Middle-lobe irregular branching and tree-in-bud appearance

37

ABPA: allergic bronchopulmonary aspergillosis; NTM: nontuberculous mycobacteria; MAC: Mycobacterium avium-intracellulare complex.

Once the diagnosis of bronchiectasis has been confirmed, a detailed clinical work-up should be undertaken in order to determine the extent of the impact on lung function, morbidity and prognosis, the underlying cause of the existing structural lung damage and the most prevalent infecting organisms. The benefits of this are that not only can treatment be tailored to the individual, but also a potentially treatable underlying condition may be uncovered [1, 24, 25]. A comprehensive clinical assessment, including a detailed history and physical examination, are required to illicit any pointers towards a specific diagnosis. This should be followed up by extensive investigation to allow determination of baseline functional status and lung function and to permit guidance of treatment. During the course of investigation, underlying conditions which are known to have an association with bronchiectasis, albeit not a causative one, may be discovered.

ASSESSMENT AND INVESTIGATION

Pulmonary function testing and other physiological factors Forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC) should be measured at the time of the diagnostic evaluation and at least annually, more frequently in the setting of PCD, immunodeficiency or connective tissue disease [21, 24]. Spirometric results may be normal in some patients, although usually show a pattern of airflow limitation, with a decreased FEV1 and a reduced FEV1/FVC ratio. FVC may be normal or slightly reduced, although this finding alone may be indicative of mucous impaction [2, 24]. Airway hyperresponsiveness has also been demonstrated. In 40% of patients, an FEV1 reversibility of .15% following administration of b-agonist can be demonstrated [29]. In addition, 30–69% of patients who do not exhibit a reduced FEV1 at baseline, show a 20% decrease in FEV1 following histamine or methacholine challenge [30, 31], indicating clinically significant hyperresponsiveness. FEV1 has the strongest correlation with severity of structural abnormality on HRCT [32, 33]; however, it correlates poorly with clinical fluctuations in disease course. Full pulmonary function testing, including lung volumes and gas transfer coefficient, should be carried out at the outset in adult presentations in order to give a picture of the overall functional status of the lungs and also to assist in the diagnosis of underlying conditions [2]. Reduced lung volumes and transfer factor should prompt consideration of underlying interstitial lung disease. Elevated lung volumes can be secondary to air-trapping or indicate mucous impaction of smallcalibre airways. Exercise testing, such as the incremental shuttle test and 6-minute walking test, are widely used tools for the assessment of functional capacity in chronic pulmonary disease patients and can be applied to bronchiectatic patients [34]. However, such tests have no value in diagnosis and there are no data to support their use outside of clinical studies [35]. Limitation of exercise capacity has not been shown to correlate with severity of airway damage on HRCT [36]. Studies in post-resection patients have shown that exercise testing is more informative as an ongoing assessment of lung function than static spirometry, particularly in patients whose performance or symptoms do not correlate with spirometric results [37].

Specific investigations Cystic fibrosis

38

CF is the single most common cause of structural bronchiectasis in children and a reasonably common diagnosis in adults [2, 7, 24]. Increasingly, CF is diagnosed later in life, with many patients now being diagnosed in their third and fourth decades of life, and some even later [38–43]. Given this, all adults presenting with bronchiectasis and other features of CF should undergo comprehensive investigation in order to rule out CF. Pilocarpine iontophoresis for sweat chloride ion concentration ([Cl-]) measurement, should be carried out in all patients with bronchiectasis and a clinical suspicion of CF [2, 44]. The results should be interpreted as detailed in table 5. A sweat [Cl-]

of ,30 mM effectively excludes CF as a diagnosis, although one CF-disease-causing mutation has been described with normal sweat [Cl-] [44]. If the sweat [Cl-] is .60 mM, a diagnosis of CF is confirmed. If the sweat test result is 30–60 mM, the identification of one or more disease-causing mutations determines which diagnostic category the patient falls into, CF or CF transmembrane conductance regulator (CFTR)-related disorder (table 5) [44]. The diagnostic category of CFTR-related disorder has recently emerged and describes single-organ disease, most frequently bronchiectasis, with an associated sweat [Cl-] of 30–60 mM or one or two disease-causing mutations of the CFTR. In some cases of diagnostic uncertainty, measurement of nasal potential difference may help to determine CFTR dysfunction. This may help to distinguish CF from a CFTR-related disorder [44].

Immunological investigations

Specific IgG subclass deficiency can be detected in serum or by checking the antibody response to vaccination with either pneumococcal or Haemophilus influenzae and tetanus toxoid vaccines. This is performed by measuring antibody levels prior to administration of a dose and again 4 weeks later in order to investigate whether or not the individual has mounted an appropriate response [45]. Specific antibody response studies should be undertaken in consultation with an immunologist as interpretation of responses is complex, and a decision to treat patients with specific deficiencies with Ig replacement requires a range of considerations and should undertaken by an immunologist with expertise in this area [2]. Replacing deficient IgG is usually effective in reducing the frequency of infection and preventing further lung damage [45–47]. Neutrophil, Tcell, B-cell and complement disorders are a rare cause of bronchiectasis, and functional studies should be discussed with a specialist immunologist. All patients with an identified immunodeficiency should be managed with a specialist immunologist [2].

Primary ciliary dyskinesia PCD is an autosomal recessive disorder leading to immotile cilia, and occurs in 1 in 15,000 to 1 in 40,000 of the population. It results in bronchiectasis and sinusitis and, in around half of cases, Kartagener’s syndrome (bronchiectasis, sinusitis and situs inversus) [10]. Diagnosis is based on exhaled nasal nitric oxide levels and electron microscopy of nasal biopsy specimens [48]. Reduced nitric oxide level has a specificity of 98% and a positive predictive value of 92% for PCD [48], and may be used as a screening tool to select those in whom nasal mucosal biopsy for electron microscopy is required. The diagnostic gold standard is transmission electron microscopy of nasal biopsy specimens to view the ultrastructural defects in the dynein arms within individual cilia [10]. Recent studies suggest that 15% of patients with functional PCD show no ultrastructural defects and so there is a high falsenegative diagnostic rate [10]. Genetic testing is now becoming more readily available and may go some way towards overcoming limitations to ultrastructure as a diagnostic method [10].

M. DRAIN AND J.S. ELBORN

A range of immunological abnormalities are associated with non-CF bronchiectasis [24]. The prevalence of each in bronchiectasis varies from study to study. Humoral immunity can be affected by low levels of any of the major immunoglobulin (Ig) classes, IgM, IgG and IgA [1, 24, 45, 46]), and, in some cases, IgG subclasses, IgG1, IgG2, IgG3 and IgG4. The specific antibody response to polysaccharide and peptide vaccines provides additional information about the innate immune response to antigenic stimulus [11].

Table 5. Sweat test diagnostic criteria for cystic fibrosis (CF) Sweat [Cl-] mM

Diagnostic conclusion

o60 30–60 f30

CF confirmed Equivocal: further investigation required: CFTR DNA test Not CF

39

[Cl-]: chloride ion concentration; CFTR: CF transmembrane conductance regulator.

Allergic bronchopulmonary aspergillosis IgE is a sensitive marker for ABPA if levels are .1,000 IU?L-1. Aspergillus precipitins or specific IgG directed against Aspergillus confirm the diagnosis. This condition responds well to a combination of high-dose oral corticosteroid and oral antifungal therapy [2, 49–51].

a1-Antitrypsin deficiency In order to diagnose a1-AT deficiency, serum levels of a1-AT should be checked with the biochemical phenotype requested in those patients with low levels, particularly if there is a family history of respiratory disease of young onset, or in family members who have never smoked or show evidence of bullous disease on HRCT [9]. Genetic tests for the different genotypes (M, Z and S) are also now available.

Connective tissue disorders

ASSESSMENT AND INVESTIGATION

Autoimmune disease covers a spectrum of conditions, which, although rare individually, can cause bronchiectasis and, depending on the condition, may respond to directed treatment. These conditions can be screened for by thorough history-taking and measurement of rheumatoid factor and other specific autoantibodies, such as antineutrophilic cytoplasmic antibody and cryoglobulin [2]. More common autoimmune conditions with a strong association with bronchiectasis are rheumatoid arthritis and ulcerative colitis. It is recommended that patients attending specialist rheumatology or gastroenterology clinics for monitoring of these conditions who develop chronic cough or respiratory symptoms should undergo lung function testing and HRCT in order to rule out bronchiectasis.

Gastro-oesophageal reflux Gastro-oesophageal reflux disease has been associated with bronchiectasis, although it is unclear whether or not there is a direct causal relationship. If suspected, barium studies and fluoroscopy are indicated [2, 24].

Infection and sputum microbiology Sputum microbiology is a key investigation in the diagnosis of patients with bronchiectasis [52]. H. influenzae is the most-frequently isolated pathogen, being found in up to 35% of patients. Staphylococcus aureus, Streptococcus pneumoniae, Moraxella catarrhalis and Pseudomonas aeruginosa are also commonly identified organisms [53]. Aspergillus sp. may also be found, and may be related to a diagnosis of ABPA. The presence of P. aeruginosa in sputum from people with bronchiectasis is associated with more-severe lung disease and may also have a negative impact upon prognosis [54, 55].

Monitoring disease activity Monitoring disease activity in bronchiectasis can be difficult as there is little fluctuation in lung function as measured by spirometry [2]. The inflammatory response to infection in bronchiectasis has been shown to be compartmentalised, with higher concentrations of inflammatory mediators being found in the airways than in the systemic circulation [3, 56]. Patients’ symptoms are a very important guide to pulmonary exacerbations, with increased cough, sputum volume and purulence, and haemoptysis and reduced energy all being common symptoms.

40

Sputum analysis plays a pivotal role in the assessment of bronchiectasis, with antibiotic therapy being directed by the results of sputum culture and antibiotic sensitivity testing. Sputum culture should be performed at all outpatient reviews and when symptoms deteriorate.

Although exacerbation rate does not clearly correlate with particular organisms, it has been shown to increase with increasing resistance of organisms to antibiotics [54]. Longitudinal studies demonstrate that subjects who carry the same organism after a 5-year period tend to carry increasingly resistant organisms, making exacerbations more difficult to treat successfully [54]. Recent studies using molecular identification techniques in the sputum of CF patients have revealed a wider spectrum of organisms in significant quantities than culture alone [57]. This has led to the discovery that the CF microbiome is much more extensive and diverse than was previously suspected. This is also likely to be the case in non-CF bronchiectasis. Molecular diagnostic methods are considerably more expensive than culture-based methods and not freely available in most clinical microbiological laboratory settings. Exacerbations are often associated with new isolates of bacteria and respond to antibiotic therapies. However, in many such episodes, no clinically significant organism can be identified as the precipitating factor. Although it may be some time before molecular diagnostics enter clinical practice, it is worth bearing in mind, in the case of an infection not responding to standard antibiotic therapy, that there are other potentially pathogenic organisms present that may require alternative treatment. As a rule of thumb, sputum culture is more likely to underestimate the prevalence of bacterial infection, and each positive culture should be treated with appropriate antibiotics. A thorough structured approach to the investigation of patients with suspected bronchiectasis will enable further learning about the natural history of the condition and improve patient outcomes by appropriate direction of treatment.

References 1. Dagli E. Non cystic fibrosis bronchiectasis. Paediatr Respir Rev 2000; 1: 64–70. 2. Pasteur MC, Bilton D, Hill AT. British Thoracic Society guideline for non-CF bronchiectasis. Thorax 2010; 65: Suppl. 1, i1–i58. 3. Li AM, Sonnappa S, Lex C, et al. Non-CF bronchiectasis: does knowing the aetiology lead to changes in management? Eur Respir J 2005; 26: 8–14. 4. Shoemark A, Ozerovitch L, Wilson R. Aetiology in adult patients with bronchiectasis. Respir Med 2007; 101: 1163–1170. 5. Scala R, Aranne P, Palumbo V, et al. Prevalence, age distribution and aetiology of bronchiectasis; a retrospective study on 144 symptomatic cases. Monaldi Arch Chest Dis 2000; 55: 101–105. 6. Valery PC, Torzillo PJ, Mulholland K, et al. Hospital-based case–control study of bronchiectasis in indigenous children in Central Australia. Pediatr Infect Dis J 2004; 23: 902–908. 7. Pasteur MC, Helliwell SM, Houghton SJ, et al. An investigation into causative factors in patients with bronchiectasis. Am J Respir Crit Care Med 2000; 162: 1277–1284. 8. DeGracia J, Rodrigo MJ, Morell F, et al. IgG subclass deficiencies associated with bronchiectasis. Am J Respir Crit Care Med 1996; 153: 650–655. 9. Parr DG, Guest PG, Reynolds JH, et al. Prevalence and impact of bronchiectasis in a1-antitrypsin deficiency. Am J Respir Crit Care Med 2007; 176: 1215–1221. 10. Noone PG, Leigh MW, Sannuti A, et al. Primary ciliary dyskinesia: diagnostic and phenotypic features. Am J Respir Crit Care Med 2004; 169: 459–467. 11. Liote H. Etiological work-up for bronchectasis in adults. Rev Pneumol Clin 2004; 60: 255–264. 12. Weycker D, Edelsberg J, Oster G. Prevalence and economic burden of bronchiectasis. Clin Pulm Med 2005; 12: 205–209. 13. Department of Health, Social Services and Public Safety. A Healthier Future. A Strategic Framework for Respiratory Conditions. Belfast, Department of Health, Social Services and Public Safety, 2006. 14. Sa¨yna¨jakangas O, Keistinen T, Tuuponen T, et al. Evaluation of the incidence and age distribution of bronchiectasis from the Finnish hospital discharge register. Cent Eur J Public Health 1998; 6: 235–237. 15. Twiss J, Metcalfe R, Edwards E, et al. New Zealand national incidence of bronchiectasis is too high for a developed country. Arch Dis Child 2005; 90: 737–740.

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None declared.

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Statement of interest

ASSESSMENT AND INVESTIGATION

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16. Chang AB, Grimwood K, Maguire G, et al. Management of bronchiectasis and chronic suppurative lung disease in indigenous children and adults from rural and remote Australian communities. Med J Aust 2008; 189: 386–393. 17. Singleton R, Morris A, Redding G, et al. Bronchiectasis in Alaska native children: causes and clinical courses. Pediatr Pulmonol 2000; 29: 182–187. 18. Nicotra MB, Rivera M, Dale AM, et al. Clinical, pathophysiologic, and microbiologic characterization of bronchiectasis in an aging cohort. Chest 1995; 108: 955–961. 19. Shields MD, Bush A, Everard ML, et al. BTS guidelines. Recommendations for the assessment and management of cough in children. Thorax 2008; 63: Suppl. 3, iii1–iii15. 20. Patel IS, Viahos I, Wilkinson TM, et al. Bronchiectasis, exacerbation indices and inflammation in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2004; 70: 400–407. 21. Ellis DA, Thornley PE, Wightman AJ, et al. Present outlook in bronchiectasis: clinical and social study and review of factors influencing prognosis. Thorax 1981; 36: 659–664. 22. Watt AP, Brown V, Courtney J, et al. Neutrophil apoptosis, proinflammatory mediators and cell counts in bronchiectasis. Thorax 2004; 59: 231–236. 23. Naidich DP, McCauley DI, Khouri NF, et al. Computed tomography of bronchiectasis. J Comput Assist Tomogr 1982; 6: 437–444. 24. O’Donnell AE. Bronchiectasis. Chest 2008; 134: 815–823. 25. Hansell DM. Bronchiectasis. Radiol Clin North Am 1998; 36: 107–128. 26. Gudbjerg CE. Roentgenologic diagnosis of bronchiectasis: an analysis of 112 cases. Acta Radiol 1955; 43: 209–225. 27. Remy Jardin M, Amara A, Campistron P, et al. Diagnosis of bronchiectasis with multislice spiral CT: accuracy of 3-mm-thick structured sections. Eur Radiol 2003; 13: 1165–1171. 28. Reiff DB, Wells AU, Carr DH, et al. CT findings in bronchiectasis: limited value in distinguishing between idiopathic and specific types. AJR Am J Roentgenol 1995; 165: 261–267. 29. Murphy MB, Reen DJ, Fitzgerald MX. Atopy, immunological changes, and respiratory function in bronchiectasis. Thorax 1984; 39: 179–184. 30. Swaminathan S, Kuppurao KV, Somu N, et al. Reduced exercise capacity in non-cystic fibrosis bronchiectasis. Indian J Pediatr 2003; 70: 553–556. 31. Pang J, Chan HS, Sung JY. Prevalence of asthma, atopy, and bronchial hyperreactivity in bronchiectasis: a controlled study. Thorax 1989; 44: 948–951. 32. Sheehan RE, Wells AU, Copley SJ. A comparison of serial computed tomography and functional change in bronchiectasis. Eur Respir J 2002; 20: 581–587. 33. Roberts HR, Wells AU, Milne DG. Airflow obstruction in bronchiectasis: correlation between computed tomography features and pulmonary function tests. Thorax 2000; 55: 198–204. 34. Lee AL, Button BM, Ellis S, et al. Clinical determinants of the 6-minute walk test in bronchiectasis. Respir Med 2009; 103: 780–785. 35. Newall C, Stockley RA, Hill SL. Exercise training and inspiratory muscle training in patients with bronchiectasis. Thorax 2005; 60: 943–948. 36. Edwards EA, Narang I, Li A, et al. HRCT lung abnormalities are not a surrogate for exercise limitation in bronchiectasis. Eur Respir J 2004; 24: 538–544. 37. Tsubota N, Yanagawa M, Yoshimura M, et al. The superiority of exercise testing over spirometry in the evaluation of postoperative lung function for patients with pulmonary disease. Surg Today 1994; 24: 103–105. 38. McCloskey M, Redmond AOB, Hill B, et al. Clinical features associated with a delayed diagnosis in CF. Ir J Med Sci 2000; 67: 402–407. 39. King PT, Freezer NJ, Holmes PW, et al. Role of CFTR mutations in adult bronchiectasis. Thorax 2004; 59: 357–358. 40. Hubert D, Fajac I, Bienvenu T, et al. Diagnosis of cystic fibrosis in adults with diffuse bronchiectasis. J Cyst Fibros 2004; 3: 15–22. 41. Gilljam M, Ellis L, Corey M, et al. Clinical manifestations of cystic fibrosis among patients with diagnosis in adulthood. Chest 2004; 126: 1215–1224. 42. Paranjape SM, Zeitlin PL. Atypical cystic fibrosis and CFTR-related disease. Clin Rev Allergy Immunol 2008; 35: 116–123. 43. Knowles MR, Durac PR. What is cystic fibrosis. N Engl J Med 2002; 347: 439–442. 44. DeBoeck K, Wilschanski M, Castellani C, et al. Cystic fibrosis: terminology and diagnostic algorithms. Thorax 2006; 61: 627–635. 45. Stead A, Douglas JG, Broadfoot CJ, et al. Humoral immunity and bronchiectasis. Clin Exp Immunol 2002; 130: 325–330. 46. Bernatowska E, Madalin´ski K, Janowicz W, et al. Results of a prospective controlled two-dose crossover study with intravenous immunoglobulin and comparison (retrospective) with plasma treatment. Clin Immunol Immunopathol 1987; 43: 153–162. 47. Eijkhout HW, van Der Meer JW, Kallenberg CG, et al. The effect of two different dosages of intravenous immunoglobulin on the incidence of recurrent infections in patients with primary hypogammaglobulinemia: a randomized, double-blind, multicenter crossover trial. Ann Intern Med 2001; 135: 165–174.

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48. Horvath I, Loukides S, Wodehouse T, et al. Comparison of exhaled and nasal nitric oxide and exhaled carbon monoxide levels in bronchiectatic patients with and without primary ciliary dyskinesia. Thorax 2003; 58: 68–72. 49. Greenberger PA, Miller TP, Roberts M, et al. Allergic bronchopulmonary aspergillosis in patients with and without evidence of bronchiectasis. Ann Allergy 1993; 70: 333–338. 50. Bahous J, Malo JL, Paquin R, et al. Allergic bronchopulmonary aspergillosis and sensitization to Aspergillus fumigatus in chronic bronchiectasis in adults. Clin Allergy 1985; 15: 571–579. 51. Wang JL, Patterson R, Rosenberg M, et al. Serum IgE and IgG antibody activity against Aspergillus fumigatus as a diagnostic aid in allergic bronchopulmonary aspergillosis. Am Rev Respir Dis 1978; 177: 917–927. 52. Angrill J, Agusti C, de Celis R, et al. Bacterial colonisation in patients with bronchiectasis: microbiological pattern and risk factors. Thorax 2002; 57: 15–19. 53. Kelly MG, Murphy S, Elborn JS. Bronchiectasis in secondary care: a comprehensive profile of a neglected disease. Eur J Intern Med 2003; 14: 488–492. 54. Wilson CB, Jones PW, O’Leary CJ, et al. Effect of sputum bacteriology on the quality of life of patients with bronchiectasis. Eur Respir J 1997; 10: 1754–1760. 55. King PT, Holdsworth SR, Freezer NJ, et al. Microbiologic follow-up study in adult bronchiectasis. Respir Med 2007; 101: 1633–1638. 56. Hill SL, Morrison HM, Burnett D, et al. Short term response of patients with bronchiectasis to treatment with amoxycillin given in standard or high doses orally or by inhalation. Thorax 1986; 41: 559–565. 57. Tunney MM, Field TR, Moriarty TF, et al. Detection of anaerobic bacteria in high numbers in sputum from patients with cystic fibrosis. Am J Respir Crit Care Med 2008; 177: 995–1001.

Chapter 5

Radiological features of bronchiectasis P.L. Perera* and N.J. Screaton#

RADIOLOGICAL FEATURES

Summary Imaging plays a crucial role in the diagnosis and monitoring of bronchiectasis and the management of complications. Chest radiography is useful as an initial screening tool and during acute exacerbations, but has limited sensitivity and specificity. High-resolution computed tomography (HRCT) is the reference standard for diagnosis and quantification of bronchiectasis, providing detailed morphological information. Computed tomography (CT) is also valuable in diagnosing and managing complications. Routine surveillance using HRCT has been mooted, particularly in cystic fibrosis (CF), where advances in treatment have increased life expectancy considerably, but cumulative radiation dose remains a concern. Pulmonary magnetic resonance imaging is an evolving technique that provides both structural and functional information. Its advantage is the lack of ionising radiation. Limitations include cost, availability and its inferior spatial resolution compared to CT. The technique requires further evaluation, but has potential benefits where serial follow-up imaging is being considered, such as in CF. Evaluation of mucociliary clearance using radionuclide scintigraphy may be of value, particularly in drug development. Keywords: Bronchiectasis, cystic fibrosis, diagnostic imaging, magnetic resonance imaging, mucociliary clearance, spiral computed tomography

B

*Dept of Radiology, Norfolk and Norwich University Hospital, Norwich, and # Diagnostic Imaging Dept, Papworth Hospital, Papworth Everard, UK. Correspondence: N.J. Screaton, Diagnostic Imaging Dept, Papworth Hospital, Papworth Everard, CB23 3RE, UK, Email [email protected]

Eur Respir Mon 2011. 52, 44–67. Printed in UK – all rights reserved. Copyright ERS 2011. European Respiratory Monograph; ISSN: 1025-448x. DOI: 10.1183/1025448x.10003510

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ronchiectasis is characterised by irreversible dilation of bronchi, which may be focal or diffuse, and usually occurs with associated inflammation. Its pathogenesis is complex and often multifactorial, with bronchial wall inflammation, bronchial wall weakness and infection often occurring in parallel and with numerous aetiological factors. Since it was first described by LAENNEC [1] in 1819, there have been considerable advances in the understanding, diagnosis and treatment of bronchiectasis. Imaging now forms the cornerstone of diagnosis of bronchiectasis and its complications and plays an increasing role in disease monitoring and therapeutic planning. The present review focuses on imaging features in bronchiectasis and their role in diagnosis and monitoring of disease. Several imaging modalities are available, with varying strengths and

limitations, which are outlined below. The choice of diagnostic/monitoring strategy shows some variation depending on access to each modality and interpretive expertise. Image-guided intervention, such as percutaneous pleural aspiration/drainage and angiography with embolisation play important roles in treating complications, but are beyond the scope of the present review.

Chest radiography Chest radiography (CXR) is usually the initial study performed in both suspected bronchiectasis and the evaluation of nonspecific respiratory symptoms, such as dyspnoea and haemoptysis, when bronchiectasis may be identified incidentally. Signs on CXR include the identification of parallel linear densities, tram-track opacities, or ring shadows reflecting thickened and abnormally dilated bronchial walls. These bronchial abnormalities form a spectrum from subtle or barely perceptible 5-mm ring shadows to obvious cysts. Tubular branching opacities conforming to the expected bronchial branching pattern may result from fluid or mucous filling of bronchi. Peribronchial fibrosis results in a loss of definition of vessel walls (fig. 1) [2–5].

The radiograph may raise the initial suspicion of bronchiectasis, triggering more definitive imaging. However, its projectional nature and limited contrast resolution lead to limited sensitivity and specificity, particularly in mild disease. CXR also plays a role in the follow-up of bronchiectasis and management of exacerbations, although, again, the relative insensitivity to change is highlighted by proponents of computed tomography (CT) and magnetic resonance (MR) imaging (MRI) [2–5]. The reported accuracy of CXR has changed over the years as the management emphasis has shifted from being reactive to complications to one of early detection and proactive management, and as the diagnostic reference standard has shifted from bronchography to CT. In 1955, GUDJBERG [6] reported only 7.1% of 114 bronchiectatic patients having a normal CXR, perhaps reflecting the more florid nature of the condition during this period. In 1987, CURRIE et al. [7] reported an overall sensitivity of 47%, and only 13% on a lobar basis, in 19 patients with bronchographically proven bronchiectasis. The same study confirmed significant interobserver variation in CXR interpretation, with two experienced readers disagreeing on the diagnosis of bronchiectasis in 22% of cases [7]. In comparison to CXR, CT is both more sensitive and provides more specific information. BHALLA et al. [8] showed that, out of a total of 162 bronchopulmonary segments reviewed, bronchiectasis was detected in 124 on high-resolution CT (HRCT) and only 71 on CXR.

P.L. PERERA AND N.J. SCREATON

Signs of complications/exacerbations, such as patchy densities due to mucoid impaction and consolidation, volume loss secondary to bronchial mucoid obstruction or chronic cicatrisation, are also seen. In the more diffuse forms of bronchiectasis, such as cystic fibrosis (CF), generalised hyperinflation and oligaemia are often present, consistent with severe small airways obstruction.

Radiographic scoring systems, such as those of CHRISPIN and NORMAN [9] and BRASFIELD et al. [10], have been developed and subsequently modified for patients with CF. These can be useful clinically, but are more commonly used for comparison in research. CLEVELAND et al. [11] showed that a score based on the scoring system of BRASFIELD et al. [10] could be used to assess the longitudinal progression of lung disease in CF, and was at least as effective as spirometry in this regard. Although CXR has limitations in specificity in diagnosing bronchiectasis and in detecting early or subtle changes, it is useful for assessing more florid cases of bronchiectasis, in CF and in follow-up of bronchiectatic patients.

Computed tomography

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The CT signs of bronchiectasis were first described by NAIDICH et al. [12] in 1982. Although initial studies using 8–10-mm slice thickness showed low sensitivity [13–15], the advent of HRCT led to markedly improved sensitivity, resulting in HRCT replacing bronchography as the diagnostic reference standard. GRENIER et al. [16] showed that HRCT with 1.5-mm collimation at 10-mm

a)

RADIOLOGICAL FEATURES

b)

intervals was accurate in the recognition of bronchiectasis using bronchography as the gold standard in 36 patients. Multidetector CT (MDCT) with volumetric acquisition further increases the sensitivity in detection of subtle nontapering airways. DODD et al. [17] compared contiguous 1-mm MDCT with 1-mm incremental HRCT with 10-mm interspaces in 61 bronchiectatic patients and 19 normal controls. Using MDCT as the gold standard, the sensitivity, specificity and positive and negative predictive values of incremental HRCT in detecting bronchiectasis were 71, 93, 88 and 81%, respectively. Interobserver agreement for the presence, extent and severity of bronchiectasis was also better for MDCT (kappa 0.64, 0.5 and 0.48, respectively) than for incremental HRCT (kappa 0.65, 046 and 0.25, respectively).

Optimal HRCT technique is important for maximising diagnostic accuracy. Importantly, thin slices of 1–2 mm and a high-resolution lung reconstruction algorithm are used to optimise spatial resolution. Incremental imaging with 10-mm slice interspace reduces radiation dose, but helical MDCT permits volumetric acquisition in a single breathhold, which is often the preferred technique. Electronic images should be viewed in stack/cine mode using the appropriate window settings (centred -400– -950 HU; width 1,000–1,600 HU). Difficulties in diagnosing bronchiectasis can arise from cardiorespiratory motion artefact, use Figure 1. Chest radiography showing a) cystic bronchiectasis with multiple cystic airspaces and b) cylindrical bronchiectasis and tramof inappropriate window widths and track opacities in a cystic fibrosis patient. levels, and the relatively thick-walled appearance of bronchial walls on expiratory scans. Tractional airway dilation associated with pulmonary fibrosis has a characteristic corkscrew appearance and should be differentiated from pathological bronchiectasis.

CT signs of bronchiectasis

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Bronchial dilation, the cardinal sign of bronchiectasis, is characterised on HRCT by a bronchoarterial ratio (BAR) of .1, lack of bronchial tapering, and visibility of airways within 1 cm of the pleural surface or abutting the mediastinal pleural surface [2, 12, 16, 18, 19].

The different morphological types of bronchiectasis corresponding to the bronchographic classification of REID [20] show differing radiological features [16, 21], but this is of little relevance in terms of aetiology and rather reflects varying severity of the disease. Although, in cylindrical bronchiectasis, there is uniform dilation of airways with nontapering walls, in varicose bronchiectasis, dilated bronchi have a beaded appearance and, in cystic bronchiectasis, grosser bronchial dilation gives the appearance of cysts (fig. 2). The BAR refers to the ratio of the internal bronchial diameter to the diameter of the accompanying pulmonary artery at an equivalent branching level. A BAR of .1 is considered abnormal [14, 15, 19] and is otherwise known as the signet-ring sign (fig. 3).

Figure 2. High-resolution computed tomography image showing cystic bronchiectasis (same patient as in fig. 1a).

Physiological influence on BAR was highlighted by LYNCH et al. [23], who showed that 59% of 27 normal volunteers living in Colorado, USA (1,600 m above sea level) had at least one bronchus that had an internal diameter larger than its accompanying artery. KIM et al. [18] confirmed this environmental influence on BAR by demonstrating that residents living at 1,600 m exhibited significantly higher BARs than those living at sea level (0.76 and 0.62, respectively; p,0.001). Physiological variation can also occur due to regional hypoxia, and so secondary vasoconstriction causing apparent bronchial dilation must be recognised in order to prevent a spurious diagnosis of bronchiectasis. Conversely, if there is arterial dilation (e.g. due to pulmonary arterial hypertension), bronchiectasis could be missed. A practical problem in assessing BAR is the need to identify the accompanying artery, which may be difficult in the presence of other lung pathology, such as consolidation. In the setting of consolidation, the presence of bronchial dilation may be a reversible phenomenon and so caution should be observed when interpreting CT data during an acute illness. Although objective measurement may be performed, visual inspection is the usual method of assessing bronchial dilation. DIEDERICH et al. [24] showed this to have good interobserver agreement for detection (kappa 0.78) and severity assessment (kappa 0.68).

Figure 3. High-resolution computed tomography image demonstrating signs of bronchiectasis, the signet-ring sign (short arrow) and peripheral airway visible within 1 cm of the pleural surface (long arrow).

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Lack of bronchial tapering or tram-track appearance of the parallel bronchial walls is a sensitive feature of bronchiectasis often identified in more horizontally orientated airways in the mid-zones. KANG et al. [25] showed that lack of tapering on HRCT was more sensitive than bronchial dilation in bronchiectasis (79 and 60%, respectively) using pathology as the reference standard. However, the sign can be difficult to interpret in nonvolumetric CT studies. KIM et al. [18] demonstrated lack of tapering on HRCT in 95% of patients with bronchiectasis, but also in 10% of normal subjects. It has been suggested that bronchial diameter should remain unchanged for at least 2 cm distal to a branching point for this sign to be robust (fig. 4) [26].

P.L. PERERA AND N.J. SCREATON

The accuracy of the BAR can be limited by a number of factors, including physiological variation and orientation of the bronchovascular bundle with respect to the imaging plane [16, 22, 23]. Comparison is best performed on perpendicularly orientated airways. When oblique to the acquisition plane, airways and vessels appear ovoid and their short axis should be compared.

Visibility of peripheral airways is another important direct sign of bronchiectasis [17]. Current HRCT techniques permit visualisation of airways of up to 2 mm in diameter and walls of around 0.2 mm in thickness. KIM et al. [18] showed that normal bronchi are not visualised within 1 cm of the costal pleura, but may be seen within 1 cm of the mediastinal pleura. Visible bronchi within 1 cm of the costal pleura or abutting the mediastinal surface were seen in 81 and 53% of HRCT images of known bronchiectatic patients (fig. 4). Ancillary signs commonly identified in bronchiectatic patients include bronchial wall thickening, mucoid tomography image showing nontapering impaction and air-trapping. Minor volume loss can be bronchi, in keeping with bronchiectasis. seen in the early stages of bronchiectasis. Larger areas of collapse secondary to mucous plugging may be seen in more advanced disease. Patchy consolidation is sometimes seen reflecting superimposed infection. Figure 4. High-resolution computed

RADIOLOGICAL FEATURES

Bronchial wall thickening is often seen in the presence of bronchiectasis [25], but is a variable nondiagnostic feature. It may result from reversible airway wall inflammation [27] or smooth muscle hypertrophy and fibroblastic proliferation. Minor bronchial wall thickening has, however, also been described in normal individuals, asthmatics, asymptomatic smokers and during lower respiratory tract infections [23, 28]. Identification of bronchial wall thickening on HRCT is often made subjectively and is associated with significant interobserver variation, with no universally agreed definition. REMY-JARDIN and REMY [28] defined a thickened bronchus as being twice as thick as a normal bronchus; however, this definition is difficult in diffuse disease DIEDERICH et al. [24] defined a thick-walled bronchus by an internal diameter of ,80% of its external diameter and showed good interobserver agreement (kappa 0.66). However, this can lead to overdiagnosis of thickening in the presence of bronchoconstriction and underestimation with marked bronchodilation. An alternative approach, used by BHALLA et al. [8] in CF and subsequently modified by REIFF et al. [29], is to compare bronchial wall thickness to the diameter of the adjacent artery. As with assessment of BAR, peribronchial fibrosis and consolidation pose practical difficulties in identifying accompanying vessels (fig. 5). Mucous plugging of dilated bronchi is readily identified, causing either complete or partial luminal filling (fig. 5). Plugging of the smaller peripheral airways and peribronchiolar inflammation are characterised by a tree-in-bud appearance, with V- and Y-shaped branching nodular opacities [30]. Mucous plugging was scored in terms of number and generation of involved bronchopulonary segments using the scoring system of BHALLA et al. [8], and may be reversible.

*

Figure 5. High-resolution computed tomo-

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graphy image demonstrating bronchiectasis with bronchial wall thickening (asterisk) and mucous plugging (arrow) in the right lower lobe.

Air-trapping results either from mucous plugging and abnormal bronchial compliance [31] or inflammation/ fibrosis of the small airways [2]. On HRCT, airtrapping is characterised by patchy lobular areas of low attenuation with regional vasoconstriction, which causes a mosaic attenuation pattern, accentuated on expiratory images, although inspiratory images are usually characteristic (fig. 6). Air-trapping and bronchiectasis coexist in the same lobe in approximately half of cases [25]. Whether it is the bronchiectasis and recurrent infections driving obliterative bronchiolitis (OB) or primary small airways disease that precedes the

onset of bronchiectasis is debated, and may vary [31]. The latter is supported both by the observation that, in patients with CT-proven bronchiectasis, expiratory HRCT identifies air-trapping in 17% of lobes with no bronchiectatic features and that, in patients with rheumatoid arthritis (RA)-associated OB, the onset of symptoms and obstructive function may predate the onset of bronchiectasis by several years [32].

Imaging and aetiology of bronchiectasis

However, in a large study comparing HRCT features in bronchiectasis of defined aetiology with idiopathic bronchiectasis, REIFF et al. [29] concluded that, although some HRCT features were more common in some aetiological groups, the differences were not sufficient to be diagnostic. LEE et al. [40] reinforced this observation in a study of 108 bronchiectatic patients in whom the correct cause was identified on CT in only 45% of cases. A confident diagnosis was asserted in a minority (9%) and was correct in only 35%. Interobserver agreement in likely aetiology was also poor (kappa 0.2) [40]. However, more recently, CARTIER et al. [41] obtained accurate diagnoses on the basis of HRCT in 61% of 82 patients with bronchiectasis of known cause, with moderately good agreement (kappa 0.53). Confident and accurate diagnosis was made in 44% of patients (kappa 0.53). Accuracy was highest in CF (68%), previous tuberculosis (67%) and ABPA (56%). Part of the reason for the higher number of accurate diagnoses was attributed to the exclusion of patients in whom the aetiology of bronchiectasis was not known. They concluded that the combination of radiological pattern and clinical scenario would have improved the accuracy of the evaluation. HRCT is important in the assessment of mycobacterial infection. This is particularly true of nontuberculous mycobacteria (NTM), where the diagnosis is often first suggested on HRCT. CT signs of NTM include bronchiectasis, nodules, tree-in-bud opacity, patchy consolidation and cavities, often affecting the upper lobes and superior segments of lower lobes in the classic subtype and middle lobe/lingula in the nonclassic subtype [42]. The presence of this combination of features with a middle lobe and lingual predominance, especially in the setting of elderly females with no underlying malignancy or immunocompromise, is particularly suggestive of nonclassic NTM [43, 44]. Bronchiectasis is more common in NTM infection, being seen in up to 94% of patients with Mycobacterium avium complex and 27% of patients with M. tuberculosis [45].

P.L. PERERA AND N.J. SCREATON

There are many aetiologies associated with bronchFigure 6. Inspiratory high-resolution computed tomography image showing bronchiectasis, but a specific underlying cause is found in iectasis and widespread areas of low ,40% of patients [33]. In some cases, the distribution attenuation, representing air-trapping. and pattern of bronchiectasis on HRCT may suggest the aetiology. Allergic bronchopulmonary aspergillosis (ABPA) typically demonstrates an upper zone and central predominance [34–37], hypogammaglobulinaemia may be associated with bronchiectasis with disproportionate bronchial wall thickening, middle lobe predominance is common in immotile cilia syndrome [38] and idiopathic bronchiectasis often has a lower lobe distribution [39].

Bronchiectasis in CF usually has a bilateral, proximal, parahilar and upper lobe predominance. Other findings include bronchial wall thickening, peribronchial interstitial thickening, mucous plugging, tree-in-bud opacification, superadded consolidation and mosaic attenuation. SHAH et al. [27] assessed the CT changes in 19 symptomatic adult CF patients before and after 2 weeks of therapy and identified air–fluid levels in bronchiectatic airways, mucous plugging, centrilobular nodules and peribronchial thickening as potentially reversible signs.

49

Bronchiectasis with a central or proximal predominance is the characteristic finding in ABPA (fig. 7a). REIFF et al. [29] showed that the prevalence of central bronchiectasis was higher in ABPA (11 out of 30) than in idiopathic bronchiectasis (26 out of 179) (p,0.005). However, the sensitivity of the observation of central bronchiectasis in diagnosing ABPA was only 37%. PANCHAL et al. [46]

a)

b)

RADIOLOGICAL FEATURES

Figure 7. High-resolution computed tomo-

demonstrated central bronchiectasis in 85% of lobes and 52% of lung segments in a series of 23 patients with ABPA. Other common findings in ABPA include mucous plugging, high-attenuation mucus, tree-inbud opacities, atelectasis, peripheral consolidation or ground-glass opacification, mosaic perfusion and airtrapping. The bronchiectasis is often cystic or varicose. WARD et al. [47] assessed CT images from 44 asthmatic patients with ABPA and 38 without and found much higher levels of bronchiectasis, centrilobular nodules and mucous impaction in ABPA. They concluded that randomly distributed predominantly central moderate-to-severe bronchiectasis affecting three or more lobes, bronchial wall thickening and centrilobular nodules in asthmatics is highly suggestive of ABPA (fig. 7b). High-attenuation mucous plugs are reported to occur in 18–28% of patients with ABPA, and, if observed, are thought to be characteristic [48–50]. In a study of 155 patients with ABPA, AGARWAL et al. [48] found this sign in 29 patients, and that it correlated with greater severity and greater likelihood of relapse.

graphy showing a) proximal bronchiectasis affecting segmental airways and b) highattenuation mucous plugs in patients with allergic bronchopulmonary aspergillosis. No intravenous contrast medium was used in (b).

In summary, there are some recognised clinical conditions in which assessment of bronchiectasis forms an important part of management. CT images in bronchiectatic patients should be examined for features suggesting ABPA, CF, immotile cilia, opportunist mycobacteria and tracheobronchomegaly, but these observation need to be correlated with clinical and laboratory findings.

CT scoring of bronchiectasis Although the extent, severity and distribution of bronchiectasis may be evaluated subjectively, more-robust objective scoring systems have been developed particularly for use in the research arena. With the development of novel software tools, it is now possible to objectively quantify parameters, such as airway wall area and volume, in a semi-automatic manner. Both subjective and objective quantification permit correlations between structure and function to be evaluated. CT scoring systems are based on collective scores for the extent and distribution of a range of morphological abnormalities, including bronchial dilation, bronchial thickening, abscesses, mucous plugging, emphysema, collapse and consolidation. The HRCT score of BHALLA et al. [8] was devised to evaluate the severity of CF in an objective manner. Severity of bronchial dilation and thickening were defined relative to the adjacent pulmonary artery, and other parameters were scored according to the number of bronchopulmonary segments involved, as shown in table 1.

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This scoring system has been modified many times, and has also been adapted for use in MRI assessment of CF. Modifications have included incorporation of additional findings, such as airtrapping/mosaic attenuation, ground-glass opacification, acinar nodules and septal thickening, with some scores being per segment and others based on lobar scoring. Each scoring system attempts to produce both a total score, by combining features, and specific morphological scores. These have been demonstrated to be more sensitive to disease and show better correlation with both clinical features and lung function than the CXR-based scoring systems. OIKONOMOU et al. [51]

Table 1. Summary of computed tomography scoring system Score 0

1

2

3

Severity of bronchiectasis

Absent

Moderate (luminal diameter 2–3 times that of adjacent blood vessel)

Severe (luminal diameter .3 times that of adjacent blood vessel)

Peribronchial thickening

Absent

Mild (luminal diameter slightly greater than that of adjacent blood vessel) Mild (wall thickness equal to diameter of adjacent vessel)

Severe (wall thickness .2 times the diameter of adjacent vessel)

Extent of bronchiectasis BP segments n Extent of mucous plugging BP segments n Sacculations or abscesses BP segments n Bronchial divisions involved (bronchiectasis/ plugging) generations Bullae Bullae n Emphysema BP segments n Collapse/consolidation

Absent

Present 1–5 Present 1–5 Present 1–5 Up to 4th

Moderate (wall thickness greater than and up to twice the diameter of adjacent vessel) Present 6–9 Present 6–9 Present 6–9 Up to 5th

Unilateral not .4 Present 1–5 Subsegmental

Bilateral not .4 Present .5 Segmental/lobar

Absent Absent Absent

Absent Absent Absent

Present .9 Present .9 Present .9 Up to 6th and distal

Present .4

BP: bronchopulmonary. Reproduced from [8] with permission from the publisher.

suggested a simplified scoring system evaluating severity of bronchiectasis, bronchial wall thickening and atelectasis consolidation. They found strong correlation between the simplified scores and the complete scores. SHAH et al. [27] used a modified Bhalla score in bronchiectatic patients undergoing HRCT at baseline and 2 weeks after exacerbation in order to identify reversible findings, and showed that air–fluid levels, centrilobular nodules, mucous plugging and peribronchial thickening improved following treatment in 100, 36, 33 and 11% of cases, respectively. DE JONG et al. [52] compared the original scoring system of BHALLA et al. [8] and four modified Bhalla systems [53–56]. Three observers reviewed thin-slice CT images of 25 children with CF using the various scoring systems. Interobserver variability was analysed using kappa coefficients and found to be generally good (kappa .0.76; p,0.05). However, inter- and intra-observer agreement was less for mild disease, as well as for parameters such as mosaic perfusion, acinar nodules and airspace disease. All five scoring systems correlated strongly with forced expiratory volume in 1 second (FEV1), forced vital capacity (FVC), forced expiratory flow between 25 and 75% of vital capacity (FEF25–75), FEV1/FVC ratio and each other.

P.L. PERERA AND N.J. SCREATON

Category

Quantitative computerised evaluation of the airways presents a number of challenges, including obtaining a plane perpendicular to the airway, exclusion of the adjacent artery, determining the borders of the bronchus, artefacts and partial volume averaging. Three different methods have been used to obtain airway measurements, full width at half maximum, model fitting approaches and boundary fitting approaches [19]. A detailed description of these techniques is beyond the scope of the present chapter.

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GORIS et al. [57] looked at automated evaluation of the extent of air-trapping in 25 patients with mild CF compared to 10 controls; six anatomically matched CT slices were obtained during

inspiration and expiration. Computerised lung segmentation was performed and automated software used to quantify air-trapping, using analysis of a histogram of the distribution of densities in the lung, and assessing contiguous low-attenuation voxel regions. In mild CF, air-trapping did not correlate with global pulmonary function test (PFT) results, except for the ratio of residual volume (RV) to total lung capacity (TLC); however, the size of the air-trapping defects was the best discriminator between patients and control subjects (p,0.005). KIRALY et al. [58] looked at fully automated methods of obtaining three-dimensional (3D) images and quantification of airway abnormalities. Working from thin-slice image acquisition and using computerised segmentation techniques, they obtained 3D images of the airways with colour-coded maps showing BAR, wall thickness and mucous plugging. These have, however, not been fully clinically validated. Although these objective tools are interesting, further studies are required to evaluate the various computerised imaging parameters and their relation to functional and clinical findings in bronchiectasis. A note of caution was raised by MATSUOKA et al. [59], who used semiautomatic image processing to assess the airways of 52 asymptomatic patients with no cardiopulmonary disease. They found that luminal area and wall area increased in 10 and 29% of subjects, respectively, suggesting caution in over-reliance on changes in airway calibre in disease monitoring.

RADIOLOGICAL FEATURES

Structure–function relationships Relationships between HRCT data and functional and clinical characteristics have been widely explored. WONG-YOU-CHEONG et al. [60] showed a clear negative correlation between FEV1 and extent of bronchiectasis on HRCT (p,0.002; r5 -0.43). SMITH et al. [61] showed correlation between extent of bronchiectasis on HRCT and both dyspnoea (p,0.01; r50.38) and FEV1 (p,0.01; r5 -0.43). More recently, DE JONG et al. [52] showed strong correlations between five scoring systems [8, 53–56] and FEV1 (r5 -0.69– -0.73), FEF25–75 (r5 -0.76– -0.82) and FEV1/ FVC ratio (r5-0.72– -0.78). Correlation with FVC was moderate (r5 -0.54– -0.58). Authors have investigated which morphological abnormalities are most strongly associated with a functional deficit. LYNCH et al. [62], in a study of 261 bronchiectatic patients, found significant correlation between severity of bronchiectasis, FEV1 (r5 -0.362) and FVC (r5 -0.362), and between bronchial wall thickening and FEV1 (r5 -0.367) and FVC (r5 -0.239). Cystic bronchiectasis was found to show worse PFT results than cylindrical or varicose disease. In a study of 100 patients with bronchiectasis, ROBERTS et al. [63] found good correlation between FEV1 and bronchial wall thickening (r5 -0.51; p50.00005) and extent of decreased attenuation on expiratory HRCT (r5 -0.55, p50.00005) on univariate analysis. These were the only factors that independently correlated with degree of airflow limitation on multivariate analysis. In this study, obstructive lung function was not strongly associated with severity of bronchiectasis, bronchodilation, or retained sections in bronchiectasis (r5 -0.42, -0.35 and -0.19, respectively, on univariate analysis). Bronchial dilation as an independent factor was positively associated with airflow obstruction on regression analysis (r250.42). The authors concluded that airflow limitation in bronchiectasis occurred mainly due to inflammatory or obstructive/constrictive bronchiolitis. They also suggested that areas of low attenuation attributed to emphysema in bronchiectatic patients in previous studies (e.g. [64]) should be interpreted with caution, suggesting that the emphysema demonstrated was often due to air-trapping related to intrinsic small airways disease rather than emphysema, as evidenced by preserved gas transfer in the both of these studies.

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HRCT scoring can also be correlated with clinical parameters. In a study of 61 CF children, baseline and follow-up PFT and HRCT scores were compared to the number of respiratory exacerbations over 2 years. Only the HRCT score (r50.91; p50.001) and bronchiectatic score (r50.083; p50.01) correlated significantly with exacerbation frequency. All HRCT parameters

progressed over this time period except for bronchial wall thickening and mucous plugging, suggesting that these are reversible features [65]. OOI et al. [66] studied 60 patients with stable bronchiectasis with HRCT. They showed good correlation between the extents of bronchiectasis, bronchial wall thickening and mosaic attenuation and FEV1 (r5 -0.43– -0.60), FVC (r5 -0.36– -0.46), FEF25–50 (r5 -0.38– -0.57) and FEV1/FVC (r5 -0.31– -0.49). Regression analysis showed that extent of bronchiectasis and wall thickening were the most significant determinants of airflow obstruction, correlating with all PFT parameters This study also demonstrated associations between bronchial wall thickening and clinical factors, such as exacerbation frequency and 24-hour sputum production (r5 -0.32 and -0.30). ALZEER [67] found that the HRCT score correlated well with FEV1 (r5 -0.51), as well as with systolic pulmonary artery pressure (Ppa,sys) (r50.23), in a study of 94 bronchiectatic patients.

The validity of the use of PFTs as the gold standard in evaluating HRCT has been questioned by several authors. BRODY et al. [69] and HELBICH et al. [53] have shown that early HRCT changes, including mosaic attenuation and bronchial dilation, can be seen early in disease in the presence of normal PFT results. LONG et al. [70] showed HRCT changes, including wall thickening and bronchial dilation in CF infants with a mean age of 2 years, further emphasising the sensitivity of HRCT. Regular low-dose HRCT for the surveillance of CF has been adopted by several centres since the late 1990s [71]. This was initially in the form of 1-mm slice incremental HRCT (with 10mm interspaces), but, more recently, of full-lung volumetric HRCT. CT is performed as early as an age of 2 years, when it is performed as controlled-ventilation CT (CVCT), requiring sedation or general anaesthesia. The rationale for using this imaging-intensive approach is that PFT results may lag behind structural CT changes, difficulty in reliably performing PFTs in young children and the ability of imaging to follow relevant objective structural markers, such as bronchiectasis, bronchial wall thickening, air-trapping and mucous plugging. Furthermore, in the modern era, improved therapy has slowed the annual decline in PFT results such that individual variability and changes due to other factors, such as technique, puberty and infections, have made PFTs even less reliable for disease monitoring. However, the benefit has to be viewed in the setting of radiation risk. DE JONG et al. [72] used a computational model to estimate mortality effects of regular CTs. The mean radiation dose for the published CT protocol was 1 mSv. Survival reduction associated with annual scans from the age of 2 years until death for CF patients with a median survival of 26 and 50 years, were approximately 1 month and 2 years, respectively. Cumulative cancer mortality was approximately 2 and 13% at age 40 and 65 years, respectively. Biannual CTs exhibit half the risk. This highlights the increasing reduction in survival with increasing age, an important point given the increasing life expectancy of CF patients.

P.L. PERERA AND N.J. SCREATON

Studies on the utility of HRCT in the follow-up of non-CF bronchiectatic patients have been limited. In a study of 48 patients, SHEEHAN et al. [68] compared serial CT studies with PFTs, showing correlation of changes in PFT results with air-trapping due to mucous plugging. Greater severity of mucous plugging, bronchiectasis and bronchial wall thickening were predictive of decreased FEV1. In a study evaluating morphological features in bronchiectasis at baseline and 2 weeks after exacerbation, SHAH et al. [27] showed that changes in HRCT score during exacerbation of bronchiectasis also correlate with improvement in FEV1/FVC (r50.39; p50.049). Severity of bronchiectasis was the component most strongly associated with PFT results (r50.4 for FEV1 and r50.5 for FVC), whereas tree in bud and mucous plugging were not strongly correlated.

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DE JONG et al. [73] studied 48 young patients with CF using serial low-dose HRCT and PFTs 2 years apart. In all children, there was progressive structural HRCT change with deterioration in HRCT scores by 2.2–3.5% overall, but particularly with peripheral extension of bronchiectasis and mucous plugging, despite stable (or, in some cases, improved) lung function. This may reflect poor PFT technique in young CF children, the use of predicted FEV1 (with reference to a global population) and/or the greater sensitivity of HRCT for detection of early and regional changes.

JUDGE et al. [74] assessed serial HRCT performed 18 months apart in 39 consecutive CF patients and found that the modified HRCT score declined faster (2.7% per year; p,0.001) than did FEV1 (2.3% per year). Six patients demonstrated worsening HRCT score with no change in PFT result. DE JONG et al. [75], in a study of 119 children and adults with CF, showed that PFT results, component and CT scores deteriorated over 2 years. The CT score (and its components) and PFT results showed similar rates of deterioration in adults and children (p.0.09). Peripheral bronchiectasis worsened by 1.7% per year in children (p,0.0001) and by 1.5% per year in adults (p,0.0001).

RADIOLOGICAL FEATURES

In view of the potential for discordance between morphology and function and the complementary nature of these variables, authors have attempted to create more-robust clinically useful composite scoring systems combining PFT results and HRCT findings [76]. Studies on assessment following therapy have been limited. ROBINSON et al. [76] used a composite scoring system using many of the HRCT parameters described above and combining them with PFT measures of obstructive function (FEV1 and FEF25–75). They showed the composite measurement to be more sensitive for assessing response to treatment in 25 CF children who were randomised to a treatment arm and a nebulised saline arm; FEF25–50 showed 13% improvement, global HRCT 5% and composite score 30%. Small studies have shown some HRCT features to be useful in post-treatment evaluation. NASR et al. [77] showed a significant improvement in total HRCT score following recombinant human (rh) deoxyribonuclease (DNAse) therapy compared to placebo. GORIS et al. [57] compared 25 CF patients with 10 age-matched controls using PFTs and automated quantitative assessment of lung density. No significant difference was seen in PFT results, but significant differences in air-trapping were seen, with the size of defects being the best discriminator. There was a significant decrease in mean HRCT score from 25 to 22 (p50.014), with improvement in peribronchial thickening (p50.007), mucous plugging (p50.002) and overall appearance (p50.025). There have been some differences in the degree of correlation between PFT results and HRCT findings of bronchiectasis in the various studies, which could be attributed to several causes, including the scoring system used, radiological interpretation, parameters assessed, population studied, reliability of PFTs and data analysis (multivariate versus univariate). However, the link between measures of obstructive lung function (FEV1 and FEF25–50) and bronchial wall thickness/ total HRCT score has been consistently shown.

Role of HRCT in bronchiectasis CT is invaluable in the diagnosis of bronchiectasis, but also plays an important role in the evaluation of complications and assessment and monitoring of disease severity. HRCT provides good clinical correlation, and the use of scoring systems holds promise for monitoring of bronchiectasis. The more robust CT scoring systems have been formulated in CF patients. HRCT is sometimes of value in identifying the aetiology of bronchiectasis. Advantages of HRCT over PFTs include its ability to identify focal changes as well as provide a global score, in addition to assessing multiple parameters, some of which are reversible and others irreversible (fig. 8). Current British Thoracic Society guidelines recommend HRCT at diagnosis and during exacerbations, but not for routine follow-up. An exception is in bronchiectasis secondary to humoral immunodeficiency, where follow-up HRCT may be beneficial [78].

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HRCT is now widely regarded as part of the standard clinical evaluation of patients with CF. The ability to quantify extent and severity of disease and to serve as a useful outcome marker, and the potential for assessing treatment response, make this a particularly valuable investigation. However, although HRCT provides valuable information on initial assessment, the role and periodicity of serial imaging in CF remains controversial in view of increasing life expectancy and cumulative radiation exposure. Some authors suggest that HRCT should form part of the routine monitoring of CF, but with due consideration to the excess radiation [71]. With appropriate

scanning technique, it is estimated that a chest CT every other year from birth to age 30 years would involve an effective dose of 15 mSv compared to the mean background radiation dose over this period of 90 mSv [79]. Thus HRCT forms an important part of the investigation and diagnosis of bronchiectasis and exacerbations. It is particularly useful in CF, where it has been shown to be a good marker of outcome and has demonstrated the ability to pick up subtle and early changes with greater sensitivity than PFTs.

Interest in the use of MRI for lung imaging arose in Figure 8. Coronal reconstruction from the mid-1980s. The inherent difficulties in pulmonary high-resolution computed tomography MRI are well documented. Lungs have an intrinsically showing a bronchial collateral vessel. low proton density, which results in poor signal generation. This low signal is further degraded by susceptibility artefact resulting from the innumerable air–tissue interfaces and cardiac and respiratory motion. However, the advent of parallel imaging and new rapid imaging sequences have permitted marked improvement in temporal and spatial resolution. Although spatial resolution using conventional proton MRI is less than that using CT, MRI offers significant morphological information and has advantages in enabling improved tissue characterisation and providing functional imaging, including vascular flow and respiratory mechanics. The use of novel imaging techniques, such as oxygen-enhanced MRI and hyperpolarised helium-3 MRI, potentially permits derivation of further functional parameters, including regional ventilation, regional oxygen concentration and evaluation of lung microstructure using the apparent diffusion coefficient (ADC). Although application of this technique currently lies largely in the research arena, a significant advantage of MRI is the lack of radiation, which is particularly important in patients who require recurrent imaging and the younger population group. This is pertinent when considering the potential cumulative lifetime radiation dose from annual/biannual low-dose CT examination in the CF population that are currently advocated by some groups [71], especially in view of the rapid improvement in life expectancy in this patient group, which is projected to continue. Thus it is unsurprising that much of the work on thoracic MRI imaging has been focused on patients with CF.

Conventional proton MRI features

P.L. PERERA AND N.J. SCREATON

Magnetic resonance imaging

Owing to the limited spatial resolution of MRI, assessment of bronchial wall thickness and bronchiectasis are dependent upon bronchial level, wall thickness and wall signal. Thirdgeneration bronchi and beyond are poorly visualised on MRI, except in pathological states, when the wall and luminal signal are raised due to wall thickening, inflammation and mucus [80]. Inflammation and oedema contribute to wall thickening. Gadolinium-enhanced images may be useful in demonstrating inflammatory change.

55

Mucous plugging on MRI results in homogeneous high T2-signal intensity in proximal airways or an abnormal branching grape-like pattern more peripherally, equivalent to the tree-in-bud opacities seen on HRCT. In contrast to CT, the improved tissue characterisation of MRI can also differentiate between mucus, haemorrhage and bronchial wall thickening [81]. Air–fluid levels may be identified on MRI, particularly in cystic or varicose bronchiectasis. Unlike CT, using contrast-enhanced MRI sequences, thickened airway walls can be differentiated from mucous plugging. Consolidation may also be identified as high T2-signal inflammatory fluid contrasts with the low airway signal, equivalent to the classical air bronchogram. Air-trapping and mosaic perfusion are not readily seen on conventional proton MRI in the absence of gadolinium (figs 9 and 10).

a)

b)

Figure 9. a) Transverse magnetic resonance (T2-weighted half-Fourier acquisition single-shot turbo spin-

RADIOLOGICAL FEATURES

echo (HASTE)) image and b) corresponding computed tomography image in a 14-year-old female with cystic fibrosis. In both images, bronchial wall thickening, bronchiectasis, peripheral mucous plugging and dorsal consolidations are demonstrated, as shown by the arrows. Reproduced from [81] with permission from the publisher.

An early study of 17 CF patients (aged 7–30 years) in 1995 [82] found MRI to be inferior to CT in the assessment of bronchiectasis. Correlations between CT and MRI (r for each observer) were good for bronchial dilation (r50.81 and 0.50), bronchial thickening (r50.82 and 0.60) and mucous plugging (r50.93 and 0.70). Progress in MRI technique in recent years has led to marked improvement in its accuracy. In a study of six paediatric patients with CF, HEBESTREIT et al. [83] found that CXR and MRI provided equal information, and considered MRI suitable for follow-up. In a more recent study in 2007, PUDERBACH et al. [84] evaluated 31 patients with CF using CXR, MDCT and MRI. MRI and MDCT were assessed using a modified Helbich score, whereas CXR was evaluated using a modified Chrispin–Norman score. Mosaic perfusion was excluded from the original scoring system as this cannot be quantified on MRI. They concluded that morphological a)

b)

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Figure 10. T1-weighted magnetic resonance imaging showing appearance a) before and b) after contrast medium in a 43-year-old cystic fibrosis patient. The post-contrast images demonstrate extensive bronchial wall enhancement and permit differentiation of a thickened wall from intrabronchial secretions, with intrabronchial fluid having an air–fluid level (arrow). Reproduced from [81] with permission from the publisher.

MRI showed comparable results to MDCT and CXR. Median extent scores for MRI, MDCT and CXR scores were 13, 13.5 and 14, and correlation between modalities ranged 0.63–0.80 (MRI/CT 0.80, p,0.0001; MRI/CXR 0.63, p,0.0018; CXR/CT 0.75, p,0.0001). The median lobe-related concordance was 80% for bronchiectasis, 77% for mucous plugging, 93% for sacculation/abscesses and 100% for collapse/consolidation. MONTELLA et al. [85] evaluated patients with primary ciliary dyskinesia using HRCT and MRI. They used a modified Helbich score for both HRCT and morphological MRI assessment, showing mean scores of 12 for both, good-to-excellent agreement between HRCT and MRI scores (r.0.8), and good correlation between both CT and MRI scores and FEV1 and FVC.

Functional MRI A significant advantage of MRI over CT is its superior ability to assess function. Within the lungs this mainly involves evaluation of haemodynamic function and perfusion and ventilation studies.

Perfusion imaging

EICHINGER et al. [90] performed morphological and contrast-enhanced MRI sequences on 11 patients with CF; 198 lung segments were scored for morphological (3 point score of none, moderate or severe) and perfusion defects (0 normal; 1 impaired perfusion). In 86% of segments considered morphologically normal, homogenous perfusion was demonstrated, whereas 97% of segments with severe morphological changes were associated with perfusion defects. Of segments with moderate morphological changes, 53%showed normal and 47% impaired perfusion. Thus contrast-enhanced MRI appears to be a feasible method of assessing regional perfusion defects as a surrogate for small airways disease, although further work is required in order to improve sensitivity in moderately affected areas of lung. In bronchiectasis, increased blood flow though bronchial and nonbronchial systemic collateral vessels results in a systemic arterial to pulmonary venous shunt. Using conventional proton MRI and phase-contrast flow-sensitive sequences, aortic and pulmonary arterial flow can be readily quantified. In a study of 10 patients with CF and 15 healthy volunteers, LEY et al. [91] found a significantly increased shunt in CF patients (1.3 L?min-1) compared to healthy volunteers (0.1 L?min-1).

P.L. PERERA AND N.J. SCREATON

In the presence of small airways obstruction, regional ventilation defects lead to impaired gas exchange and reflex hypoxic vasoconstriction [86]. Perfusion imaging can thus serve as a marker of airway obstruction. ITTI et al. [87] used radionuclide imaging to show that the degree of abnormal lung perfusion correlates well with disease severity in CF, as measured by PFTs and the Shwachman radiographic score. Contrast-enhanced pulmonary MRI imaging can be used to acquire a 3D data set in just 1.5 seconds [88, 89]. This also has advantages over scintigraphy in terms of radiation dose and provides regional information.

Oxygen-enhanced MRI exploits the weak paramagnetic properties of oxygen, which cause a shortening of T1 at high concentration and can be used as a gaseous contrast agent. The solubility of oxygen means that images represent a combination of ventilation and perfusion. A limitation is the low signal-to-noise ratio [81]. JAKOB et al. [92] studied five CF patients and five healthy volunteers using oxygen-enhanced MRI, showing inhomogeneity of the lung parenchyma in the CF patients.

Hyperpolarised noble-gas-enhanced MRI

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Imaging of hyperpolarised noble gases using MRI is a relatively new imaging technique that shows promise in the research arena in the evaluation of several ventilatory functional parameters. Hyperpolarised helium-3 and hyperpolarised xenon-129 are gaseous contrast agents that provide a very high MR signal [35]. Since oxygen promotes depolarisation, the polarised helium-3 is mixed

with nitrogen rather than air before being administered by active inhalation via a device such as a plastic Tedlar bag or respirator-driven gas delivery system. The bag method uses a mixture of 300 mL helium-3 and 700 mL nitrogen and requires a single anoxic breathhold. The respiratordriven system provides an accurate single dose followed by an air chaser and hence no anoxic breathhold is required. Polarisation is not renewable and has to be used carefully during the scan, with use of sequences to maximise use of finite magnetisation [93]. Dedicated receiver coils for the relevant resonance frequency of helium-3 and xenon-129 are also required. The different physical properties of helium-3 and xenon-129 present different opportunities. Although helium-3 provides a better signal and greater polarisation levels have been obtained, its larger diffusion coefficient results in signal loss. In addition, although helium-3 is virtually insoluble in water, xenon-129 is highly soluble and hence has potential for use in assessing perfusion [93]. On a purely practical basis, the limited supply of helium-3, estimated at 200 kg globally [94], compared to that of xenon-129 is likely to lead to xenon-129 eventually emerging as agent of choice [95].

RADIOLOGICAL FEATURES

The main techniques used in hyperpolarised noble gas imaging are static ventilation imaging and dynamic ventilation imaging, as well as assessment of lung microstructure using ADC and regional oxygen tension imaging. MCMAHON et al. [96] showed that static helium-3 MRI ventilation in CF correlated strongly with HRCT assessment of structural abnormalities (R5¡0.89; p,0.001), and that the correlation was higher between helium-3 MRI and PFT results than helium-3 MRI and HRCT. In a further observational study of 18 patients aged 5–17 years with CF undergoing hyperpolarised helium-2 MRI, VAN BEEK et al. [97] confirmed moderate correlation between a visual score of ventilation on MRI and global assessment of pulmonary function (FEV1 r5 -0.41 and FVC r5 -0.42). In a study comparing healthy volunteers and CF patients, MENTORE et al. [98] performed spirometry and hyperpolarised helium-3 imaging at baseline in all cases, and following various interventions in the eight CF patients. Treatments in the CF group included bronchodilators, DNAse and chest physiotherapy. The number of ventilation defects was scored. The helium-3 ventilation score correlated moderately with spirometry, and was higher in CF patients than controls (mean 8.2 and 1.6, respectively). The helium-3 ventilation score was raised in comparison to controls even in CF patients with normal spirometric results. Defects in the eight treated patients decreased in response to bronchodilator therapy (p50.025). WOODHOUSE et al. [99] demonstrated reproducibility of regional and total lung volume measurements using hyperpolarised helium-3 MR in two examinations performed 30 minutes apart in a group of five young CF sufferers. The ADC of helium-3 or xenon-129 in the lung and the paramagnetic effect of oxygen are two novel methods with the potential for extracting clinically relevant data. The ADC provides a measure of the diffusion of gas and thus an assessment of the degree to which free diffusion is restricted. Helium-3 has a very high self-diffusion coefficient, but, in the lung, diffusion becomes restricted by the boundaries of the airspaces, and thus the ADC can be used to interrogate the microstructure of the lung [93].

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The oxygen-induced depolarisation of helium-3 or xenon-129 results in signal decay proportional to the concentration of oxygen [100], permitting estimation of regional oxygen concentration and uptake and regional pulmonary perfusion, and providing a regional ventilation/perfusion (V/Q) map at a much higher resolution than that of radionuclide imaging [101]. PATZ et al. [95] measured regional oxygen concentrations using xenon-129, and, although this is inherently more complex than with helium-3 due to diffusion into septal tissue and vasculature, an oxygen tension (PO2) equivalent can be calculated, which can provide valuable functional information.

A limitation of hyperpolarised noble gas MRI is that the signal is also influenced by factors other than ventilation, including the sensitivity of the MR coil and local oxygen concentration in the lung. The need for noble gas isotopes and both polarisation hardware and additional MRI hardware, together with considerable physical and technical support, mean that hyperpolarised noble gas imaging remains expensive and limited to the research environment. However, the potential to perform noninvasive evaluation of regional ventilation, diffusion, regional oxygen concentration, lung microstructure and perfusion without the use of ionising radiation has potential, especially in the research setting.

Summary MRI has potential in the imaging of bronchiectasis, particularly in conditions such as CF, in which young patients may require serial imaging for disease monitoring and assessment of response to treatment. Compared to HRCT, the ability of MR to provide functional imaging and lack of radiation could compensate for its limited spatial resolution. With improvement in MRI techniques, recent studies have shown good reproducibility and good correlation with PFT results. Further work is required to improve spatial resolution, develop robust validated scoring systems and evaluate correlations with clinical outcomes. Currently cost, limited availability and limited spatial resolution limit the use of MRI in bronchiectasis largely to the research arena. Although hyperpolarised noble gas imaging has great potential in terms of provision of functional data, technical issues and set-up and ongoing costs suggest its role will be limited to research for the foreseeable future.

Prior to the advent of HRCT, ventilation (with or without perfusion) scintigraphy was used to aid disease evaluation in bronchiectasis. DOLLERY and HUGH-JONES [102] studied the physiological implications of bronchiectasis and found reduced blood flow and impaired ventilation in bronchiectatic areas. V/Q scintigraphy typically demonstrates matched ventilation and perfusion defects, reflecting abnormal ventilation secondary to bronchiectasis and associated small airways obstruction [103]. PIFFERI et al. [104] studied 16 children aged 4–18 years with clinical and CXR evidence of bronchiectasis, performing HRCT and V/Q scintigraphy. The extent of bronchiectasis, degree of air-trapping on expiratory HRCT and ventilation and perfusion scores from V/Q scintigraphy were assessed. HRCT scores for bronchiectasis and air-trapping showed a strong correlation with perfusion (r50.82; p,0.001) and ventilation scores (r50.72; p,0.01). There was a moderate negative correlation between FEV1 and HRCT bronchiectatic scores (r5 -0.53; p50.02), airtrapping (r5 -0.64; p50.007) and atelectatic score (r5 -0.54; p50.03).

P.L. PERERA AND N.J. SCREATON

Scintigraphy

The authors concluded that HRCT provides a comprehensive assessment of children with bronchiectasis, and V/Q scintigraphy and lung function are additive tools to aid diagnosis and guide therapeutic management. The ongoing issue of radiation dose and absence of useful anatomical information, however, limit the value of V/Q scintigraphy in routine practice.

Mucociliary clearance

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The interaction between the cilia on respiratory epithelium and the periciliary mucous layer (periciliary liquid (PCL))/overlying mucous layer, together known as the airway surface liquid (ASL) layer, has been widely investigated. Coordinated function is responsible for the constant clearance of foreign material, including microorganisms and other debris, towards the pharynx and ultimate expectoration or swallowing. Impaired mucociliary function has been implicated in many disease processes, but particularly bronchiectasis. Techniques to objectively measure mucociliary clearance (MCC) in vivo have been sought in order to improve understanding of the disease processes and evaluate therapeutic response.

Techniques for measuring MCC In vivo assessment of MCC relies on the inhalation of radiolabelled particulate material that becomes trapped in the mucous layer and can be imaged scintigraphically. Data acquired from the gamma camera over time can be presented either as a series of images for visual inspection or, more commonly, as time–activity curves (fig. 11). Technetium-99m-labelled human albumin, iron oxide and technetium-99–sulfur colloid are some of the aerosols used. Sulfur colloid is nondiffusible, remains extravascular and is expelled by MCC/ swallowing. Deposition of particles is affected by many factors. Some, such as particle size and breathing pattern, can be controlled for, whereas others reflect the underlying lung condition (e.g. obstruction and lung size). Thus, in order to make comparisons, it is important to standardise the nature of the aerosolised particles (size and distribution) and provide a consistent nebulised flow in order to produce a reproducible deposition rate [105]. In order to define the margins of the lung and differentiate central (C) and peripheral (P) lung regions, an initial ventilation study utilising a xenon-133 or krypton-81 scan [106] can be performed immediately prior to administration of particulate material. Following this, the patient b)

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RADIOLOGICAL FEATURES

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Figure 11. Measurement of mucociliary clearance (MCC). a) A xenon-133 equilibrium scan was used to identify

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the left (L) and right (R) lung boundaries in a normal subject, and assign central (C) and peripheral (P) regions of interest (b). c) Deposition image obtained immediately after inhalation of technetium–sulfur colloid in the same subject. d) Mean rate of clearance of technetium–sulfur colloid from 12 subjects with cystic fibrosis at baseline (&) h and immediately after inhalation of hypertonic saline ($) [16]. The fast phase (approximately 0–20 minutes; – – – – –), reflecting clearance from large airways, and slow phase (from 40 minutes to start of cough clearance measurement; --------), reflecting smaller airway clearance, are highlighted. e) Effect of ratio of radioactive counts measured in the C and P regions on rate of MCC, as denoted by particle retention at 120 minutes, in a cohort of normal study subjects. VC: voluntary cough. Reproduced from [105] with permission from the publisher.

inhales nebulised radiolabelled aerosol. Various adjuncts are used during nebulisation in order to provide consistent reproducible dosing, including pneumotachographic devices with visual feedback to control inhalation flow rate and tidal volume within specific ranges, metronomes to guide the timing of inhalation and exhalation, and aerosol dosimetric equipment to pulse aerosol delivery during specific portions of the breathing cycle [105].

The rate of clearance from central airways is up to 100–1,000 times faster than that from peripheral airways [108, 109]. A two-phase MCC pattern is typically seen, with an initial rapid phase lasting approximately 30 minutes and reflecting clearance from the central airways and a prolonged slower phase. The latter occurs over 1–2 hours and is thought to represent movement of particles to compartments that are more difficult to clear (e.g. absorption of PCL) or slow clearance from peripheral airway/alveolar deposition. 24-hour measurement of clearance has also been used to assess the pattern of clearance during the slower phase, which could also be of value in assessing response to treatment. This, however, requires a higher administered radiation dose due to the 6-hour half-life of technetium. A static measurement at 24 hours can be used as a marker of deposition in the nonciliate airways or alveoli [110]. The relative contributions to the 24-hour measurement of slow clearance from peripheral airways, alveolar deposition and mixing in a poorly cleared part of the ASL, are not fully understood [105]. The static 24-hour measurement is useful in aiding calculation of other parameters, such as the tracheobronchial retention (TBR) curve, which is derived by subtracting the 24-hour retention from the corrected lung retention (LR) curve. A potentially more accurate means of assessing peripheral clearance is inhaling particles of different sizes, smaller (4 mm) particles being deposited more peripherally than larger (7.5 mm) ones [111]. YEATES et al. [108] proposed labelling the differently sized aerosolised particles with different radioisotopes to permit simultaneous measurement of central and peripheral regional clearance. This method is not widely used due to practical difficulties. Some authors [106] advocate measurement of activity solely in the lung periphery, where uptake is more homogenous. This avoids the potential errors caused by differential uptake in central and peripheral airways and confounding by variability of initial deposition. It is, however, limited by a low signal-to-noise ratio due to lower deposition peripherally and intrinsically slower clearance in these regions. It is also not possible to assess response to therapy in the central airways using this method.

P.L. PERERA AND N.J. SCREATON

Following inhalation, the patient is positioned in front of the gamma camera and the gamma radiation emitted is detected and recorded. The results are analysed graphically with reference to the zones defined on the initial ventilation scan. At this stage of analysis, it is important to account for decay of radioisotope and background radiation level. Given the variability of deposition of radiolabelled aerosol in various parts of the airways, it is important to measure the initial deposition pattern. The deposition pattern is usually presented as a ratio between C and P or as the penetration index (PI), which is the ratio of radioactive counts per pixel in P to counts per pixel in C [107]. A high initial C/P deposition ratio or low PI is associated with a higher clearance rate in the central airways, making this a potential confounding factor in analysing final clearance data.

Additional imaging following various interventions, such as cough clearance (CC) assessed after a standardised pattern of coughing, can also be performed. This has some limitations, as performing this late in the study makes it less sensitive as the central airways would have been largely cleared of radioisotope. A further normalisation measurement of C/P ratio must be performed prior to CC.

Clinical applications

61

Measurement of MCC has important research applications in both understanding disease processes and assessing therapeutic response. To date, the technique has not been broadly adopted clinically, being cumbersome to establish.

Disorders that impair MCC can seriously affect respiratory function, with build-up of thick mucus in the airways/lungs and inability to expel harmful material. This can predispose to complications, such as infection and structural lung disease. Other factors may influence MCC, which is faster in nonsmokers and enhanced by b2-agonists, particularly in nonsmokers [112]. CF is a prominent example of a condition in which measurement of MCC could prove useful. Scintigraphic evaluation has also been used to demonstrate impaired MCC in primary ciliary dyskinesia [110] and following lung transplantation [113]. There have been limited studies in idiopathic bronchiectasis [114].

RADIOLOGICAL FEATURES

In CF, disordered ion transport leads to dehydration of the ASL layer [115], impaired ciliary motion and decreased mucus clearance, ultimately leading to degradation of cilia [105], exacerbating the cycle of frequent infections. Ongoing research is focused on the earliest stages of disease pathogenesis and therapeutic interventions to target defective mucus clearance. Biomarkers objectively measuring MCC have the potential to assess response to treatment at an early stage in contrast to longer-term end-points, such as clinical or functional parameters, and thus to expedite drug development. In a study of 24 patients with CF, DONALDSON et al. [116] showed improved MCC, measured using technetium-99-labelled iron oxide, at both 1 and 24 hours after inhalation of hypertonic saline, and that pretreatment with amiloride reduced the magnitude of this improvement. Using radiolabelled iron oxide BENNETT et al. [106] demonstrated significantly reduced baseline MCC at 40 minutes in CF patients compared to healthy volunteers. In the CF group, treatment with uridine 5’-triphosphate and amiloride in combination improved peripheral MCC to near-normal levels. Similar studies have used technetium–sulfur colloid to demonstrate improved MCC and CC following inhaled hypertonic saline and mannitol in CF patients [117]. In summary, MCC can be measured using radiolabelled particulate materials, such as technetium99–sulfur colloid. In the research setting, this provides a potential biomarker for evaluation of mucociliary dysfunction and, in particular, assessment of the impact of targeted therapies. This is especially true in conditions such as CF, in which impaired MCC plays a significant part in the pathophysiology of the disease and where treatment is targeted at improving this.

Conclusions Imaging plays a central role in the diagnosis, characterisation and quantification of disease severity in bronchiectasis, as well as the evaluation of complications. Currently CXR and CT are the main modalities. CXR is the initial screening tool, but has well-documented limitations in sensitivity and specificity, particularly in early disease. Radiography also plays an important role in the diagnosis of complications. HRCT is the reference standard in identifying airway dilation, permitting detection of disease and quantification of extent. Routine surveillance CT has potential for the diagnosis of structural disease at an early stage and impact on patient care, particularly where these is discordance with functional parameters. Radiation dose, however, remains an area of concern requiring further elucidation, particularly in the cohort of CF patients given their ever-increasing life expectancy and the potentially large cumulative radiation dose. Although the present review concentrates on the monitoring of disease, CT is an excellent problem-solving tool, permitting the diagnosis of both infective complications, such as abscess, empyema and aspergilloma, as well as identification of small pneumothoraces or enlarged systemic collateral vessels (fig. 11) and aiding relevant image-guided intervention.

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MRI offers opportunities to image the lung structure and its function without the use of ionising radiation. The spatial resolution is inferior to that of CT but has improved substantially over recent years. An increasing role for structural (proton) MRI is anticipated, but widespread adoption will require further evidence to support its effectiveness. Although hyperpolarised noble gases permit interrogation of a range of physiological parameters, the set-up costs of this technique

are likely to ensure it remains a predominantly research tool for at least the foreseeable future. Evaluation of MCC using scintigraphy is another area in which there is great potential, particularly in order to expedite and reduce costs of drug development.

Statement of interest None declared.

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87. Itti E, Fauroux B, Pigeot J, et al. Quantitative lung perfusion scan as a predictor of aerosol distribution heterogeneity and disease severity in children with cystic fibrosis. Nucl Med Commun 2004; 25: 563–569. 88. Fink C, Bock M, Puderbach M, et al. Partially parallel three-dimensional magnetic resonance imaging for the assessment of lung perfusion – initial results. Invest Radiol 2003; 38: 482–488. 89. Ley S, Fink C, Puderbach M, et al. Kontrastmittelversta¨rkte 3D-MR-Perfusion der Lunge: Einsatz paralleler Bildgebungstechniken bei gesunden Probanden. [Contrast-enhanced 3D MR perfusion of the lung: application of parallel imaging technique in healthy subjects.] Rofo 2004; 176: 330–334. 90. Eichinger M, Puderbach M, Fink C, et al. Contrast-enhanced 3D MRI of lung perfusion in children with cystic fibrosis – initial results. Eur Radiol 2006; 16: 2147–2152. 91. Ley S, Puderbach M, Fink C, et al. Assessment of hemodynamic changes in the systemic and pulmonary arterial circulation in patients with cystic fibrosis using phase-contrast MRI. Eur Radiol 2005; 15: 1575–1580. 92. Jakob PM, Wang T, Schultz G, et al. Assessment of human pulmonary function using oxygen-enhanced T1 imaging in patients with cystic fibrosis. Magn Reson Med 2004; 51: 1009–1016. 93. van Beek EJ, Wild JM, Kauczor HU, et al. Functional MRI of the lung using hyperpolarized 3-helium gas. J Magn Reson Imaging 2004; 20: 540–554. 94. Kauczor H-U, Surkau R, Roberts T. MRI using hyperpolarized noble gases. Eur Radiol 1998; 8: 820–827. 95. Patz S, Hersman W, Muradian I. Hyperpolarized 129Xe MRI: a viable functional lung imaging modality? Eur J Radiol 2007; 64: 335–344. 96. McMahon CJ, Dodd JD, Hill C, et al. Hyperpolarized 3helium magnetic resonance ventilation imaging of the lung in cystic fibrosis: comparison with high resolution CT and spirometry. Eur Radiol 2006; 16: 2483–2490. 97. van Beek EJ, Hill C, Woodhouse N, et al. Assessment of lung disease in children with cystic fibrosis using hyperpolarized 3-helium MRI: comparison with Shwachman score, Chrispin–Norman score and spirometry. Eur Radiol 2007; 17: 1018–1024. 98. Mentore K, Froh DK, de Lange EE, et al. Hyperpolarized HHe 3 MRI of the lung in cystic fibrosis: assessment at baseline and after bronchodilator and airway clearance treatment. Acad Radiol 2005; 12: 1423–1429. 99. Woodhouse N, Wild JM, van Beek EJR, et al. Assessment of hyperpolarized 3He lung MRI for regional evaluation of interventional therapy: a pilot study in pediatric cystic fibrosis. J Magn Reson Imag 2009; 30: 981–988. 100. Eberle B, Weiler N, Markstaller K, et al. Analysis of intrapulmonary O2 concentration by MR imaging of inhaled hyperpolarized helium-3. J Appl Physiol 1999; 87: 2043–2052. 101. Kauczor H-U. Hyperpolarized helium-3 gas magnetic resonance imaging of the lung. Top Magn Reson Imaging 2003; 14: 223–230. 102. Dollery CT, Hugh-Jones P. Distribution of gas and blood in the lungs in disease. Br Med Bull 1963; 19: 59–63. 103. Singh MM, Talwar D, Jena A, et al. Ventilation–perfusion studies in bronchiectasis. Ind J Tub 1987; 34: 182–186. 104. Pifferi M, Caramella D, Bulleri A, et al. Pediatric bronchiectasis: correlation of HRCT, ventilation and perfusion scintigraphy, and pulmonary function testing. Pediatr Pulmonol 2004; 38: 298–303. 105. Donaldson SH, Corcoran TC, Laube BL, et al. Mucociliary clearance as an outcome measure for cystic fibrosis clinical research. Proc Am Thorac Soc 2007; 4: 399–405. 106. Bennett WD, Olivier KN, Zeman KL, et al. Effect of uridine 5’-triphosphate plus amiloride on mucociliary clearance in adult cystic fibrosis. Am J Respir Crit Care Med 1996; 153: 1796–1801. 107. Robinson M, Eberl S, Tomlinson C, et al. Regional mucociliary clearance in patients with cystic fibrosis. J Aerosol Med 2000; 13: 73–86. 108. Yeates DB, Gerrity TR, Garrard CS. Characteristics of tracheobronchial deposition and clearance in man. Ann Occup Hyg 1982; 26: 245–257. 109. Wilkey DD, Lee PS, Hass FJ, et al. Mucociliary clearance of deposited particles from the human lung: intra- and inter-subject reproducibility, total and regional lung clearance, and model comparisons. Arch Environ Health 1980; 35: 294–303. 110. Marthin JK, Mortensen J, Pressler T, et al. Pulmonary radioaerosol mucociliary clearance in diagnosis of primary ciliary dyskinesia. Chest 2007; 132: 966–976. 111. Pavia D, Sutton PP, Agnew JE, et al. Measurement of bronchial mucociliary clearance. Eur J Respir Dis 1983; 64: Suppl. 127, 41–56. 112. Mortensen J, Lange P, Nyboe J, et al. Lung mucociliary clearance. Eur J Nucl Med 1994; 21: 953–961. 113. Laube BL, Karmazyn YJ, Orens JB, et al. Albuterol improves impaired mucociliary clearance after lung transplantation. J Heart Lung Transplant 2007; 26: 138–144. 114. Isawa T, Teshima T, Hirano T, et al. Mucociliary clearance in pulmonary vascular disease. Ann Nucl Med 1988; 2: 41–47. 115. Rowe S, Accurso F, Clancy JP. Detection of CFTR activity in early phase clinical trials. Proc Am Thorac Soc 2007; 4: 387–398.

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116. Donaldson SH, Bennett WD, Zeman KL, et al. Mucus clearance and lung function in cystic fibrosis with hypertonic saline. N Engl J Med 2006; 354: 241–250. 117. Robinson M, Daviskas E, Eberl S, et al. The effect of inhaled mannitol on bronchial mucus clearance in cystic fibrosis patients: a pilot study. Eur Respir J 1999; 14: 678–685.

Chapter 6

Microbiology of nonCF bronchiectasis J.E. Foweraker* and D. Wat#

MICROBIOLOGY

Summary Non-cystic fibrosis (CF) bronchiectasis is a complex disorder characterised by recurrent chest infections and poorly regulated respiratory innate and adaptive immunity. These lead to a ‘‘vicious cycle’’ of impaired mucociliary clearance, chronic infection, bronchial inflammation and progressive lung injury. The most prevalent pathogenic bacteria are Haemophilus influenzae, Pseudomonas aeruginosa, Streptococcus pneumoniae, Staphylococcus aureus and Moraxella catarrhalis although variations in sampling techniques and detection methods have influenced their isolation rates. These organisms can inhibit mucociliary clearance, destroy respiratory epithelium and produce biofilms that promote persistent infection by blocking innate immune defences and increasing antibiotic resistance. While numerous studies have examined the role of different bacteria in CF and chronic obstructive pulmonary disease, little is known about how they contribute to the pathogenesis of nonCF bronchiectasis. There is also a paucity of data regarding the role of respiratory viruses in this condition. This chapter describes the microbiology of non-CF bronchiectasis, defines the bacterial mechanisms that may contribute to persistent infection and airway damage and discusses the potential role for respiratory viruses in this condition. Understanding the pathogenic properties of these microorganisms may allow the development of novel therapies for the management of respiratory exacerbations. Keywords: Anaerobes, Haemophilus, Moraxella, Pseudomonas, Streptococcus, viruses

P

*Dept of Microbiology, and # Lung Defence Unit, Papworth Hospital, Cambridge, UK. Correspondence: J.E. Foweraker, Dept of Microbiology, Papworth Hospital, Papworth Everard, Cambridge, CB23 3RE, UK, Email [email protected]

Eur Respir Mon 2011. 52, 68–96. Printed in UK – all rights reserved. Copyright ERS 2011. European Respiratory Monograph; ISSN: 1025-448x. DOI: 10.1183/1025448x.10003610

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atients with bronchiectasis are commonly colonised with potentially pathogenic microorganisms in the airways [1]. These microorganisms can cause lung infections and may produce a number of inflammatory mediators that can lead to progressive tissue damage and bronchial obstruction. The phenomenon of chronic infection, bronchial inflammation and progressive lung injury is a ‘‘vicious cycle’’ and is also the reason why prompt evaluation of infection is important [2].

Being able to identify the causative bacterium may allow appropriate antibiotic administration to break this vicious cycle. The most prevalent microorganisms found in non-cystic fibrosis (CF) bronchiectasis are discussed in this chapter and we have included the role of viruses, as well as some recent studies that have investigated microorganisms that are not usually considered to be pathogens in the respiratory tract. Surprisingly, there is little published data on the epidemiology and pathogenesis of infections in non-CF bronchiectasis. However, there are similarities with infections in CF bronchiectasis and chronic obstructive pulmonary disease (COPD). Where the literature for non-CF bronchiectasis is sparse, studies in CF and COPD have been drawn upon as these may aid the understanding of the microbiology; in particular the adaptations that take place to enable microorganisms to establish and maintain chronic infection and the role taken in the development of exacerbations. Fungal infections, including allergic bronchopulmonary aspergillosis (ABPA), are discussed further by HILVERLING et al. [3], while nontuberculous mycobacteria infections are discussed further by DALEY [4] in this Monograph.

Several studies have reviewed the bacteria found in patients with non-CF bronchiectasis (table 1). A similar range of organisms is found in most studies, but the prevalence of each varies. Age, ethnicity, the underlying causes of bronchiectasis and the proportion of patients that were stable or had cultures taken during an exacerbation varies between the different studies and would be expected to affect the microbial flora found. The pattern of antibiotic usage, including long-term prophylaxis, may vary between different centres and could also have an affect on the type of microorganisms cultured. The type of respiratory specimen tested may also determine the rate of positive cultures found. The use of a protected specimen brush to take samples at bronchoscopy yielded the highest positivity rate when compared with sputum specimens in one study [7]. Finally the methodology used for analysis (quantification, culture and identification techniques) will vary between centres and could also affect the result. Haemophilus influenzae and Pseudomonas aeruginosa were the most common bacteria found in the majority of the studies and the most likely to cause long-term colonisation [12]. No potentially pathogenic microorganisms were cultured from 18–24% of the patients investigated and an absence of a potentially pathogenic microorganism was associated with the milder disease [9, 13].

Haemophilus influenzae

J.E. FOWERAKER AND D. WAT

Range of bacteria in patients with non-CF bronchiectasis

H. influenzae has been reported in 14–52% of patients with non-CF bronchiectasis. It is a Gramnegative coccobacillus with specific growth requirements, which can be difficult to isolate in the laboratory if mixed with other flora. Some H. influenzae possess a polysaccharide capsule and can be typed using type-specific anticapsule antisera. Those with the type B capsule (Hib) can cause invasive infection with bacteraemia, and are most familiar as a cause of meningitis or epiglottitis. The use of Hib vaccine has greatly reduced the incidence of these life-threatening conditions. H. influenzae with capsule types other than type B are relatively rare and are far less pathogenic. The nonencapsulated strains, referred to as nontypeable H. influenzae (NTHi), are also less pathogenic than Hib and only rarely cause bacteraemia. They live as commensals in the human upper respiratory tract but can cause otitis media, sinusitis and conjunctivitis, often following a primary viral infection. NTHi are a common cause of lower respiratory infection in patients with underlying respiratory abnormalities including non-CF bronchiectasis [9]. The Hib vaccine does not prevent infection with NTHi as it only contains the H. influenzae type B capsule antigen.

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NTHi could be an oral contaminant in expectorated sputum; however, studies using a protected specimen brush (PSB) at bronchoscopy found NTHi in significant numbers in non-CF bronchiectasis, confirming its presence in the lower respiratory tract [7]. In contrast

15 (10) 9 (6) 47 (33) 47 (33) Colonised subgroup"

13 (9)

21 (14) 39 (27) 30 (20) 39 (27) 20 (13) 42 (30) 46 (31) 62 (43) 52 (35) 75 (52) ND ND Sputum Sputum PASTEUR [10] MACFARLANE [11]

UK UK

150 143

60.6 (16–90)

3 (4) 7 (8) 6 (7) 11 (12) 42 (47) Stable Sputum 89 KING [9]

Australia

57¡14

9 (7) 3 (2) 13 (11) 38 (31) 37 (30) ND Sputum 123 USA NICOTRA [8]

57.2¡16.7

2 (3) 3 (4) 6 (8) 12 (16) 24 (32) Stable 58 (16–76) PSB 75 Spain ANGRILL [7]

Data are presented as mean (range), mean¡SD or n (%), unless otherwise stated. RTFlora: upper respiratory tract flora; PPM: potentially pathogenic microorganisms; ND: not described; K. pneumoniae: Klebsiella pneumoniae; GNB: Gram-negative bacilli; GPB: Gram-positive bacilli; PSB: protected specimen brush; A. xylosoxidans: Achromobacter xylosoxidans: E. coli: Escherichia coli; S. maltophilia: Stenotrophomonas maltophilia; #: some had more than one PPM cultured; ": bacteria were isolated on at least two occasions, 3 months apart, in 1 year.

7 (14) K. pneumonia 7 (14) GNB 2 (4) GPB 18 (24) RTFlora 1 (1) A. xylosoxidans 1 (1) E. coli ND 16 (13) GNB 2 (3) Anaerobes 19 (21) no PPM 2 (2) E. coli 1 (1) A. xylosoxidans ND 28 (20) 12 (8) S. maltophilia 4 (3) A. xylosoxidans 42 (30) Coliforms 2 (1) S. maltophilia 2 (1) A. xylosoxidans 13 (9) Coliforms

Sputum Sputum 92 50 Ireland Thailand ZAID [5] PALWATWICHAI [6]

,18 58 (30–85)

ND ND

50 (46) 7 (14)

8 (9) 10 (20)

34 (37) 3 (6)

9 (10) 2 (4)

14 (15)

ND 9 (18) RTFlora

Other RTFlora/ no PPM

Haemophilus Pseudomonas Streptococcus Moraxella Staphylococcus influenzae aeruginosa pneumoniae catarrhalis aureus

Patients with organisms cultured from respiratory tract# Stable or at exacerbation Age yrs Sample Subjects n Country First author [ref.]

Table 1. Range of microorganisms cultured from patients with non-cystic fibrosis bronchiectasis

MICROBIOLOGY

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Haemophilus parainfluenzae, a common commensal organism found in the upper respiratory tract, may be cultured from sputum but was not found in PSB samples. In patients with COPD the presence of NTHi in sputum was associated with raised inflammatory cytokines, whereas patients with H. parainfluenzae in sputum had similar levels of cytokines to those who had no microorganisms cultured from their sputum, suggesting that even if present in the lower tract it does not have a direct pathogenic role [14, 15]. There is little published data on the epidemiology of H. influenzae in non-CF bronchiectasis. It may be cultured repeatedly from the same patient over several years, but without typing data it is not known if this is the persistence of a single strain or repeated episodes of infection [9]. In COPD patients, NTHi were found in higher numbers (.106 colony forming units (CFU)?mL-1) during exacerbation compared with when the patient was stable, and exacerbations in COPD may be associated with the appearance of a new strain [16, 17]. A prospective study in COPD using molecular typing of H. influenzae and direct analysis of amplified DNA from sputum showed persistence of the same strain over prolonged periods [18]. This suggests either long-term colonising infection in the lung or persistence in the upper respiratory tract with repeated deposition followed by clearance from the lung. In CF, sequential infection with different strains of H. influenzae was found in some patients and persistence of the same clone in other individuals [19]. NTHi have various properties that can help explain their pathogenicity and ability to persist in the lung. They can adhere to mucus and to various cell types in the human respiratory tract using pili and other adhesion molecules. Virulence factors include the endotoxin lipo-oligosaccharides

(LOS), and immunoglobulin (Ig)-A protease [20]. NTHi possess mechanisms to vary the structure and activity of LOS and these may explain variations in the pathogenicity of different isolates [21]. Studies comparing the proteome of H. influenzae grown in vitro, either in pooled sputum or chemically defined media, have shown that the organism is able to adapt to oxidative stress and limited nutrients [22]. This is thought to be because H. influenzae has the ability to generate a diverse population that allows rapid adaptation to changes in the environment by expansion of the clonal members that express the phenotypic characteristics needed to survive. Mechanisms used by H. influenzae to generate phenotypic diversity include altered gene expression, e.g. by phase variation, and altered gene content by mutation or by horizontal gene transfer, e.g. direct DNA uptaketransformation, or via bacteriophage [23].

H. influenzae (along with other bacteria infecting a bronchiectatic lung) may exist in biofilms in the respiratory tract. These are co-operative populations of bacteria surrounded by an amorphous matrix and could help the organism to survive in a hostile environment by resisting both host defences and antibiotics. The antibiotic resistance observed for bacteria growing in biofilms is in part attributable to its electrolyte content but also by reduced bacterial growth or even dormancy within the biofilm matrix. NTHi from patients with COPD can form biofilms in vitro, and NTHi biofilms were seen in the chinchilla model of otitis media [28, 29]. NTHi cultured from CF patients could form biofilms in vitro and on the surface of cultured airway epithelial cells. Structures consistent with biofilms containing H. influenzae were also found in bronchoalveolar lavage (BAL) samples from children with CF [30]. NTHi in biofilms were more resistant to antibiotics in vitro. Sub-inhibitory concentrations of azithromycin were found to reduce the size of both growing and established biofilms [31]. The prevalence of antibiotic resistant NTHi increases over time in patients with non-CF bronchiectasis [9]. Many are resistant to amidopenicillins (e.g. amoxicillin, ampicillin) either due to production of b-lactamase or alteration of penicillin binding proteins. Quinolone resistance is now recognised and resistance rates to trimethoprim and tetracycline are rising.

J.E. FOWERAKER AND D. WAT

NTHi may be able to evade the immune response by varying its surface antigens. Mechanisms include phase variation of LOS [24], and changes to outer membrane proteins (OMP) either by horizontal gene transfer or point mutations of the immuno-dominant OMP, i.e. P2. Antigenic drift, resulting from change in the P2 gene, has been observed in persistent infections in patients with COPD [25]. NTHi could be protected within host cells as they have been found inside macrophages in the chinchilla otitis media model and in macrophage-like cells in human adenoids. NTHi were able to enter cultured nonciliated respiratory epithelial cells and cross the respiratory epithelium [26]. Using in situ hybridisation, NTHi were identified inside cells in bronchial biopsies taken from patients with COPD [27].

Antibiotic resistance may occur by horizontal transfer of genetic material from other organisms in the complex polymicrobial environment of the mouth and upper respiratory tract. It may also take place in the lower respiratory tract, which may be polymicrobial in non-CF bronchiectasis. Alternatively, resistance may result from gene mutation. Some NTHi have a higher than usual mutation rate due to a mutation in mutS, which is one of the methyl-directed mismatch repair genes (MMR) that corrects errors in DNA. This hypermutability is not usually thought to be advantageous, as many random mutations can reduce bacterial fitness. However, if mutations lead to antibiotic resistance, the hypermutable state may become beneficial to the bacterial population. Hypermutators are generally rare in acute infection but hypermutable H. influenzae have been found in patients with CF and these strains have more resistance to antibiotics compared with normo-mutators [19, 32]. Hypermutability is seen in other species causing chronic infection (as is discussed later in this chapter) and may be a general adaptation to long-term survival in the lung. The prevalence and role of hypermutable H. influenzae in non-CF bronchiectasis has yet to be assessed.

Pseudomonas aeruginosa

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P. aeruginosa is a versatile nonfermentative Gram-negative bacillus that is found in a range of environments. It is an opportunistic human pathogen that can cause severe, acute and invasive

infections, such as necrotising ventilator-associated pneumonia and infections in immunocompromised patients often with bacteraemia [33]. It is one of the most common causes of infection in non-CF bronchiectasis and other chronic lung diseases, most notably CF, but may also be important in severe COPD. The epidemiology of P. aeruginosa, the mechanisms of pathogenicity and the genotypic and phenotypic changes in chronic infection have been extensively studied in CF, with fewer publications in non-CF bronchiectasis and COPD. There are many similarities between the infections in these different conditions, suggesting a common route of adaptation to chronic infection in the lung.

MICROBIOLOGY

P. aeruginosa in CF Early infections in CF are caused by genotypically distinct isolates, suggesting repeated episodes of acquisition. These early P. aeruginosa have the typical phenotype of isolates causing acute infections and environmental strains [34]. As chronic infection with P. aeruginosa can lead to an accelerated deterioration in lung function, antibiotic treatment regimens were developed to clear early infection and delay the onset of chronic infection [35]. CF patients eventually developed a persistent infection that seldom cleared despite aggressive antibiotic therapy. While there are some mixed infections, most CF patients carry a single genotype of P. aeruginosa, often for many decades [36, 37], and exacerbations do not appear to be due to the acquisition of a new strain of P. aeruginosa [38]. Early studies in CF showed that individual patients were infected with distinct strains that were thought to have been acquired from the environment. Some siblings shared strains but it was not known whether this was cross-infection or exposure to a common environmental source. More recently there have been reports in several countries of crossinfection between CF patients with what are termed ‘‘epidemic’’ strains. Some, in particular the Liverpool epidemic strain (LES), have been associated with increased morbidity [39, 40]. LES is now the most common epidemic strain in the UK affecting as many as 11% of patients in England and Wales [41].

P. aeruginosa in COPD P. aeruginosa has been cultured from 4–15% of patients with COPD and was more prevalent in patients with advanced disease, particularly those requiring mechanical ventilation for severe exacerbations. P. aeruginosa infection was associated with steroid use, prior antibiotics and a low forced expiratory volume in 1 second (FEV1) [42]. In a study of 126 patients with moderate-tosevere COPD over an 11-year period, 39 patients grew P. aeruginosa from one or more sputum culture. There was a significant association with the culture of a new strain of P. aeruginosa and symptoms of an exacerbation. However, of interest, two-thirds of new infections that later cleared from the sputum, did so without the use of specific antibiotic treatment [43]. Only 13 patients had carriage of the same clone for more than 6 months with four patients infected with mucoid strains. Chronic infection is therefore rare in COPD, but when it does occur P. aeruginosa has a range of colony forms (morphotypes) and adaptations including increased mutability, reduced motility, reduced protease production and increased antibiotic resistance, similar to those seen in CF [44].

P. aeruginosa in non-CF bronchiectasis P. aeruginosa is one of the most common isolates found in 12–43% of non-CF bronchiectasis patients (table 1). Stable patients with P. aeruginosa have poorer lung function and more sputum production when compared with patients with other potentially pathogenic microorganisms (PPM) [45] and it has been associated with a poorer quality of life and more frequent hospital admissions [46]. There is debate over whether infection with P. aeruginosa leads to a faster decline in lung function as is seen in CF, or whether it is a marker of more damaged lungs [47, 48].

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A recent study compared long-term colonisation with P. aeruginosa in 21 patients, of which six had CF, 10 had non-CF bronchiectasis and five had COPD. The authors typed 125 sequential

isolates from sputa taken at least 1 month apart. The authors found a similar pattern of colonisation in all three diseases, with a dominant persistent clone, showing that the pattern of infection found in CF could also be shown in other conditions [49]. There have been no studies using genomic typing methods to investigate whether patients with nonCF bronchiectasis share strains of P. aeruginosa, but there has been one report of a patient with nonCF bronchiectasis acquiring LES from a relative with CF [50]. Interestingly, early studies using pyocin typing and examining mucinophilic and chemotactic properties of P. aeruginosa suggest that specific subpopulations may have a predilection to infect bronchiectatic lungs [51, 52].

Pathogenicity of P. aeruginosa P. aeruginosa possesses a range of virulence factors, although their expression may differ between isolates that cause acute infection and those responsible for chronic infection. Flagella, type IV pili, lipopolysaccharide and exopolysaccharides contribute to the adherence to cells and surfaces. Type I and type II secretion systems export protein toxins, such as alkaline protease, elastase, exotoxin A and phospholipase C, while type III secretion systems inject exoenzymes directly into eukaryotic cells. Other extra-cellular virulence factors include rhamnolipids, pyocyanin and hydrogen cyanide [53, 54]. Another pathogenicity factor is the ability to form alginate-enhanced biofilms [55], which contributes to the persistence of the organism rather than acute tissue damage and, together with other adaptations, promotes chronic infection (refer to later section).

P. aeruginosa is intrinsically resistant to many commonly used antibiotics and easily acquires resistance by chromosomal mutation or the acquisition of new genes from other microorganisms by horizontal transfer [56]. In addition, the biofilm mode of growth also protects P. aeruginosa from antibiotics by a variety of mechanisms [57]. Little has been published specifically on antibiotic susceptibility of P. aeruginosa from patients with non-CF bronchiectasis. In CF the prevalence of resistant P. aeruginosa is increasing as a result of repeated antibiotic courses. Resistance rates are significantly higher than for strains originating from patients without CF [58] and pan-resistant bacteria that are resistant to all antibiotics other than the polymixins have been described. P. aeruginosa can develop resistance by either: 1) producing enzymes that destroy the antibiotic, such as AmpC b-lactamase, carbapenemases or aminoglycoside modifying enzymes; 2) modifying the antibiotic target, such as gyrA for quinolone resistance; or 3) reducing exposure either by a decrease in permeability or increased removal of the antibiotic from the bacterial cell (efflux). Efflux mechanisms often affect more that one class of antibiotics and therefore contribute to multi-drug resistance [56].

J.E. FOWERAKER AND D. WAT

Antibiotic resistance

Antibiotic resistance and its regulation can be complex in P. aeruginosa and various mechanisms that affect resistance to a single antibiotic may be present in the same organism. For example, low level resistance to meropenem may be due to reduced permeability following changes to the membrane porin OprD. More resistance can result from an increase in an efflux pump that can remove the meropenem from the cell. Both mechanisms may be present and additive, leading to high-level resistance. Enzymes that can destroy meropenem (penemases such as VIM) do occur but are currently rare [56].

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AmpC codes for an inducible cephalosporinase which, when production is increased, can result in resistance to nearly all b-lactam antibiotics except the penems. Treatment with piperacillin or ceftazidime can lead to the selection of bacteria that produce the enzyme constitutively rather than just on induction. These are called derepressed mutants and offer a survival advantage. Imipenem induces the AmpC b-lactamase, even though it is not affected by the enzyme, and it also induces genes involved with alginate production [59]. The regulation of ampC is exceedingly complex and

is intimately linked to cell wall recycling [60–62]. Some mutations can reduce biological competitiveness and more work is needed to assess the link between antimicrobial resistance and fitness [61]. AmpR does not just regulate ampC but is a global transcriptional regulator that regulates another b-lactamase PoxB, as well as proteases, quorum sensing and other virulence factors [63]. Antibiotic resistance may therefore be associated with a change in virulence and/or fitness. This could explain why some CF patients respond to treatment for acute exacerbation, even though some of the P. aeruginosa are resistant to the antibiotic used [64]. P. aeruginosa possesses multi-drug efflux pumps that can expel a wide range of antibiotics and are responsible for much of the organism’s intrinsic resistance to antimicrobials. For example, substrates for efflux pump MexAB-OprM include ticarcillin, aztreonam, piperacillin, ceftazidime and tetracycline [65]. MexXY uses the same exit duct OprM and can export aminoglycosides, cefepime and ciprofloxacin. Antibiotic resistance may arise from an increase in the efflux pump activity, e.g. MexXY-OprM over-expression may be due to mutation in the regulatory gene mexZ and/or to mutations in the MexXY translocase genes [66].

MICROBIOLOGY

Conversely, P. aeruginosa may be hyper-susceptible in vitro to some anti-pseudomonal antibiotics and susceptible to agents such as tetracycline and chloramphenicol to which P. aeruginosa would normally be intrinsically resistant. This has been described in chronic infection in both CF and non-CF bronchiectasis [67, 68], but the clinical relevance of these findings has not been investigated. It was found that 25 out of 46 CF patients had strains hyper-susceptible to ticarcillin due to deficiencies in MexAB-OprM efflux activity, resulting from various gene defects including reduced or abnormal expression of MexB and OprM [66]. It is unclear why this phenomenon exists. Efflux pumps do not just expel antibiotics and therefore reduced efflux may give a selective advantage under certain physiological conditions in the chronically infected lung.

Adaptations to chronic infection One of the characteristics of chronic infection with P. aeruginosa is the appearance in vitro of a variety of colony forms (morphotypes) that differ from those seen in environmental strains or those causing acute infection (fig. 1). Several different morphotypes may be found in the same sputum, even though the isolates are clonally related. These can include colonies lacking the typical pigmentation, mucoid forms, some that look like coliforms, ‘‘dwarf’’ forms and very slow growing ‘‘small colony variants’’. One of the most easily recognised is the mucoid morphotype. This results from over-production of the polysaccharide alginate, due to mutation in the regulatory genes. Hyper-alginate producers were originally thought unique to CF but they are also found in non-CF bronchiectasis and COPD [45, 49, 69]. They are thought to be an adaptation to chronic infection irrespective of the underlying cause. Alginate may protect against phagocytosis [70] and contribute to the formation of biofilms [71, 72]. Small colony variants (SCVs) have enhanced ability to form biofilms and may also contribute to persistence [73]. SCVs have only been described so far in CF but are easily missed unless cultures are prolonged.

Figure 1. Different pseudomonal morpho-

74

logical types of Pseudomonas aeruginosa found in a single sputum sample taken from a chronically infected individual.

The phenotypic changes found in chronic infection have been studied extensively in CF but not in non-CF bronchiectasis. They include loss of acute virulence factors, such as toxin production (e.g. elastase, phospholipase C, pyoverdin, hydrogen cyanide) and type III secretion [74]. Many virulence factors are regulated by the quorum sensing (QS) system. These are signalling

molecules that act on the regulators of gene transcription. Some QS molecules depend on population density, and only have their effect when the number of organisms reaches a critical concentration (or quorum). P. aeruginosa QS molecules comprise acyl-homoserine lactones and molecules of the PQS system. They can affect a large number of functions including pathogenicity, metabolic adaptation and persistence [75]. P. aeruginosa with mutations in QS genes, most frequently las R, do not respond to QS molecules and are surprisingly common, they were found in 19 out of 30 CF patients in a study by SMITH et al. [76]. Las R mutants form characteristic iridescent colonies and have also been cultured from patients with non-CF bronchiectasis (J.E. Foweraker, Papworth Hospital, Cambridge, UK; personal communication). These mutants do not produce the toxins elastase, phenazines or hydrogen cyanide. Las R mutants can use a more diverse range of compounds as a source of carbon, nitrogen, phosphorus or sulphur and have a growth advantage over the wild type when grown with phenylalanine, isoleucine or tyrosine. They therefore appear to be less pathogenic but better able to adapt to the local environment. Again, different phenotypes can co-exist so sputum may contain Las R mutants and organisms without the mutation. Longitudinal studies in CF have analysed strains from patients over several years. It is thought that with time the bacteria adapt to a form that is less virulent but better able to persist in the damaged lung [76]. Multiple phenotypic variants of the underlying clonal population of P. aeruginosa coexist and form a complex population in the chronically infected lung. This is described as ‘‘adaptive radiation’’ and is thought to give the bacteria an advantage in that they can rapidly respond to changes in the environment, as individual organisms that have the necessary adaptation may already be present in the population.

P. aeruginosa is thought to grow in biofilms in chronic infections in both CF and non-CF bronchiectasis. Biofilm formation is thought to be a general adaptation to a hostile environment and may allow persistence of infection by protecting the bacteria from the host response and the effects of antibiotics. Biofilm fragments have been seen in CF sputum [77] and may contain a mixture of P. aeruginosa plus other bacteria and even fungi, such as Candida spp. The extracellular matrix comprises alginate produced by P. aeruginosa plus proteins and DNA from other microorganisms and host cells. The biofilm contains a steep oxygen gradient and is anaerobic just below the surface. Different concentrations of nutrients and waste products will also be found in different areas of the biofilm. Therefore, the biofilm contains a wide range of physiological conditions, by which the bacteria possess a variety of adaptations that enable them to survive within these microniches [78].

J.E. FOWERAKER AND D. WAT

Biofilms

Alginate protects P. aeruginosa in biofilms from interferon (IFN)-c activated macrophages [79]. Neutrophils have been observed immobilised in the extra-cellular matrix, unable to penetrate the biofilm [80]. It is thought that neutrophils may actually enhance early biofilm formation, as biofilms formed in vitro in the presence of neutrophils are thicker and contain more bacteria [81]. If P. aeruginosa and neutrophils are combined, the bacteria aggregate around necrotic dying neutrophils. If neutrophil apoptosis is induced before the bacteria are added, the neutrophils are intact and the P. aeruginosa remain dispersed. Neutrophils can release DNA and F-actin complexed with histones and other cations, and these may form the framework for the biofilm. The combination of DNAse and anionic polymers has a synergistic effect in clearing early neutrophil-associated biofilms in vitro and is being studied as a potential treatment to prevent or disrupt early biofilm formation [81]. Neutrophil lysis is thought to be caused by rhamnolipid, a toxin produced by P. aeruginosa under QS control. Rhamnolipid may therefore help to protect the biofilm from disruption by neutrophils, especially in the early stages of formation [82].

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Azithromycin is a macrolide antibiotic that does not directly inhibit or kill P. aeruginosa, but it can block QS and alginate polymer formation in vitro [83]. It can disrupt early biofilms formed by nonmucoid strains but has less effect on early biofilms formed by hyper-alginate producers

MICROBIOLOGY

(mucoid strains) or on established biofilms [84]. Azithromycin also has an anti-inflammatory effect in chronic lung infection and the relative importance of its diverse actions is yet to be established. Other antibiotics may also influence bacterial virulence. For example ciprofloxacin can suppress alginate biosynthesis at concentration well below minimum inhibitory concentration (MIC) [85]. Bacteria cultured in biofilms in vitro are more resistant to most antibiotics than when they are dispersed (planktonic). Several mechanisms have been proposed to explain this resistance [86]. It was thought that the extra-cellular matrix formed a physical barrier but there are channels within the biofilm through which most antibiotics can permeate. Positively charged antibiotics such as colistin may bind to free anionic DNA and therefore not reach the bacteria, and an anionic antibiotic, such as an aminoglycoside (e.g. tobramycin) may bind to the alginate. If the AmpC b-lactamase is over produced by some P. aeruginosa it may form a high local concentration and protect bacteria that can only produce basal levels of the enzyme. Mutability is increased in biofilms, partly because of the presence of hypermutators but also because DNA can be damaged by the increased amounts of reactive oxygen species within the biofilm. The range of metabolic conditions in the biofilm may affect antibiotic susceptibility. P. aeruginosa can survive in the anaerobic environment just below the surface of the biofilm by using nitrogen rather than oxygen as a terminal electron acceptor and aminoglycosides, such as tobramycin, cannot act on organisms that are metabolising anaerobically. Organisms within a biofilm may become dormant and therefore resist quinolones and b-lactam antibiotics [87]. These affects have been shown in an in vitro model of a young biofilm in a flow chamber using live/dead staining. Ciprofloxacin kills organisms on the surface of the biofilm but cannot kill those deeply set within the biofilm, whereas colistin can kill the non-dividing cells in the centre [88]. The two antibiotics appear to be very effective against young biofilms in vitro and may explain why that combination is particularly effective in eliminating early infection with P. aeruginosa in CF.

Hypermutators One of the drivers of variability and adaptation seen in persistent infection in bronchiectasis is thought to be the presence of hypermutator (HM) bacteria [89]. These are P. aeruginosa with a higher than usual spontaneous mutation rate and are thought to accelerate bacterial evolution. P. aeruginosa usually mutates at a frequency of one in 108–109, while mutation rates in HM bacteria can be as high as one in 100. HM P. aeruginosa were found in 37% of chronically infected CF patients. This was the highest prevalence that had been described for a naturally occurring population [90]. In comparison a HM prevalence of 1% in Escherichia coli and Salmonella spp. had previously been considered high [91]. In a longitudinal study of CF patients in Denmark, none of the bacteria from early infections were found to be HMs but after 20 years of colonisation 65% of patients were infected with HM P. aeruginosa [92]. HM P. aeruginosa were described in 57% of chronically infected patients with COPD or non-CF bronchiectasis, suggesting that hypermutability is a general adaptation to long-term survival in the lung [93]. KENNA et al. [94] suggests that hypermutability is an extremely rare finding in environmental P. aeruginosa and in isolates from newly infected CF patients. Most of the information on HM P. aeruginosa comes from work on isolates from CF [95]. Hypermutability usually results from a primary mutation in genes of the MMR system, most commonly mutS and mutL, or defects in the GO system (mut M, Y and T). The function of these systems is to detect and repair DNA replication errors and repair oxidative damage. MMR also inhibits recombination between moderately diverged sequences and therefore reduces the acquisition of exogenous DNA through horizontal gene transfer [96].

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HMs are uncommon in most bacterial populations because many of the mutations are deleterious. HM P. aeruginosa had reduced virulence and fitness both in vitro and in an animal model [97, 98]. However, in changing environments or stressful conditions HM bacteria may be selected because they have adaptive mutations, such as antimicrobial resistance (referred to as ‘‘hitchhiking’’).

The sequential acquisition of resistance to multiple antibiotics is seen in infection with P. aeruginosa in CF, and several studies in CF, non-CF bronchiectasis and COPD have shown that HM are more likely to be antibiotic resistant than isolates with normal mutation rates [93, 99]. In a study of 29 CF patients over a 5-year period, mutations accumulated at an average mutations rate of three per year in HM P. aeruginosa compared with 0.25 per year in non-mutators. HM had more mutations leading to antibiotic resistance but also more mutations in other genes such as lasR [89]. Therefore, other adaptations may provide a selective advantage for HM isolates, not just antibiotic resistance. Two recent studies have shown that CF patients with HM had poorer lung function (FEV1 predicted), but longitudinal studies are needed to determine if this was due to infection with a HM or just an association, both being the result of prolonged infection [99, 100]. Work is needed on the role of HM in non-CF bronchiectasis.

Chronic P. aeruginosa infection and the clinical microbiology laboratory One practical implication of the range of phenotypic diversity of P. aeruginosa from non-CF bronchiectasis is that some isolates may be difficult to identify. Colonies of P. aeruginosa from chronic infection may lack pigmentation, grow very slowly and may mimic other species.

Another consequence of phenotypic diversity is that a range of antimicrobial susceptibility patterns can be found in a population of P. aeruginosa in a single sputum sample (fig. 2). Bacteria with the same morphotype may have different susceptibility and therefore resistant subpopulations may be missed, depending on which colony is picked for testing [68]. In CF, once a chronic infection is established the range in the antibiotic susceptibility of P. aeruginosa in a single sputum is so diverse that susceptibility testing methods are unreliable [102, 103]. It is currently unclear whether these findings can equally be applied to chronic infection in non-CF bronchiectasis. Finally it has been questioned whether current methods used for testing antimicrobial susceptibility are relevant for bacteria that may be present in biofilms in the chronically infected lungs. A variety of methods are being developed for testing biofilm susceptibility; however, their clinical relevance still needs to be determined.

J.E. FOWERAKER AND D. WAT

Commercial identification schemes that use biochemical reactions and assimilation tests are not reliable in identifying atypical P. aeruginosa and some of the other nonfermenting Gram-negative bacilli found in chronic infection, and therefore identification methods, such as species-specific PCR or sequencing of the 16S ribosomal RNA gene may be required [101].

Streptococcus pneumoniae

Figure 2. Variation in antimicrobial susceptibility testing. The figure shows the results from testing two bacteria with identical colony form in sputum taken from a patient with non-cystic fibrosis bronchiectasis. The same six different antimicrobial discs were used for both cultures. The susceptibility is proportional to the diameter of the zone of diffusion around the antibiotic disc.

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S. pneumoniae is a Gram-positive coccus appearing in pairs and in short chains. It may be a harmless commensal in the oro-pharynx but can cause severe and invasive disease (pneumonia or meningitis). It can also cause otitis media or sinusitis, or lower airway infections in patients with damaged lungs such as non-CF bronchiectasis or COPD, but it is rare in CF. Although S. pneumoniae can be found in up to 37% of patients with non-CF

bronchiectasis, very little has been published on its role in this condition. In COPD, S. pneumoniae has been cultured from both stable patients and those with exacerbation [20, 104]. The patient with non-CF bronchiectasis due to an underlying antibody deficiency may be particularly susceptible to recurrent infections with S. pneumoniae [105]. Bronchiectasis in primary and secondary immunodeficiency patients is discussed further in the chapter by BROWN et al. [106]. S. pneumoniae has a polysaccharide capsule that helps evade opsonisation, and isolates lacking the capsule are avirulent. There are over 90 capsule types and the capsule type may be one of several factors that determine the pathogenicity of an individual strain [107]. A polyvalent vaccine containing the most common serotypes is available and recommended for use in patients with chronic lung disease. S. pneumoniae can use a wide variety of molecules to adhere to host cells and produces an IgA protease and a toxin, pneumolysin that can promote invasion, inflammation and tissue damage [108]. Pneumolysin is proinflammatory and has many actions including cytolysis, inhibition of cilial beating, and direct activation of the classical complement cascade. Although it is not a common pathogen in CF, isolates of S. pneumoniae from CF sputum have characteristics that may be associated with adaptation to persistence in the lung, i.e. hypermutability and the ability to form biofilms [109, 110]. Further work is needed to clarify the role of the different virulence factors in order to understand why S. pneumoniae may be a harmless commensal or cause noninvasive respiratory tract infection (in COPD or bronchiectasis) or produce severe invasive disease with bacteraemia.

MICROBIOLOGY

The prevalence of antibiotic resistant S. pneumoniae has increased and in some countries very high rates of resistance to penicillin, macrolides and tetracyclines limit the treatment options. Penicillin resistance is due to modifications to penicillin binding proteins not by the production of a b-lactamase and, therefore, amoxicillin–clavulanate is ineffective.

Moraxella catarrhalis M. catarrhalis is a Gram-negative diplococcus that was previously named Branhamella or Neisseria catarrhalis. Like NTHi it is a common commensal organism in the upper respiratory tract and can cause otitis media or sinusitis. It was not reported in studies of non-CF bronchiectasis in the 1960s as it was considered an oral contaminant rather than a PPM. However, it can be cultured in significant numbers from sputum or PSB in up to 27% of patients with non-CF bronchiectasis [7]. It is also considered a significant pathogen in COPD but is only rarely isolated in CF. A longitudinal study of M. catarrhalis in 29 patients with non-CF bronchiectasis found that patients were colonised with a variety of strains with average colonisation duration of 2.3 months for each strain. No association between strain acquisition and exacerbation was found and as M. catarrhalis was often in mixed culture with other PPMs (H. influenzae or S. pneumoniae), it was difficult to determine whether it had an independent pathogenic role [111]. In a study of 50 patients with COPD, the average time from acquisition to clearance of a new strain of M. catarrhalis was 1 month and re-infection with the same strain was rare, suggesting that there was an effective immune response. Of the new acquisitions, 47% were associated with an exacerbation [112]. Acquisition of M. catarrhalis led to an increase in airway inflammation, characterised by a rise in sputum neutrophil elastase, interleukin (IL)-8, tumour necrosis factor (TNF)-a and a reduction in secretory leukocyte protease inhibitor (SLPI) [113]. Putative virulence factors of M. catarrhalis include several outer membrane proteins plus LOS and these affect cell adhesion, epithelial cell invasion, serum resistance and biofilm formation [114]. More work is needed to understand the pathogenesis of infection in both COPD and non-CF bronchiectasis.

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More than 90% of M. catarrhalis produce a b-lactamase (BRO-1 or BRO-2) and are resistant to ampicillin. Acquired resistance to other antibiotics is rare with most remaining susceptible to macrolides, tetracyclines, amoxicillin-clavulanic acid and quinolones [115].

Staphylococcus aureus S. aureus is a Gram-positive coccus found in clusters that may be part of the normal flora in the anterior nares, throat and on moist skin sites such as groin and axilla. Infection is characterised by abscess formation, particularly in skin and soft tissues. It is a rare cause of respiratory tract infection, but can cause severe pneumonia after influenza. It is a common cause of early infection in CF but is less common in non-CF bronchiectasis where its presence may indicate undiagnosed CF [10]. There is also an association of S. aureus with ABPA in non-CF bronchiectasis [116].

The ability of S. aureus to rapidly adapt and persist in the lung may be a result of genomic instability due to mobilisation of bacteriophages. Isolates from the anterior nares of CF patients had a higher frequency of genomic alterations than those from healthy controls [120]. A higher proportion of hypermutable strains of S. aureus were found in CF patients when compared with isolates from bacteraemia or other respiratory infections. As with other species with high mutation rates, many of these had defects in mutS [121]. Meticillin resistant S. aureus (MRSA) are resistant to all penicillins, cephalosporins and penems and are often also resistant to other classes of antibiotics (macrolides, fluoroquinolones and aminoglycosides). They can be difficult to treat, partly because oral options are limited but also because the active parenteral options (glycopeptides) may be less effective compared with the use of a b-lactam antibiotic to treat a susceptible isolate. It may be difficult to clear MRSA carriage from patients with bronchiectasis, but there is data from CF that shows that a combination of systemic treatment with skin antisepsis and inhaled antibiotics may be effective [122].

J.E. FOWERAKER AND D. WAT

S. aureus produces a range of exotoxins that can cause tissue damage. It is also thought to form biofilms on prosthetic devices and thereby evade the host response and resist antimicrobial therapy [117]. Biofilm-like aggregates of S. aureus surrounded with the polysaccharide poly-Nacetyl-glucosamine have been observed in anaerobic conditions in CF mucus and can resist nonoxidative killing [118]. Persistence of S. aureus in CF and prosthetic infections has also been related to the presence of small colony variants. These tiny colonies are difficult to identify in vitro. They are associated with treatment with trimethoprim/sulphamethoxazole or aminoglycosides, are more antibiotic resistant than the typical forms in the same sputum and may survive within host cells [119].

Burkholderia spp. and other non-fermenters Burkholderia spp are plant pathogens and are a major cause of morbidity and mortality in CF but are rarely encountered in other conditions. In spite of the frequent presence of these bacteria in the environment, and their propensity for spread between CF patients, there are only two case reports of infection in non-CF bronchiectasis, one with Burkholderia cepacia complex (not speciated) and another with Burkholderia gladioli [123, 124].

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A wide variety of other nonfermentative Gram-negative bacilli can occasionally act as opportunistic pathogens in the human lung. Species of the genera Achromobacter, Stenotrophomonas, Ralstonia, Pandoraea and Inquilinus can cause infection in the CF lung, and S. maltophilia and Achromobacter (previously Alkaligenes) xylosoxidans have been reported in non-CF bronchiectasis (table 1). Many are both intrinsically resistant to some antibiotics and easily acquire resistance. They can be difficult to identify in the laboratory and molecular methods are recommended to ensure accurate identification [101]. In particular it is important to differentiate these organisms from the Burkholderia spp. because of the need to prevent cross infection. There is too little experience with these microorganisms to comment on their propensity for colonisation, infection, or role in exacerbation of non-CF bronchiectasis.

Anaerobes and other bacteria considered normal upper respiratory tract flora Sputum may contain microorganisms other than the PPM. These have been considered either contaminants from the upper respiratory tract or harmless commensals colonising the sputum. This assumption has been challenged following studies using both conventional culture methods and culture-independent techniques. Sputum is not routinely cultured for anaerobes partly because they are present in large numbers in saliva and can easily contaminate expectorated sputum, but also because some are very difficult to culture. Following the observation of a rapid drop in oxygen partial pressure just below the surface of a CF sputum plug, investigators began to look for anaerobes in the sputum from CF and nonCF bronchiectasis [125]. Obligate anaerobes in particular Prevotella spp. were found in significant numbers, far more than would be expected from oral contamination [126–128]. Their significance in disease has been questioned as high numbers of bacteria were found in a stable CF patient in one study, and the numbers of anaerobes remained constant during successful treatment of a clinical exacerbation [129].

MICROBIOLOGY

It has been proposed that members of the Streptococcus milleri group may have a role in chronic lung infection. One study followed the changes in the microbial flora during and between pulmonary exacerbations of CF using both culture and culture-independent methods. The group identified members of the S. milleri group as of potential importance in exacerbations both in CF and in two patients with non-CF bronchiectasis [130]. Following an observation that a range of upper respiratory tract flora were seen in large numbers in sputum from CF patients, a Staphylococcus sp. (not S. aureus) and a viridans-type Streptococcus sp. were further studied. While not intrinsically pathogenic, they were able to enhance the virulence of P. aeruginosa in an animal model and increase the expression of certain virulence genes of P. aeruginosa in vitro. This could be reproduced using an inter-species QS molecule, Auto Inducer-2 (AI-2) [131]. Of interest, the oral anaerobe Prevotella also produces AI-2 [127]. The complex pattern of interaction between microorganisms in ecosystems other than the lung has been described and it is known that microorganisms can enhance or inhibit growth of other cohabitants [132]. The studies in CF show that interactions may also enhance pathogenicity [133]. The CF lung, therefore, may contain a mixture of microorganisms that includes those that are directly pathogenic, those that behave as commensals and those that are not directly pathogenic but may increase the virulence of other organisms. Although this has not been studied in non-CF bronchiectasis, microorganisms other than PPMs are regularly observed in sputum cultures in combination with PPMs and further work on these potential interactions is needed.

Culture-independent studies of microbial flora in the lung

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There have been attempts to describe the composition and diversity of the microbes in the lung irrespective of the ability to culture individual microorganisms. One approach is to analyse the gene encoding 16S rRNA. This is present in all true bacteria and the sequence variation is sufficient to identify most genera and many species. Genetic material can be extracted from a clinical sample, and the 16S rRNA gene amplified by PCR. The product may be analysed looking at terminal restriction fragment length polymorphisms (TRFLP). This compares the size of the terminal fragment of rRNA after cutting with a restriction enzyme; the length of the fragment being characteristic of certain species. Alternately the PCR product can be cloned, sequenced and compared with databases containing sequence data from a wide range of microorganisms. These methods and other variations have been applied to patients with CF and non-CF bronchiectasis to describe the diversity of microorganisms, and have revealed species not previously found in respiratory samples using traditional culture methods [12, 134–136]. While the presence of nucleic acid does not necessarily indicate the presence of viable organisms, a comparison of RT-TRFLP

with TRFLP showed that a high proportion of the bacterial species detected in CF sputum were metabolically active [137]. There have been major technical advances facilitated by the development of next generation sequencers plus developments in bioinformatics. These have allowed direct analysis of amplified DNA without the cloning step, and greater depth of sequencing of 16S rRNA DNA [138, 139]. An alternative approach is to attempt whole genome sequencing directly from the clinical sample [140]. This could include analysis of nucleic acid from eukaryotes and viruses in sputum as well as bacteria [141]. Such techniques can provide an enormous amount of information that is a great challenge to process. However, they offer the potential for a far more sophisticated analysis of the genetic variability found in single species and the variety of microorganisms in chronic lung infection.

Respiratory viruses The role of viruses in non-CF bronchiectasis is not known and remains an important area for future research. Some data exists that viral infections in childhood may predispose to the development of bronchiectasis in later life [142], whether it is through the development of bronchiolitis, disruption of small airway associated innate/adaptive immunity, damage of airway epithelia or compromise of mucociliary clearance, it is unclear.

Viral infections in asthma, COPD and CF Viral exacerbation of asthma has been well published. In a study by JOHNSTON et al [143] using PCR and viral culture, viruses were detected in 80% of episodes of wheeze or reduced peak expiratory flow in children aged 9–11 years with asthma. Rhinovirus accounted for 61% of the viruses detected, coronavirus 16%, influenza 9%, parainfluenza 9% and respiratory syncytial virus (RSV) 5%. Similarly, NICHOLSON et al. [144] found that respiratory viruses accounted for 44% of asthma exacerbations in adults. Respiratory viruses were also present in most patients hospitalised for life-threatening asthma and acute not life-threatening asthma [145]. The application of molecular diagnostic methods has improved the understanding of viral epidemiology. Respiratory viruses may induce asthma exacerbations via direct effects on the airway epithelium as well as through a systemic immune reaction.

J.E. FOWERAKER AND D. WAT

What is also unclear is the role that viral infection plays in triggering infective exacerbations and progressive lung damage in patients with non-CF bronchiectasis where no studies have to date been carried out. Therefore, only cautiously can parallels be drawn from studies examining the role of viruses in asthma, COPD and CF.

Rhinovirus is the most common respiratory virus and represents two-thirds of all upper respiratory tract infections. It also accounts for 50% of asthma exacerbations in children [146]. Traditionally, rhinovirus is thought to infect the upper respiratory epithelium. However, rhinovirus is also capable of replicating in the lower airway cells during experimental infection [147]. PAPADOPOULOS et al. [148] showed that both rhinovirus genomic material and replicative strand RNA were detectable in bronchial biopsies using in situ hybridisation in 50% of adult volunteers subjected to an experimental rhinovirus upper respiratory infection.

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The mechanism by which viruses cause bronchoconstriction is not fully understood, but it is likely to involve cytokine production in response to viral replication in the lower airways, which includes upregulating the expression of a range of proinflammatory mediators. The proinflammatory cytokine IL-1b is detectable in experimental infected individuals. IL-8, a key mediator in neutrophil-mediated acute inflammation, is also detected in naturally occurring infections correlating with neutrophilia in blood and nasal samples in children with virally precipated asthma or experimental infection [149]. Other mediators induced by rhinovirus infections include neutrophil-activating peptide (which induces neutrophil migration), eotaxin and RANTES

(regulated on activation, T-cell expressed and secreted), IL-16 and monocyte chemoattractant protein (MCP-1). All of these can lead to enhance airway inflammation. RSV was detected in only 5% of asthma episodes in the study by JOHNSTON [143]. However, it is known to be a potent cause of wheezing, particularly in infancy. It has been shown that Gglycoprotein of RSV appears to stimulate T-helper cell (Th) type 2 immune response in the upper airway, whether or not if the infant is atopic [150]. Th2 cytokine patterns are known to be associated with viral immunopathology and allergic-type responses, in contrast to Th1 cytokine patterns, which are classically associated with viral elimination. Interestingly, the nasal cytokine responses to other viruses are of the predominant Th1 type (except RSV). This could explain the tendency for RSV to cause wheezing, but not the association between other respiratory viruses and wheezing.

MICROBIOLOGY

Influenza A infection induces large amounts of intrapulmonary IFN-c and enhances both later allergen specific asthma and dual Th1/Th2 responses [151]. TERAN et al. [152] also demonstarted that the eosinophil product, major basic protein (MBP), and RANTES increased with viral infections, and there was a correlation in the concentration of RANTES with clinical symptoms. In addition, epithelial cells infected with influenza in vitro were associated with an increase in eotaxin [153]. Eotaxin can in turn lead to an exaggerated inflammatory response by being an agonist for chemokine receptor 3, which can be found on eosinophils, T-cells and basophils. These are all key factors in asthma exacerbation. Patients with asthma are no more susceptible to upper respiratory tract rhinovirus infections than healthy people, but suffer from more severe consequences of the lower respiratory tract infection. Recent epidemiological studies suggest that viruses provoke asthma attacks by additive or synergistic interactions with allergen exposure or with air pollution. An impaired antiviral immunity to a rhinovirus may lead to impaired viral clearance and hence prolonged symptoms. Indirect prevention strategies focus on the reduction of overall airway inflammation to reduce the severity of the host response to respiratory viral infections. There is a lack of specific antiviral strategies in the prevention or reduction of viral-triggered asthma exacerbations. Recent advances in the understanding of the epidemiology and immunopathogenesis of respiratory viral infections in asthma may provide opportunities or the identification of specific targets for antiviral agents and strategies for management and prevention. COPD is the fourth leading cause of mortality worldwide and is an important cause of global burden of disease [154]. The disease is associated with intermittent exacerbations characterised by acute deterioration in symptoms, lung function, and quality of life [155, 156]. Exacerbations have major effects on health status and are associated with considerable morbidity and mortality that can lead to hospital admission with high treatment costs [157]. Infectious agents are recognised as a major pathogenic factor in exacerbations. Bacteria have a role in the pathogenesis [158, 159] and the exacerbations of COPD. However, bacteria are absent in about 50% of exacerbations and the frequency of isolation does not increase during exacerbation [160].

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Early studies looking at respiratory viruses and COPD have stated a 20% detection rate in COPD exacerbations [161, 162]. However, these studies were limited by using less sensitive methods in viral detection. SEEMUNGAL et al. [163] detected respiratory viruses from nasal samples and blood of patients with COPD using a combination of culture, serology and PCR. They showed that 64% of COPD exacerbations were associated with a cold occurring up to 18 days before exacerbation. In total, there were 168 episodes of COPD exacerbation in 53 patients and 77 viruses (39 were rhinoviruses) were detected. Viral exacerbations were associated with frequent exacerbatons, increased symptoms, a longer median symptom recovery period (up to 13 days) and a tendency towards higher plasma fibrinogen and serum IL-6 levels. RSV has also been shown to be an important virus in COPD exacerbations and was detectable in 11.4% of patients admitted into hospital [164]. Patients with stable COPD may carry respiratory viruses. Non-RSV respiratory viruses were detected in 11 (16%), and RSV in 16 (23.5%) out of 68 stable COPD patients, with RSV detection being associated with higher inflammatory marker levels [161, 164].

Early studies looking at respiratory viruses in CF relied on repeated serological testing, either alone [165], or in combination with viral cultures for viral detection [166–170]. These methods are relatively insensitive and more recent studies have utilised molecular based methodologies [171–175]. All these studies produced different results in terms of prevalence of respiratory viruses in CF, these differences could be due to the different methodologies utilised. It is also likely that there are differences in the populations studied, as the prognosis for CF has improved with each successive birth cohort.

More recent studies suggest no difference in the frequency of either upper respiratory tract illness episodes [166] or proven respiratory viral infections [168] between children with CF and healthy controls; however, children with CF have significantly more episodes of lower airway symptoms than controls [166, 168]. RAMSEY et al. [168] prospectively compared the incidence and effect of viral infections on pulmonary function and clinical scores in 15 school children with CF aged 5–21 years and their unaffected siblings. Over a 2-year period, samples were taken at regular 2-month intervals and during acute respiratory illnesses for pharyngeal culture and serology for respiratory viruses. There were a total of 68 acute respiratory illness (ARI) episodes that occurred in the patients with CF, in 19 of these episodes an associated virus identified. A total of 49 infective agents were identified either during ARIs or at routine testing in the patients with CF; 14 were identified on viral isolation (rhinovirus on 11 occasions), whilst 35 were isolated on seroconversion (parainfluenza virus on 12, RSV on nine and Mycoplasma pneumoniae on six occasions). There was no significant difference in the rate of viral infections between the patients with CF and their sibling controls, as measured either by culture or serology. The rate of viral infections was higher in younger children (both CF and controls), and the rate of decline in pulmonary function was greater in the younger children with CF with more viral infections. At the time of an ARI, the virus isolation and seroconversion (four-fold increase in titres) rates were 8.8% and 19.1%, respectively, in children with CF compared with 15% and 15%, respectively, for the siblings not affected. In contrast the rates for virus isolation and seroconversion at routine 2 monthly visits were 5.6% and 16.2%, respectively, for children with CF and 7.7% and 20.2%, respectively, for the siblings not affected.

J.E. FOWERAKER AND D. WAT

It has now been 25 years since WANG et al. [169] described the relationship between respiratory viral infections and the deterioration in clinical status in CF patients. Viruses were identified through repeated serology and nasal lavages for viral isolation in 49 patients with CF (mean age 13.7 years) over a 2-year period. Although the CF patients had more respiratory illnesses than the sibling controls (3.7 per year versus 1.7 per year), there were no differences in virus identification rates (1.7 per year). The rate of proven virus infection was significantly correlated with the decline in forced vital capacity (FVC) and FEV1, Shwachman score, and frequency and duration of hospitalisation.

Similarly HIATT et al. [166] assessed respiratory viral infections over three winters in 22 infants less than 2 years of age with CF (30 patient seasons) and 27 age matched controls (28 patient seasons). The average number of acute respiratory illness per winter was the same in the control and the CF groups (5.0 versus 5.0). However, only four of the 28 control infants had lower respiratory tract symptoms in association with the respiratory tract illness, compared with 13 out of the 30 infants with CF (OR -4.6, 95% CI 1.3–16.5; p-value ,0.05). Seven of the infants with CF cultured RSV, of whom three required hospitalisation. In contrast, none of the controls required hospitalisation. Pulmonary function measured by rapid chest compression technique was significantly reduced in the infants with CF after the winter months and was associated with two interactions; RSV infection with lower respiratory tract infection and male sex with lower respiratory tract infection.

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From previous reports, two viral agents appear to have the greatest effect on respiratory status in CF, namely RSV and influenza, possibly because the uses of viral culture and serology have underestimated the effects of rhinovirus (due to the vast amount of serotypes). In younger children, RSV is a major pathogen resulting in an increased rate of subsequent hospitalisation. ABMAN et al. [176] prospectively followed up 48 children with CF diagnosed through newborn

screening and documented the effect of RSV infection. 18 of the infants were admitted into hospital a total of 30 times over a mean follow-up period of 28 months (range 5–59 years). In seven of these infants RSV was isolated, and their clinical course was severe with three requiring mechanical ventilation and five necessitating chronic oxygen therapy. Over the next 2 years these infants had significantly more frequent respiratory symptoms and lower chest radiograph scores than non-RSV identified infants. In another prospective study of repeated BAL in 80 infants identified through CF newborn screening over a 5-year period, 31 infants were hospitalised for a respiratory exacerbation, 16 (52%) of which had a respiratory virus identified with the most common being RSV (n57).

MICROBIOLOGY

In older children and adults with CF, influenza seems to have the greatest effect. PRIBBLE et al. [167] assessed acute pulmonary exacerbation isolates from 54 patients with CF. Over the year of the study 80 exacerbations were identified, of which 21 episodes were associated with an identified viral agent (influenza A: five episodes; influenza B: four episodes; RSV: three episodes) with most agents identified by serology. Compared with other agents, infection with influenza was associated with a more significant drop in pulmonary function (FEV1 decreased by 26% compared with 6%, respectively). A retrospective study in older patients with chronic P. aeruginosa infection reported an acute deterioration in clinical status in association with influenza A virusl infection [177]. COLLINSON et al. [171] followed 48 children with CF over a 15-month period using a combination of viral culture and PCR for picornaviruses alone [178]. 38 children completed the study and there were 147 symptomatic upper respiratory tract infections (2.7 episodes per child per year), with samples available for 119 episodes. Picornaviruses were identified in 51 (43%) of these episodes, of which 21 (18%) were rhinoviruses. In those children old enough to perform spirometry there were significant drops in both FVC and FEV1 in association with upper respiratory tract infection, with little difference in the severity of drop whether a picornavirus was identified or not. Maximal mean drop in FEV1 was 16.5%, at 1–4 days after onset of symptoms, but a deficit of 10.3% persisted at 21–24 days. Those with more upper respiratory tract infections appeared to have a greater change in total Shwachman and Crispin–Norman scores over the study. Six children isolated a P. aeruginosa for the first time during the study, five at the time of a upper respiratory tract infection and only one was asymptomatic at the time of first isolation. The data from this study has to be handled with care as the term ‘‘upper respiratory tract illness (URTI)’’ did not necessarily imply a positive viral isolation. PUNCH et al. [173] used a multiplex RT-PCR assay combined with an enzyme-linked amplicon hybridisation assay (ELAHA) for the identification of seven common respiratory viruses in the sputum of 38 CF patients. 53 sputum samples were collected over two seasons and 12 (23%) samples from 12 patients were positive for a respiratory virus (influenza B n54, parainfluenza 1 n53, influenza A n53, RSV n52). There were no statistical associations between virus status and demographics, clinical variables or isolation rates for P. aeruginosa, S. aureus or Aspergillus fumigatus. OLESEN et al. [174] obtained sputum/laryngeal aspirated from children with CF over a 12-month period in outpatient clinics. They achieved a viral detection rate of 16%, with rhinovirus being the most prevalent virus. However, this virus did not seem to have any devastating impact on lung function. However, the other viruses detected were associated with significant reduction in lung function. The authors failed to show a positive correlation between respiratory viruses and bacterial infections in their studied population, as the type or frequency of bacterial infection during or after viral infections were not altered. They also demonstrated that clinical viral symptoms had a very poor predictive value (0.39) for a positive viral test.

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WAT et al. [179] utilised ‘‘real-time’’ nucleic acid sequence-based amplification (NASBA) to examine the role of respiratory viruses in CF. They achieved a rate of 46% for respiratory viruses in their paediatric CF cohort during reported episodes of respiratory illness. The results compare favourably with previous studies, this may be due to earlier studies relying heavily on repeated serological testing either alone [165] or in combination with viral isolation [166–170]. These traditional

methods are relatively insensitive and once again may have underestimated the prevalence of viruses in CF.

Detection of respiratory viruses The principal laboratory methods utilised for the diagnosis of respiratory viruses, rely upon the detection of the virus in respiratory secretions and therefore an important factor in respiratory viral diagnosis is the necessity for the submission of an appropriate sample for testing. Inadequate or improper specimen collection and transport account for the largest source of error in the accuracy of viral detection results [180]. Nasal swabs, nasopharyngeal aspirates, nasal wash and sputum specimens are generally considered as the specimens of choice for the detection of respiratory viruses [173, 180–183]. A prospective study by HEIKKINEN et al. [184] showed that the sensitivity of nasal swabs was comparable to nasopharyngeal aspirates for the detection of all major respiratory viruses by tissue culture, with the exception of RSV. Molecular techniques have superseded many ‘‘conventional’’ methods utilised for respiratory viral detection, such as viral culture and serology analysis, due to the rapid turn-around time for the results. Molecular assays have particular advantages where the starting material available is acellular (swab) or where surveillance samples have a low copy number of the viral target. The rapid turn-around time of results allows diagnostic virology to have an impact on patient management, thereby avoiding prescribing the inappropriate use of antibiotics and allowing the correct prescription for anti-virals. It may also play an important role in infection control in the hospital setting.

There is very little known about the interaction between respiratory viruses and bacteria in non-CF bronchiectasis but a number of publications suggest that respiratory viruses may precipitate secondary bacterial infection in CF. In a 25-year retrospective review from the Danish CF clinic, the most likely first isolation of P. aeruginosa was found to be occur between October and March [185], coinciding with the peak of the RSV season. This observation implies a causal relationship between respiratory viral and bacterial infection. The first bacterial isolation of a given organism in CF has also been shown to often follow a viral infection. In the 17-month prospective study reported by COLLINSON et al. [171], five of the six first isolations of P. aeruginosa were made during the symptomatic phase of an upper respiratory tract infection or 3 weeks thereafter. In contrast only one of the six initial infections with P. aeruginosa was identified during the asymptomatic period. Similarly, H. influenzae was recovered for the first time from three children within 3 weeks of an upper respiratory tract infection and the one new S. aureus infection was identified immediately following a viral infection.

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Interaction between bacteria and viruses

ARMSTRONG et al. [170] have reported that 50% of CF respiratory exacerbations requiring hospitalisation are associated with the isolation of a respiratory virus. In their prospective study of repeated BAL in infants over a 5-year period, a respiratory virus was identified in 52% of the infants hospitalised for a respiratory exacerbation, most commonly RSV. 11 of the 31 hospitalised infants (35%) acquired P. aeruginosa in the subsequent 12–60-month follow-up period, compared with three out of 49 (6%) non-hospitalised infants (relative risk 5.8).

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Respiratory viruses can disrupt the airway epithelium and precipitate bacterial adherence. For example influenza A infection results in epithelial shedding to basement membrane with submucosal oedema and neutrophil infiltrate [186], while both influenza and adenovirus have a cytopathic effect on cultured nasal epithelium leading to the destruction of the cell monolayer [187]. This epithelial damage results in an increase in the permeability of the mucosal layer [188, 189], possibly facilitating the bacterial adherence. Bacteria can also utilise viral glycoproteins and other virus-induced receptors on host cell membranes as bacterial receptors, in order to adhere to virus infected cells [190, 191].

The lower respiratory tract is protected by local mucociliary mechanisms that involve the integration of the ciliated epithelium, periciliary fluid and mucus. Mucus acts as a physical and chemical barrier onto which particles and organisms adhere. Cilia lining the respiratory tract propel the overlying mucus to the oropharynx where it is either swallowed or expectorated. Influenza viral infection has been shown to lead to the loss of cilial beat, and shedding of the columnar epithelial cells generally within 48 hours of infection [192]. PITTET et al. [193] showed that a prior influenza infection of tracheal cells in vivo does not increase the initial number of pneumococci found during the first hour of infection, but it does significantly reduce mucociliary velocity, and thereby reduces pneumococcal clearance during the first 2 hours after pneumococcal infection at both 3 and 6 days after an influenza infection. The defects in pneumococcal clearance were greatest 6 days after an influenza infection. Changes to the tracheal epithelium induced by influenza virus may increase susceptibility to a secondary S. pneumoniae infection by increasing pneumococcal adherence to the tracheal epithelium and/or decreasing the clearance of S. pneumoniae via the mucociliary escalator of the trachea, and thus increasing the risk of secondary bacterial infection.

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DE VRANKRIJKER et al. [194] showed that mice that were co-infected with RSV and P. aeruginosa had a 2,000 times higher CFU count of P. aeruginosa in the lung homogenates compared with mice that were infected with P. aeruginosa alone. Co-infected mice also had more severe lung function changes. These results suggest that RSV can facilitate the initiation of acute P. aeruginosa infection.

RSV has also been shown to increase adherence of NTHi and S. pneumoniae to human respiratory epithelial cells in vitro [195]. This increase adherence could be explained by an upregulation of cell surface receptors for bacteria, such as intercellular adhesion molecule-1 (ICAM-1), carcinoembryonic adhesion molecule 1 (CEACAM1) and platelet activating factor receptor (PAFr). Another study also showed that NTHi and S. pneumoniae bind to both free RSV virions and epithelial cells transfected with cell membrane-bound G protein, but not to secreted G protein. Pre-incubation with specific anti-G antibody significantly reduce bacterial adhesion to G protein-transfected cells [196]. STARK et al. [197] showed that mice that were exposed to RSV had significantly decreased S. pneumoniae, S. aureus or P. aeruginosa clearance between 1 to 7 days after RSV exposure. Mice that were exposed to both RSV and bacteria had a higher production of neutrophils induced peroxide, but less production of myeloperoxidase compared with mice that were exposed to S. pneumoniae alone. This suggests that functional changes in the recruited neutrophils may contribute to the decreased bacterial clearance. More recently, CHATTORAJ et al. [198] demonstrated that acute infection of primary CF airway epithelial cells with rhinovirus liberates planktonic bacteria from biofilm. Planktonic bacteria, which are more proinflammatory than their biofilm counterparts, stimulate increased chemokine responses in CF airway epithelial cells which, in turn, may contribute to the pathogenesis of CF exacerbations. Collectively, these findings suggest that respiratory viruses may lead to epithelial disruption, destruction of mucociliary escalator, increased cytokine production, neutrophil influx and increased neutrophil induced peroxide release, indirectly facilitating bacterial infection of the airway. Whether these are the mechanisms for infective exacerbations in the context of non-CF bronchiectasis remains to be seen.

Prevention and treatment of infection with respiratory viruses

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Influenza associated death is between 13,000 to 20,000 incidents per year throughout the winter months in the UK [199], though some of the deaths may be attributed to RSV. Influenza vaccines are the only commercially available vaccines against respiratory viruses. Recent vaccines contain antigens of two influenza A subtypes, strains of the currently circulating H3N2 and H1N1 (Swine flu) subtypes, and one influenza B virus. The waning of vaccine-induced immunity over time requires annual re-immunisation even if the vaccine antigens are unchanged. Influenza vaccination

is recommended to those with chronic respiratory diseases including non-CF bronchiectasis. Despite this recommendation, there is neither evidence for, nor against, routine annual influenza vaccination for children and adults with non-CF bronchiectasis from a recent Cochrane review [200]. Although there is no licensed RSV vaccine to date, prophylaxis using a humanised mouse monoclonal antibody, Palizivumab, which has been shown to reduce the rate of RSV associated hospitalisation in premature infants [201]. Amantadine has been the conventional anti-viral against of influenza. Its mode of action involves interfering with viral protein M2, thereby inhibiting the replication of influenza viruses by interfering with the uncoating of the virus inside the cell. However, it is strain specific as it is only effective against influenza A and has common side-effects such as insomnia, poor concentration and irritability. Amantadine has now been almost completely replaced by neuraminidase inhibitors (NI), except for some NI-resistant influenza.

NIs such as Zanamivir and Oseltamivir are licensed for the treatment of influenza A and B, avian flu (H5N1) and Swine flu (H1N1). They work by inhibiting the function of the viral neuraminidase protein, thus preventing the release of the progeny influenza virus from infected host cells, a process that prevents infection of new host cells and thereby halts the spread of the infection in the respiratory. Early initiation of these therapies within 48 hours from the onset of symptoms can reduce the duration of common cold symptoms by 1–2 days [202, 203]. Zanamivir has a poor oral bioavailability and intranasal application has been shown to be effective in treating experimental influenza infection, by the reduction in symptoms caused, virus shedding and the development of otitis media [204]. A phase III study is currently underway that looks at the efficacy of intravenous Zanamivir preparation. However, compassionate use of i.v. Zanamivir could be considered to treat critically ill adults and children having a life-threatening condition, due to suspected or confirmed pandemic Influenza A (H1N1) infection or infection due to seasonal Influenza A or B virus, who are not responding to oral or inhaled neuraminidase inhibitors. A recent systematic review metaanalysis showed that neuraminidase inhibitors only have modest effectiveness (Oseltamivir and Zanamivir 61 and 62%, respectively) against flu-like symptoms in previously healthy subjects [205].

Ribavarin Ribavarin, a synthetic guanosine nucleoside that has a broad spectrum of antiviral activity, is approved treatment for lower respiratory tract disease caused by RSV [206]. It can be incorporated into RNA as a base analog of either adenine or guanine, it pairs equally well with either uracil or cytosine, inducing mutations in RNA-dependent replication in RNA viruses. Controlled studies also show that the use of ribavarin is effective in reducing the clinical severity score, duration of mechanical ventilation, supplemental oxygen use and days of hospitalisation [207].

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Neuraminidase inhibitors

Macrolides Although rhinovirus is the major cause of colds, its vast amount of serotypes has made development of anti-virals against it problematic. 90% of rhinovirus serotypes gain entry into epithelial cells using ICAM-1 cellular receptors and blockade of these receptors in experimental studies have shown reduced infection severity [208]. Macrolide antibiotics, bifilomycin A1 and erythromycin, have been shown to inhibit ICAM-1 epithelial expression and hypothesis about their potential as anti-virals have yet to be proven, more clinical proof is required [209].

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Recently there has been a report regarding the use of an anti-rhinoviral agent known as Plecoranil. This anti-viral binds to a hydrophobic pocket of the VP1, the major shell protein for the

rhinoviruses, thereby preventing the virus from exposing its RNA and also prevents the virus from attaching itself to the host cell [210]. The rhinovirus 3C protease inhibitors, Ruprintrivir [211] and soluble recombinant ICAM-1 Tremacamra [212], have shown promising results but they are currently not widely available.

Conclusions The role of bacteria and viruses in non-CF bronchiectasis is not presently fully understood. Through necessity, evidence from studies in CF and COPD is used and applied to bronchiectasis. More research using both conventional microbiological techniques as well as newer molecular diagnostic approaches, is urgently required to address a number of important questions in non-CF bronchiectasis. 1) What is the cause of infective exacerbations? 2) What is the role of anaerobic bacteria and how do normal commensal bacteria interact with pathogenic bacteria? 3) How can we clear chronic infection? 4) What proportion of exacerbations is triggered by viral infection? 5) How do viruses influence bacterial behaviour in chronically infected airways? A greater understanding of bacterial communal behaviour and their interaction with epithelial cells and viruses will be critical in further developments in the management of non-CF bronchiectasis.

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Statement of interest J.E. Foweraker received a consultancy fee from Novartis Pharma AG for advice on a submission to the European Medicines Agency for licensing of Tobramycin inhaled powder and a consultancy fee from Gilead Sciences International Ltd for advice on an application to European Medicines Agency for licensing of Aztreonam lysine.

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Chapter 7

Allergic bronchopulmonary aspergillosis and other fungal diseases B. Hilvering*, J. Speirs#, C.K. van der Ent# and J.M. Beekman#

Fungal spores are ubiquitously present in the air. Inhalation of these spores by humans causes disease in susceptible patients; most prevalent are invasive aspergillosis and allergic bronchopulmonary aspergillosis (ABPA). This chapter provides an overview of the pathogenecity, clinical appearance, diagnosis and treatment of ABPA. ABPA is a hypersensitivity lung disease limited to patients with asthma or cystic fibrosis (CF) with a prevalence of 1–2% and 2–15%, respectively within these groups. It is triggered by the exposure to Aspergillus fumigatus. Although it is not clear what initiates this hypersensitivity response, polymorphisms in genes that drive innate and adaptive immune mechanisms as well as loss-of-function mutations in the CF transmembrane conductance regulator (CFTR) are associated with ABPA development. The chronic inflammatory conditions in ABPA eventually result in airway remodelling and functional impairment. The diagnosis of ABPA is based both on clinical symptoms, laboratory testing and diagnostic imaging. Treatment consists of a two tiered approach, glucocorticoids to control immunological activity and antifungal agents to suppress fungal load. Keywords: ABPA, aspergillosis, Aspergillus fumigatus, CFTR, hypersensitivity

*Dept of Pulmonology, University Medical Center Utrecht, and # Dept of Paediatric Pulmonology, Wilhelmina Children’s Hospital, University Medical Center Utrecht, Utrecht, The Netherlands. Correspondence: C.K. van der Ent, Dept of Paediatric Pulmonology, University Medical Center Utrecht, Lundlaan 6, 3584 EA Utrecht, the Netherlands, Email [email protected]

B. HILVERING ET AL.

Summary

Eur Respir Mon 2011. 52, 97–114. Printed in UK – all rights reserved. Copyright ERS 2011. European Respiratory Monograph; ISSN: 1025-448x. DOI: 10.1183/1025448x.10003710

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umans continuously inhale fungal spores. Only some fungal species cause invasive, allergic or toxic disease, most prevalent of which are invasive aspergillosis in immunocompromised patients and allergic bronchopulmonary aspergillosis (ABPA) in asthmatics and patients with cystic fibrosis (CF). This chapter provides an overview of the current knowledge concerning the

role of fungi in the pathogenesis of bronchiectasis and describes the clinical appearance, immunological background, diagnosis and treatment of ABPA. Centrally located, cylindrical bronchiectasis is a major characteristic of ABPA; however, 5–25% of patients with ABPA are diagnosed without the presence of bronchiectasis [1–3]. ABPA is predominantly observed in asthmatic and CF patients. Its prevalence among asthmatics and CF patients is 1–2% [4] and 2–15% [5–11], respectively.

ABPA AND FUNGAL DISEASES

In patients with ABPA, Aspergillus fumigatus antigens provoke a strong allergic reaction, characterised by the dominance of T-helper cell (Th) type 2 mediated responses, high numbers of eosinophils, a high total immunoglobulin (Ig)E level and high levels of Aspergillus specific IgE and IgG levels. Although it is not clear what initiates this hypersensitivity response, polymorphisms in genes that drive innate and adaptive immune mechanisms as well as loss-of-function mutations in the CF transmembrane conductance regulator (CFTR) are associated with ABPA development. The chronic inflammatory conditions in ABPA eventually result in airway remodelling, which is characterised by mucoid impaction, bronchial inflammation and obstruction. When left untreated fibrosis bronchiectasis and eventually respiratory insufficiency are the final pathophysiological stages in this remodelling process. The diagnosis of ABPA is complex and difficult to discriminate from chronic inflammatory episodes already observed in patients with asthma or CF. It has been estimated that on average 10 years elapse between the onset of ABPA and its eventual diagnosis [12]. Criteria for the diagnosis ABPA in asthmatics include a history of asthma with immediate skin reactivity, elevated serum IgE, precipitating antibodies against Aspergillus sp., peripheral blood eosinophilia, current or previous infiltrates on chest radiographs and central bronchiectasis on high-resolution computed tomography (HRCT) scans. CF patients are chronically exposed to multiple microorganisms and discrimination of ABPA is difficult in these patients. The main diagnostic criteria are similar to those described above, except for higher total IgE levels. ABPA treatment aims at reducing the fungal burden and dampening the immune response. Antifungal agents are effective in reducing IgE levels and improving clinical outcome within a 16week period; however, their long-term clinical effects are unknown [13]. The role of antifungal agents in the eradication of A. fumigatus hyphae is limited. Immune suppression is mainly achieved by oral glucocorticoid therapy that reduces the total serum IgE levels and correlates with a reduction in symptoms and radiological findings. However, the long-term use of steroids is associated with serious side-effects. Therapy that targets individual components of the hypersensitivity reaction is being developed and tested. The identification of crucial immunological components and associated molecular targets is essential for the design of novel drugs. Bronchiectasis due to other fungal disease is mainly limited to the immunocompromised host. Only limited studies are available on the role of fungi in otherwise healthy subjects. Both groups are briefly summarised in this chapter.

Figure 1 shows the structure and appearance of A. fumagatis under light microscopy.

History and epidemiology of ABPA In 1952, HINSON et al. [14] provided the term ABPA for the description of three patients who suffered from pulmonary eosinophilia in the UK, and in 1969 ABPA was first reported in the USA. In 1971, immunoserological features were discovered that supported hypersensitive immune reactivity as a disease mechanism in ABPA. From that time onwards the diagnostic possibilities rapidly improved and in the early 1980s ABPA was reported throughout the world.

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Still, the true population prevalence of ABPA remains highly speculative: ABPA was not acknowledged by the World Health Organization (WHO) as a disease entity in their 2007 International Classification of Disease (ICD-10) [15] and the diagnostic criteria for ABPA vary greatly within international medical societies. It has been generally assumed that there is an

estimated population ABPA prevalence of 1–2% in asthmatics [16, 17]. Overviews on the prevalence of ABPA show a spectre of 1% prevalence in the general population of asthmatics to 38.6% in patients with acute severe asthma [18]. In patients with CF the prevalence is estimated to be 1–15% [16, 19]. NOVEY [16] found an average of 7% among a total of 1,096 patients, taken from eight studies. Despite the differences in diagnostic criteria, laboratory methodology, demographical and geographical features, the range of prevalence was narrow in these studies ranging from 3–11%. MASTELLA et al. [20], on behalf of the European Registry of Cystic Fibrosis, reported data for 12,447 patients with CF in nine European countries. The overall prevalence among the European CF patients was found to be 7.8%, with a range of 2.1% in Sweden and 13.8% in Belgium. Age was found to be an important factor; in the group aged ,6 years the prevalence was 6% and a stable 10% thereafter [20].

Figure 1. Light microscopy of Aspergillus fumigatus hyphae. The stalk is called a hypha, the end of the hypha is swollen and small strings emerge from it called philiades. Chains of conidia (seen as small blue balls) emerge from the philiades. Reproduced with permission from K. Makimura (Teikyo University Institute of Medical Mycobiology, Tokyo, Japan; personal communication) and the Pathogenic Fungi Database (www. pfdb.net/).

Bronchiectasis is a morphological disorder, defined as the irreversible dilatation of the cartilage containing airways or bronchi. Approximately half of the patients with bronchiectasis is classified as having idiopathic bronchiectasis. In 7–8% of patients with bronchiectasis, ABPA is the causative factor [21, 22]. ABPA can be subclassified into three groups based upon radiological features indicating the presence or absence of central bronchiectasis and other radiological features. Approximately 75–95% of ABPA patients display both centrally located, cylindrical bronchiectasis (ABPA-CB) with or without other radiological features (ABPA-CB-ORF). The remaining 5–25% of the patients with ABPA are diagnosed without the presence of bronchiectasis, in these patients the diagnosis is based on seropositivity (ABPA-S) [1–3].

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Bronchiectasis and ABPA

The presence of central bronchiectasis is associated with disease severity. The small group of patients with ABPA-S appear to suffer from a less aggressive form of the disease when compared with ABPA-CB and ABPA-CB-ORF patients. Whether ABPA-S is able to progress into ABPA-CB or whether it is a pathogenetically different form of the disease is unclear. In a 3-year prospective cohort study in 11 patients, KUMAR and CHOPRA [23] described better lung function and a lower number of exacerbations in the ABPA-S group compared with an ABPA-CB control group. GREENBERGER et al. [24] included 28 patients in a 2-year prospective cohort study and found different immunological parameters in the ABPA-S group compared with the ABPA-CB group. The study found significantly lower serum specific anti-A. fumigatus IgG subclasses in patients with ABPA-S, and a trend towards lower levels of total serum IgE and specific anti-Aspergillus IgE and IgA [24]. The radiological differences between the groups are, therefore, also reflected by clinical and immunological differences. The question remains whether early recognition and treatment of ABPA-S can prevent progression into ABPA-CB [23].

Pathogenesis of ABPA

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The pathophysiological mechanisms underlying the development of ABPA are still poorly understood, but clearly share important immunological mechanisms with other hypersensitivity diseases. ABPA primarily develops in patients with asthma or CF, and is caused by an Aspergillusdriven strong hypersensitivity response [25]. Immunological features include highly elevated levels

of total IgE and Aspergillus-specific IgE and IgG, increased eosinophil numbers, and a Th2-dominated antigen-specific CD4+ T-cell response. Hypersensitivity to Aspergillus and colonisation by Aspergillus appear to be required but are not sufficient to develop ABPA alone. Between 31 and 59% of CF patients display sensitisation towards Aspergillus and up to 40% are colonised; however, only 1–10% of CF patients develop ABPA [26]. It appears that unique characteristics within Aspergillus itself in combination with patient-specific environmental and genetic factors facilitate the chronic colonisation and development of a deteriorating immune response, which ultimately induces the irreversible airway remodelling associated with fibrosis, pulmonary obstruction and bronchiectasis.

A. fumigatus virulence

ABPA AND FUNGAL DISEASES

Fungal spores (fig. 2) or conidia are ubiquitously present in our environment. A cubic meter of air typically contains approximately 104–105 conidia, predominantly of the Cladosporium and Alternaria genera, and of a lesser amount Aspergillus and Penicillium. The genus Aspergillus consists of 250 subspecies of which A. fumigatus is considered the most prevalent airborne fungal human pathogen, its conidia are present at approximately 1–100 m-3 air [27]. A. fumigatus causes life threatening, invasive disease in immunocompromised patients and is associated with multiple hypersensitivity responses including allergic asthma, hypersensitivity pneumonitis and ABPA [28]. Many molecular subtypes of A. fumigatus exist, 85% of analysed A. fumigatus in air samples were unique; however, in general none of these subtypes were found to be selectively enriched in patients suggesting that most subtypes are equally pathogenic [29, 30]. The development of novel antifungal reagents may, however, select for some subtypes [31, 32]. The presence of specific subtypes of A. fumigatus in ABPA remains unknown, but these may be prime candidates to study ABPA-related disease mechanisms. In recent years insights into the mechanisms by which A. fumigatus regulates its pathogenic potential or virulence have progressed significantly. These mechanisms regulate the rapid growth characteristics of A. fumigatus at 37uC (A. fumigatus conidia germinate within 4–5 hours on nutrient rich media in vitro), the overall mechanical fitness of conidia to withstand environmental pressure, and its capacity to extract nutrients of dead organic matter for growth. Selective mechanisms have also co-evolved. These selective mechanisms directly impair the epithelial barrier function and host immune defence, facilitating its infection. The particularly small size of a A. fumigatus conidia range between 1 mm and 3 mm, thereby facilitating its ability to be airborne and allowing it to reach the alveolar spaces upon inhalation. The cell wall of A. fumigatus conidia consists of a thick internal layer of structural polysaccharides enriched for branched b(1,3)/(1,6) glucans linked to chitin as observed in most fungi [33, 34]. Additional bonds to this backbone are species specific, in the case of A. fumigatus this core polysaccharide backbone is further linked to galactomannan and linear b(1,3)/ (1,4)-glucans. This large polysaccharide complex is embedded in a cement-like mixture consisting of a1,3glucan, galactomannan and polygalactosamine. A thin hydrophobic protein layer, termed surface hydrophobin, is composed of cross-linked proteins (including RodA) that form a regular pattern of rodlet structures and melanin that confers pigment, which further Figure 2. Low temperature scanning shields and protects the polysaccharide shell.

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electron microscopy image illustrating a tuft of Aspergillus spores arranged in rows. A spore is approximately 1 mm in size (M.H. Umar, Maasstad Hospital, Rotterdam, the Netherlands; personal communication).

Germination initiates an asexual developmental growth programme. It starts with conidial swelling followed by a polar growth programme that results in the protrusion of an elongating germ tube, termed hyphae, from

the conidium cell [35]. Hyphae are covered by a newly synthesised polysaccharide coat without the typical protein coat present on conidia [35]. Simultaneously with polar growth comes nuclear division by mitosis, resulting in further elongating hyphae with each cycle. In immunocompromised patients, Aspergillus grows into large hyphal networks termed mycelia and forms extracellular matrices termed biofilms that contain a1,3-glucan, galactomannan and galactosaminogalactan, and possibly other components that promote growth [36]. Interestingly, germination of A. fumigatus conidia is increased compared with Aspergillus flavus and Aspergillus niger at 37uC but not at 20uC [37]. This increased growth rate at 37uC likely contributes to the prevalence of A. fumigatus in fungal diseases, such as ABPA.

Interestingly, when comparing fungal proteases of A. fumigatus with those of Alternaria alternata and Cladosporium herbarum, KAUFFMAN et al. [41] reported an increase in the activity of A. fumigatus-derived proteases, as indicated by the shrinking and desquamation of epithelial cells and pro-inflammatory cytokine production. Although the role of isolated components from A. fumigatus in conferring virulence as a human pathogen remains difficult to establish, it is clear that their combined activity contributes to the strong association of A. fumigatus with fatal human diseases.

Innate mechanisms underlying ABPA The innate defence mechanisms involved in the clearance and inflammatory response to A. fumigatus, and how these may impact on the development of ABPA will be discussed here. The majority of conidia are cleared without inflicting a strong inflammatory response associated with tissue destruction. Most inhaled conidia are efficiently trapped by mucus and removed by mucociliary clearance systems that are affected in CF and asthma patients. Nevertheless, ABPA is generally not observed in patients with primary ciliary dyskinesia in which impaired mucociliary clearance leads to the accumulation of mucus and primarily bacterial infections, suggesting additional mechanisms contribute to the development of ABPA [42].

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A. fumigatus expresses multiple factors to evade host immune defence mechanisms, which in total contribute to the virulence of A. fumigatus in humans. These factors may be part of the growth cycle of A. fumigatus, but may also be uniquely expressed as secondary metabolites during specific phases of growth. For example, the binding of conidia to various extracellular matrix (ECM) proteins prevents its mucociliairy clearance and the oxidative mechanisms of phagocytes are counteracted by the production of superoxide dismutases, mannitol and three types of catalases. A range of other toxins and proteases further inhibit immune responses and promote epithelial cell penetration including ribotoxin [38], phospholipases [39], haemolysins, gliotoxins, metalloproteinase, alkaline proteinase and elastase [40].

Beyond the mucociliary system, resident cells of the lungs, such as alveolar macrophages (AM) and type II pneumocytes, destroy conidia by phagocytosis and the production of reactive oxygen species (ROS) upon activation of the membrane-bound NADPH-oxidase complex. It was recently shown that RodA in the protein coat surrounding conidia inhibits the inflammatory response to conidia by masking the highly immunogenic polysaccharide cell wall [43]. This may promote survival of conidia by escaping host immunity, but may also be beneficial to the host by limiting inflammatory responses upon inhalation of conidia.

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However, during germination the extending hyphae expose their polysaccharide wall and start to produce metabolites that trigger a strong inflammatory response. Pattern recognition receptors, e.g. Toll-like receptors (TLRs) and carbohydrate-binding proteins termed C-type lectins, are expressed by lung epithelial and resident immune cells, such as AM and dendritic cells (DCs), which recognise the b-glucans, chitin and galactomannan of the cell wall. Controversy still exists over the exact functional role of individual TLRs in the recognition of fungi, but it appears that TLR2, TLR4 and TLR9 do signal in a fungal morphotype-specific manner [44]. Activation of TLR2 and inhibition of TLR4 signalling during hyphal growth has been proposed to promote the development of a Th2 response [45, 46].

In addition to TLRs, members of the C-type lectin family, e.g. the mannose receptor, DC-specific intercellular adhesion molecule-grabbing nonintegrin (DC-SIGN), Dectin-1, and Dectin-2, recognise carbohydrate structures of the fungal wall and play an important role in fungal recognition, killing, and inflammatory signalling. Dectin-1 binds b-(1,3)-glucan and is prevalent on neutrophils, AM and DC. Neutrophils are the first cells to enter an inflammatory site and are short-lived phagocytic effector cells. Neutrophils produce ROS and release proteolytic enzymes, upon apoptosis their DNA traps pathogens but also increase mucus viscosity [47, 48]. Mice lacking Dectin-1 are highly susceptible to A. fumigatus infection; their macrophages and DC produce low levels of inflammatory cytokines and have limited recruitment of neutrophils to the site of infection with reduced killing capacity [49].

ABPA AND FUNGAL DISEASES

Triggering these pattern-recognition receptors induces the release of multiple inflammatory networks that recruit cells from the blood to the infected area, and play a crucial role in shaping the adaptive immune response at later stages [50–52]. Human polymorphisms in these systems can affect fungal load, growth properties and the balance of inflammatory mediators produced by innate cells that can impact on the quality and quantity of the adaptive response. ABPA is correlated with polymorphisms in TLR9 [53]. The mechanism by which TLR9 predisposes to ABPA in humans remains uncertain; however, pulmonary hypersensitivity induced by A. fumigatus in TLR9 -/- mice is significantly reduced [54]. DCs of these mice have lower Dectin-1 levels and produce low amounts of interleukin (IL)-17, which was associated with pulmonary infection of A. fumigatus. Multiple polymorphisms in other innate recognition systems including TLR2 and TLR4 and humoral pattern recognition factors, such as mannose binding lectin and surfactant protein A, have also been associated with ABPA and other different types of fungal diseases [55–59]. Collectively, it is clear that a complex multi-layered innate response to A. fumigatus has evolved to prevent infection and subsequent invasive disease. Genetic variations in innate systems that impact on pathogen recognition, fungal infection and induction of hypersensitivity responses have been associated with fungal diseases including ABPA. The extent to which genetic variation within these systems affects the development of ABPA in subgroups of CF or asthmatic patients requires further attention and may have prognostic value for patient subgroups.

Adaptive immunity in ABPA DCs are specialised cells that take up antigens at local inflammatory sites and then migrate to draining lymph nodes or bronchus associated lymphoid tissue (BALT) where they activate naı¨ve T-cells by presentation of antigenic peptides in the context of major histocompatibilty complex (MHC) [50]. Upon activation of naı¨ve Th cells, these cells acquire distinct cytokine-secreting properties that impact on the developing immune response. Multiple subsets of committed antigen-experienced Th cells are recognised including Th1, Th2, Th17 and induced T-regulatory (T-reg) cells [60]. In general, interferon-c producing Th1 and IL-17 producing Th17 subsets are important inflammatory cells associated with cell-mediated immunity against viral infections and intracellular bacteria, and are associated with multiple autoimmune diseases. IL-4 producing Th2 cells are typically associated with strong immune responses against large extracellular organisms that cannot be cleared through phagocytosis, such as intestinal parasites, and are associated with allergic diseases and ABPA in humans. Induced T-reg cells and natural T-reg cells are important to dampen immunological responses by the production of IL-10 and transforming growth factor (TGF)-b [61].

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Th responses are skewed towards Th2 in ABPA as indicated by in vitro lymphocyte responses against secreted proteins from A. fumigatus and animal models [26, 62–66]. Th1 and Th17 responses against A. fumigatus appear protective against hypersensitivity and are associated with clearance of Aspergillus [67–69]. Why ABPA patients mount such a vigorous Th2-response is not known and remains a key question. Activation of specific pattern recognition receptors and cytokine receptors at the site of inflammation induces DCs to express surface molecules and

cytokines, which help to commit naı¨ve Th cells; however, T-cell intrinsic factors, such as T-cell receptor (TCR) avidity for its antigen, also appear important. Recently, epithelial products such as IL-25 and thymic stromal lymphopoietin (TSLP) have been shown to alter DCs function and subsequent Th responses [70–72]. TSLP stimulated DCs from ABPA patients use ligand OX40 to potently induce Th2 responses [71]. Other ABPA-associated polymorphisms in genes, e.g. TLR9, IL-4Ra subunit and the IL-10 promotor, may all affect DC maturation and or induction of Th differentiation, but these proteins are expressed by many cells and thus it remains difficult to pinpoint at which level these polymorphisms affect disease [73].

Th2 cells and their cytokines play a crucial role in B-cell class switching and the recruitment of IgE-responding innate cells such as eosinophils, basophils and mast cells. Early studies indicate that supernatants of lymphocytes incubated with A. fumigatus antigens regulate IgE production by B-cells [74]. Cytokines, such as IL-4, IL-5 and IL-13, by Th2 cells facilitates B-cell class switching to IgA and IgE in BALT, and induce the production and recruitment of eosinophils to the inflammatory site [19]. IgE levels are quantitatively higher in ABPA compared with other atopic conditions, though little is known about the width of the antibody response against A. fumigatus and possible bystander antigens including self antigens. However, recent evidence indicates the existence of a Th2-mediated immune response without the presence of IgE [75]. To place this contradiction into perspective, data from a recent study indicated that out of 66 proteins present in the cytosol of A. fumigatus, which were recognised by pooled serum of ABPA patients, 63 were targeted by IgE and only three by IgG antibodies [76]. The prevalence of A. fumigatus-specific IgE over IgG antibodies suggests BALT to be a primary site for development of high-specific Aspergillus IgE and not the peripheral lymphoid system. Upon comparison of atopic and ABPA patients, ABPA B-cells were found that expressed higher levels of the low-affinity IgE receptor CD23 and the co-stimulatory molecule CD86 that is crucial for positive reinforcement by Th cells, a phenotype associated with in vitro IL-4 responsiveness [77]. Indeed, polymorphisms in IL-4Ra have been found to be enriched within ABPA patients in comparison with non-ABPA patients. Furthermore, CF patients with ABPA are more sensitive to IL-4 than CF patients without ABPA, a finding that was not observed for IL-13 [78, 79].

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TCRs that bind with low affinity to their cognate antigen may also confer Th2 properties in ABPA. Variants of human MHC class II, such as HLA-DR2 and HLA-DR5 alleles, are associated with ABPA and promote the expansion of T-cells with selective ab TCR chains. Although expression of these MHC class II variants is not sufficient for ABPA disease, peptides of a dominant allergen of A. fumigatus, termed Asp f1, are presented by these molecules and are recognised by lowaffinity, TCR-expressing Th2-skewed cells [74]. Other MHC class II alleles also appear to protect against ABPA.

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These antibodies trigger hypersensitivity responses by interacting with specialised innate immune cells. IgA and IgE-responsive granulocytes, such as eosinophils, basophils, and mast cells are activated by the Th2 response and recruited to the inflammatory site by a network of soluble mediators and cell-surface molecules [80]. Ligation of IgE on mast cells releases histamine and chemokines such as leukotriene B4 and platelet-activating factor, which induce smooth muscle contraction, vascular permeability and attract eosinophils. RANTES (regulated on activation, normal T-cell expressed and secreted), eotaxin and monocyte chemotactic protein (MCP)-3 are other important chemoattractants for eosinophils. The receptor for eotaxin, chemokine receptor 3 (CCR3) is selectively expressed by Th2, eosinophils and basophils, and is upregulated by IL-4. Th2-derived IL-5 is essential for increased eosinophil production from the bone marrow and their activation but appears dispensible for A. fumigatus-induced hyperreactivity in mice [81]. Nevertheless, these cells are a prominent feature of ABPA and are highly present in bronchial alveolar lavages suggesting their products to inflict tissue damage under chronic conditions [19]. Chemokines are implicated in various allergic conditions; however, their exact role in ABPA requires further refinement as the blockade of these by therapeutics may control the inflammatory cellular composition and local tissue destruction.

It has long been recognised that mucosal-associated immunity, especially in the gut, appears to be regulated by T-lymphocytes expressing IL-10 and or TGF-b [82]. Recently, CF patients colonised by A. fumigatus were shown to have increased levels of FoxP3-positive T-reg cells that expressed higher levels of surface TGF-b upon A. fumigatus stimulation, and confer tolerance to oral antigens in mice [61, 71]. The role of IL-10 producing T-reg cells (sometimes termed Tr1 cells) in ABPA is not clear; however, IL-10 promotor polymorphisms have been associated with fungal diseases and ABPA [73]. Adoptive transfer of T-reg cells is effective in lowering inflammatory conditions in multiple animal models suggesting that modulation of the number and activation of these cells in humans may control excessive inflammation in ABPA. In conclusion, inflammatory mediators of Th2 cells including IL-4, IL5 and IL-13 play a dominant role in the induction and maintenance of the hypersensitivity response in ABPA. These promote IgE and IgA isotype switching and attract typical innate effector cells associated with hypersensitivity responses such as eosinophils, basophils and mast cells. Genetic variation in these pathways predispose for ABPA; however, ranking these for their role in ABPA disease development will prove difficult considering the impact of environmental variables and limited patient numbers. Based on homology with other hypersensitivity disorders, the mechanisms that underlie Th differentiation in ABPA can begin to be understood; however, the characterisation of Th subsets and their role in ABPA development has only just started.

ABPA AND FUNGAL DISEASES

CFTR-related immunological disease mechanisms in ABPA In general, the immune mechanisms in CF are normal; however, there is evidence to support that ABPA, specifically, may also result from the abnormal function of CFTR in immune cells next to the epithelial cells. The association between CF patients and allergic disease was reported in 1949 [83]. CF patients have mutations in CFTR that encodes an adenosine triphosphate (ATP)- and cyclic adenosine monophosphate (cAMP)-regulated anion channel that regulates the composition of excretions [84]. CFTR in the lung epithelium regulates the air–surface liquid layer that underlies the mucus layer, which impacts the mucociliary clearance and functions of humoral components [85]. CFTR is expressed in multiple other tissues including the immune system, suggesting that the hyperinflammatory status of CF patients that was previously believed to be secondary to infection may result from a dysregulated immune response caused by a CFTR mutation [86–89]. Genetic studies in mice support a role for CFTR in macrophages, DCs and lymphocytes [90, 91]. In human innate cells the impaired bacterial clearance by phagocytes has been observed; however, the capacity of these cells to present antigens to T-cells has not been thoroughly assessed [92, 93]. MULLER et al. [91] reported that CFTR deficiency in mice provokes a stronger hypersensitivity response to A. fumigatus, and a shift from a predominant cytokine profile of IL-5 to IL-4. Recently, CD3 lymphocytes were implicated in the hypersensitivity response towards A. fumigatus by adoptive transfer experiments [94]. Conditional knockouts that lack CFTR in lymphocytes have enhanced basal and A. fumigatus-induced IgE levels, further supporting that CFTR is functional in murine CD4+ lymphocytes by limiting Th2-skewing.

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Asthmatic non-CF individuals with ABPA frequently carry a mutant CFTR allele [95–98]. A recent study, which involved the extensive CFTR sequencing of ABPA patients with normal sweat chloride levels and pancreatic function, found that the CFTR mutation frequency in patients with ABPA was approximately 48 higher compared with the general population [98]. Whether certain CFTR mutations specifically cluster with ABPA remains to be seen, and as this is difficult to study due to low patient numbers it remains undetermined. The strong correlation of ABPA with CFTR heterozygocity is remarkable, as it has been generally accepted that approximately 20% residual function is sufficient for epithelial functioning. This may point out that other tissues are more strongly affected by CFTR deficiency, but cannot rule out epithelial involvement. The hypothesis that CFTR mutant lymphocytes are intrinsically Th2-primed, as may be expected from mice studies, requires further thorough investigation and should carefully address confounding factors, such as genetic background, infectious status and therapeutic regimen.

Summary To summarise, ABPA is mostly prevalent in CF patients compared with a small percentage of asthma patients, and is a result of complex interactions between the invasive pathogen A. fumigatus and the human immune system. Th2-skewing of Th cells followed by a strong humoral IgE response and activation of IgE-responding effector cells are clear hallmarks of ABPA. To date, genetic variation in CFTR itself appears to be the strongest genetic factor associated with ABPA, also in asthmatics. A. fumigatus-driven hypersensitivity mouse models reflecting ABPA strongly support a role for CFTR within the T-cell compartment [91, 94]. The strong relationship between ABPA and CF may, therefore, not only result from impaired epithelial functioning but may also result from lymphocyte defects that only become apparent upon strong Th2 stimuli a) Infectious site

c) Infectious site Mycelium β-glucans Chitin Galactomannan Protruding hyphae

Release of toxins, proteases and other secondary metabolites

Activation of pattern recognition receptors in lung epithelium and innate immune cells

Non-inflammatory clearance; phagocytosis CFTR-dependent mucociliary system

Cellular damage; release of danger signals

TLR-4

TLR-2

Phagocytosis and antigen processing MHC class II

NADPH oxidase Endosomal proteases

Fungus-associated inflammatory signalling Inflammatory clearance by resident cells and attracted innate cells; CFTR-dependent killing?

b) Lymphoid tissue

IL-25, TSLP, ATP

Fungus-associated inflammatory signalling

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Exposure of cell wall polysaccharides: β-glucan, chitin, galactomannan

Immune inhibitory protein-rich outer shell: RodA and melanin

Epithelium Toll-like receptors

Dectin-1

Dendrtic cell

Mannose receptor

DC-SIGN

C-type lectins

TLR-9

Germination

Conidium

Aspergillus fumigatus

Induction of Th skewing conditions Nucleus

d) Lymphoid tissue Tissue-derived dendritic cells Dendritic cell

Antigen presentation MHC class II TCR

Naïve T-cells

Naïve B-cells

IL-25, IL-4

lgE+secreting plasma cell

CD40 CD40L T-helper cell

B-cell

MHC class II

BCR CFTR inhibits skewing towards Th2 cells?

Co-stimulation

Th2 cells Stimulation of lgE-responsive eosinophils

CD23

lgE class switching

TCR

Th2 skewing Eosinophil

CD40L CD40 IL-4 IL-5 IL-13

Mast cell Basophil

Figure 3. Immunopathogenesis of allergic bronchopulmonary aspergillosis. a) Aspergillus fumigatus asexual life

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cycle and the interactions of innate epithelial and immune components with dormant conidia or germinating conidia at the site of infection. b) Interactions between adaptive components of the immune system in the lymphoid tissue showing proven or possible (&) h involvement of cystic fibrosis transmembrane conductance regulator (CFTR). Detailed schematic representation of molecular interactions between A. fumigatus and various immune cell subsets at c) the infectious site or d) lymphoid tissue are shown. Local priming of dendritic cells (DCs) by fungus and epithelial-derived products is important for T-helper (Th) cell skewing. DCs at the lymphoid tissue stimulate naı¨ve Th cells by upregulation of major histocompatibilty complex (MHC) class II antigen complexes in combination with specific co-stimulatory pathways that activate Th2 cells. These facilitate immunoglobulin (Ig)E class switching of B-cells and the activation and recruitment of eosinophils, basophils and mast cells. TLR: toll-like receptors; TSLP: thymic stromal lymphopoietin; IL: interleukin; ATP: adenosine triphosphate; TCR: T-cell receptor; BCR: B-cell receptor.

associated with A. fumigatus. Therefore, it appears that next to environmental factors such as nutritional status, co-infection and long-term immune suppression, genetic variations in the systems underlying A. fumigatus recognition, clearance and Th2 skewing may also drive patientspecific ABPA susceptibility. The identification of ABPA-related disease mechanisms will be crucial for future development of therapeutics that control immune-related tissue destruction without impairment of fungal clearance. Figure 3 illustrates the immunopathogenecity of ABPA.

Clinical features and diagnostic approach

ABPA AND FUNGAL DISEASES

Patients with ABPA typically present with symptoms such as a low-grade fever, productive cough, bronchial hyperreactivity, chest pain, wheezing, haemoptysis and expectoration of brownish sputum plugs. Sometimes patients are asymptomatic and diagnosed during routine screening tests in patients with asthma or CF. Physical examination can reveal wheezing or coarse crackles on auscultation, clubbing of the fingers in 15% of patients and complications such as pulmonary hypertension and/or respiratory failure [99, 100]. The diagnostic criteria for patients with asthma are summarised in table 1. Because the primary disease symptoms in patients with CF can closely resemble ABPA, adapted criteria for ABPA have been formulated within this patient category (table 2). In CF, ABPA is diagnosed in the presence of the following: 1) acute or subacute clinical deterioration not attributable to another aetiology; 2) total serum IgE concentration of .500 IU?mL-1; 3) immediate cutaneous reactivity to A. fumigatus or in vitro demonstration of IgE antibody to A. fumigatus; and 4) either precipitins to A. fumigatus or in vitro demonstration of IgG antibody to A. fumigatus or new or recent abnormalities on radiological tests (CT scan or chest radiograph).

Skin testing In patients with bronchial asthma Aspergillus skin testing is recommended for screening purposes. Intradermal injection is more sensitive in comparison to the skin-prick test [64, 102, 103]. A positive reaction to recombinant antigens of A. fumigatus termed rAsp f 4 and/or 6 reached a sensitivity of 86.8% (95% CI 73.5–100%) and a specificity of 92% (95% CI 83.9–100%) in a study with 50 CF patients [102]. Of those 50 patients, 12 suffered from ABPA, 21 were sensitised for A. fumigatus and 17 were control patients. However, less promising results were obtained by DE OLIVEIRA et al. [104] who subjected 65 patients with asthma and a positive skin-prick test to Table 1. Criteria for the diagnostis of allergic bronchopulmonary aspergillosis (ABPA) in patients with asthma Criteria For ABPA central bronchiectasis Asthma Central bronchiectasis, inner two thirds of chest CT field Immediate cutaneous reactivity to Aspergillus sp. or A. fumigatus Total serum IgE concentration .417 kU?L-1/1000 ng?mL-1 Elevated serum IgE and or IgG to A. fumigatus Chest roentgenographic infiltrates Serum precipitating antibodies to A. fumigatus For ABPA seropositive Asthma Immediate cutaneous reactivity to Aspergillus sp. or A. fumigatus Total serum IgE concentration .417 kU?L-1/1000 ng?mL-1 Elevated serum IgE and or IgG to A. fumigatus Chest roentgenographic infiltrates

Minimal essential criteria Yes Yes Yes Yes Yes No No Yes Yes Yes Yes No

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CT: computed tomography; A. fumigatus: Aspergillus fumigatus: Ig: immunoglobulin. Reproduced from [101] with permission from the publisher.

Table 2. Diagnostic criteria for allergic bronchopulmonary aspergillosis (ABPA) in cystic fibrosis (CF) patients as proposed during the 2003 CF Foundation Consensus Conference (Bethesda, MD, USA) Classic case 1. Acute or subacute clinical deterioration (cough, wheeze, exercise intolerance, exercise-induced asthma, decline in pulmonary function, increased sputum) not attributable to another aetiology. 2. Serum total IgE concentration of .1000 IU?mL-1 (2400 ng?mL-1), unless the patient is receiving systemic corticosteroids (if so, retest when steroid treatment is discontinued). 3. Immediate cutaneous reactivity to Aspergillus (prick skin test wheal of 3 mm in diameter with surrounding erythema while the patient is not being treated with systemic antihistamines) or in vitro presence of serum IgE antibody to A. fumigatus. 4. Precipitating antibodies to A. fumigatus or serum IgG antibody to A. fumigatus by an in vitro test. 5. New or recent abnormalities on chest radiograph (infiltrates or mucus plugging) or chest CT (bronchiectasis) that have not cleared with antibiotics and standard physiotherapy. Minimal diagnostic criteria 1. Acute or subacute clinical deterioration (cough, wheeze, exercise intolerance, exercise-induced asthma, change in pulmonary function, or increased sputum production) not attributable to another aetiology. 2. Total serum IgE concentration of .500 IU?mL-1 (1200 ng?mL-1). If ABPA is suspected and the total IgE level is 200–500 IU?mL-1, repeat testing in 1–3 months is recommended. If the patient is taking steroids, repeat when steroid treatment is discontinued. 3. Immediate cutaneous reactivity to Aspergillus (prick skin test wheal of 13 mm in diameter with surrounding erythema, while the patient is not being treated with systemic antihistamines) or in vitro demonstration of IgE antibody to A. fumigatus. 4. One of the following: precipitins to A. fumigatus or in vitro demonstration of IgG antibody to A. fumigatus; or new or recent abnormalities on a chest radiograph (infiltrates or mucus plugging) or chest CT (bronchiectasis) that have not cleared with antibiotics and standard physiotherapy.

recombinant antigen testing. 19 patients tested positive for at least one recombinant antigen; however, only six of them met the classical criteria for ABPA.

Essential laboratory testing Total serum IgE is the most important laboratory test for ABPA and is essential for the diagnosis and monitoring of the disease. Normal levels of total serum IgE in patients that do not receive glucocorticoid therapy exclude ABPA as a diagnosis. In patients with asthma the total IgE levels should be .1,000 IU?mL-1, whereas in CF patients IgE levels of .1,500 IU?mL-1 can be detected. IgE levels are also used to monitor treatment. A reduction of 35–50% during treatment with systemic steroids is considered as a remission [105].

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Ig: immunoglobulin; A. fumigatus: Aspergillus fumigatatis; CT: computed tomography. Reproduced from [19] with permission from the publisher.

Increased levels of specific serum IgE antibodies to A. fumigatus distinguish ABPA from A. fumigatus hypersensitivity (AH), which is defined as a positive skin test, and other allergic conditions in asthmatics [106, 107]. The serum levels of Aspergillus-specific IgE are at least twice as high in ABPA compared with AH [108]. In patients with CF, specific serum IgE antibodies Asp f 3 and Asp f 4 are specific for ABPA and not for Aspergillus hypersensitivity [109].

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The presence of serum precipitins, i.e. precipitating IgG antibodies, are supportive to the diagnosis of ABPA [110, 111]. Peripheral eosinophilia is also regarded important in diagnosis; however, it may have relatively low specificity or sensitivity [112]. A total of .1,000 cells?mL-1 has been set as a cut-off value. The differential diagnosis of peripheral eosinophilia includes a range of other disorders such as tuberculosis, sarcoidosis, drug-induced eosinophilia and Churg–Strauss syndrome that should all be carefully ruled out. Sputum cultures are rarely used for diagnosing ABPA as fungi can be prevalent in the lungs of many immunocompromised patients. Pulmonary function testing is not suitable as a diagnostic test and is only useful as a rough indicator for the

severity of lung disease in general [113]. A promising serological test is for thymus and activationregulated chemokine (TARC). Diagnostic accuracy was proven to be greater for TARC (93%) than for total IgE (74%), rAsp f 4 (75%) or rAsp f 6 (79%) in a small diagnostic study with 12 CF patients with ABPA and 36 control patients [114]. The definition of the diagnostic accuracy was the number of correctly positively categorised patients plus the correctly negatively categorised patients as a percentage of the total.

Radiology Radiological imaging in most patients with ABPA shows centrally located, cylindrical bronchiectasis, while the presence of distal bronchiectasis is rare [115]. The radiological classification has predominantly prognostic implications as it cannot distinguish between bronchiectasis caused by ABPA or another factor [116]. HRCT scanning is regarded as the gold standard to identify bronchiectasis as a morphological diagnosis and correlates with the functional lung capacity of patients [117, 118]. Chest radiography lacks the sensitivity needed to rule out bronchiectasis and, therefore, HRCT is required if no abnormalities appear and ABPA is suspected. In ABPA, HRCT can be used to monitor disease progression and is directive for the therapeutic strategy.

Treatment

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The treatment of ABPA depends upon two important factors: 1) glucocorticoids to dampen the immunological activity, and 2) antifungal agents to suppress the antigenic load. Although glucocorticoids are the mainstay in ABPA treatment, no well-designed studies have been carried out. Neither the optimal dose regimen nor the optimal duration of therapy has ever been determined [119]. In asthmatics the optimal dose and treatment scheme as regarded by expert opinion is prednisone 0.5–1.0 mg?kg-1?day-1 for 2 weeks, followed by an alternate day regimen, which is tapered to zero during a 3–6-month period. In CF patients the prolonged use of glucocorticoids may induce severe side-effects such as glucose intolerance, growth suppression, cataracts and osteoporosis [120–122]. Therefore, the use of monthly pulses with methylprednisolone has been suggested as a treatment for ABPA in CF patients. Two small studies with 13 CF patients showed clinical and laboratory improvement after 0.3–1 mg?kg-1?day-1 and 10– 15 mg?kg-1?day-1, respectively [123, 124]. Figure 4 illustrates the effect of systemic steroids in a CF patient with ABPA.

Inhaled glucocorticoids

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Recently it was reported that inhaled glucocorticoids are significantly linked with the prevalence of Aspergillus in lungs of CF patients [125], which might increase their risk of suffering from ABPA. The efficacy of inhaled steroids in patients with ABPA has never been docua) b) mented and hence this treatment is not recommended in patients with CF. Some small case series in patients with asthma and ABPA indicate some beneficial effects of inhaled glucocorticoids [126, 127]. However, the single largest study with inhaled beclomethasone shows no beneficial effect at all [128]. Therefore, the use of inhaled glucocortiFigure 4. Chest radiograph of a 12-year-old cystic fibrosis patient coids seems limited in CF patients with allergic bronchopulmonary aspergillosis a) before and b) after a and implicates limited value for 6-week course of systemic steroids. patients with asthma.

Antifungal agents It has been suggested that itraconazole modifies the immunological activation associated with ABPA and can improve clinical outcome, at least over a 16-week period. The largest multicentre randomised controlled trial found significantly lower need for steroids decreased serum IgE concentrations and improved clinical findings in patients using itraconazole when compared with those who did not [129]. The most recent Cochrane review (updated in 2010) on the efficacy of itraconazole in the treatment of patients with CF concluded that evidence is limited and that further research is required [13]. Itraconazole might be used as an adjuvant to glucocorticoid treatment, presumably lowering the required dosage and thereby the side-effects of systemic steroids. The dosage of itraconazole is generally accepted to be 200 mg twice a day with a start dosage of 200 mg three times a day for 3 days. Liver function tests should be monitored monthly to prevent toxicity. A potential concern in patients using both inhaled corticosteroids and itraconazole is adrenal suppression due to an increase in steroid levels in serum [130].

With the progressing knowledge in the immunological mechanisms involved in patients with ABPA, the possibility of developing a more cause-related therapy becomes ever more apparent. In experimental settings some successes have been achieved. For example Asp f 1-derived peptide P1, prophylactically and therapeutically administrated to BALB/c mice is effective in regulating an allergic response to allergens/antigens of A. fumigatus [131]. The first results obtained by the administration of allergen-derived peptides to shift an Aspergillus specific Th2 response to a protective Th1 are promising. An example of immunomodulative therapy in a clinical setting is the introduction of omalizumab in children with CF and ABPA. Omalizumab is a humanised monoclonal antibody against IgE. Currently, as documented in case reports, a total of seven children who were described as irresponsive to glucocorticoid treatment were found to have improved lung function after using 300–375 mg omalizumab subcutaneously every 2 weeks [132–135]. However, in order to introduce omalizumab in daily clinical routine, more clinical trials are warranted.

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Immunomodulatory therapy

Conclusion The aim of this chapter was to provide an overview of the clinical features of ABPA, the diagnostic criteria and the underlying pathophysiological immune mechanisms. ABPA consists of an A. fumigatus-driven hypersensitivity reaction in predominantly asthmatic and CF patients. Polymorphisms in genes that drive innate and adaptive immune mechanisms, as well as lossof-function mutations in CFTR, are associated with the development of a strong Th2 response and ABPA. Continuous inhalation of A. fumigatus and resulting chronic infections, in combination with genetic predisposition, fuel a chronic inflammatory hypersensitivity response that eventually results in airway remodelling and functional impairment of the lung. The diagnostic process is characterised by a combination of tests evaluating lung function, serum hypersensitivity parameters (aspecific and specific for A. fumigatus), and radiological characteristics such as bronchiectasis. Treatment consists of dampening the immune response by the use of glucocorticoids and suppressing the fungal burden by antifungal agents.

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Recent insights into the pathogenesis, diagnostic measures and treatment possibilities illustrate the ongoing effort aimed at preventing ABPA from causing invalidating lung disease. Promising examples are the establishment of CFTR mutations in ABPA pathogenesis, the superior test characteristics of TARC regarding the diagnosis of ABPA in CF patients, and the beneficial role of itraconazole to glucocorticoids in treatment.

Statement of interest None declared.

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121. Rosenstein BJ, Eigen H. Risks of alternate-day prednisone in patients with cystic fibrosis. Pediatrics 1991; 87: 245–246. 122. Cheng K, Ashby D, Smyth R. Oral steroids for cystic fibrosis. Cochrane Database Syst Rev 2000: 2; CD000407. 123. Cohen-Cymberknoh M, Blau H, Shoseyov D, et al. Intravenous monthly pulse methylprednisolone treatment for ABPA in patients with cystic fibrosis. J Cyst Fibros 2009; 8: 253–257. 124. Thomson JM, Wesley A, Byrnes CA, et al. Pulse intravenous methylprednisolone for resistant allergic bronchopulmonary aspergillosis in cystic fibrosis. Pediatr Pulmonol 2006; 41: 164–170. 125. Van GN, Conseil V, Leroy S, et al. [The fungal risk in cystic fibrosis: a pilot study.] Ann Biol Clin (Paris) 2010; 68: 157–162. 126. Imbeault B, Cormier Y. Usefulness of inhaled high-dose corticosteroids in allergic bronchopulmonary aspergillosis. Chest 1993; 103: 1614–1617. 127. Wark PA, Gibson PG. Allergic bronchopulmonary aspergillosis: new concepts of pathogenesis and treatment. Respirology 2001; 6: 1–7. 128. Inhaled beclomethasone dipropionate in allergic bronchopulmonary aspergillosis. Report to the Research Committee of the British Thoracic Association. Br J Dis Chest 1979; 73: 349–356. 129. Stevens DA, Schwartz HJ, Lee JY, et al. A randomized trial of itraconazole in allergic bronchopulmonary aspergillosis. N Engl J Med 2000; 342: 756–762. 130. Patterson R, Greenberger PA, Radin RC, et al. Allergic bronchopulmonary aspergillosis: staging as an aid to management. Ann Intern Med 1982; 96: 286–291. 131. Chaudhary N, Mahajan L, Madan T, et al. Prophylactic and therapeutic potential of Asp. f1 epitopes in nai¨ve and sensitized BALB/c Mice. Immune Netw 2009; 9: 179–191. 132. Kanu A, Patel K. Treatment of allergic bronchopulmonary aspergillosis (ABPA) in CF with anti-IgE antibody (omalizumab). Pediatr Pulmonol 2008; 43: 1249–1251. 133. Lebecque P, Leonard A, Pilette C. Omalizumab for treatment of ABPA exacerbations in CF patients. Pediatr Pulmonol 2009; 44: 516. 134. Zirbes JM, Milla CE. Steroid-sparing effect of omalizumab for allergic bronchopulmonary aspergillosis and cystic fibrosis. Pediatr Pulmonol 2008; 43: 607–610. 135. van der Ent CK, Hoekstra H, Rijkers GT. Successful treatment of allergic bronchopulmonary aspergillosis with recombinant anti-IgE antibody. Thorax 2007; 62: 276–277.

Chapter 8

Nontuberculous mycobacterial infections C.L. Daley

Summary

Keywords: Bronchiectasis, mycobacteria, Mycobacterium avium complex, nontuberculous mycobacterial infections

Correspondence: C.L. Daley, Division of Mycobacterial and Respiratory Infections, National Jewish Health, 1400 Jackson Street, Denver, CO 80206, USA, Email [email protected]

C.L. DALEY

Nontuberculous mycobacteria (NTM) represent a large group of bacteria that have been isolated from environmental sources. When NTM are inhaled by a susceptible individual, infection can occur and lead to progressive lung disease. Epidemiological studies have described increases in the prevalence of NTM disease in multiple areas worldwide. Risk factors for disease include chronic lung diseases, such as bronchiectasis and chronic obstructive pulmonary disease, as well as various forms of immune deficiency. Patients typically present with either fibrocavitary or nodular bronchiectatic disease. Isolation of NTM from respiratory specimens does not always indicate disease so clinicians must evaluate clinical, radiographic and microbiologic information in order to diagnosis NTM-related lung disease. The American Thoracic Society has developed diagnostic criteria that can aid clinicians but the criteria cannot account for all clinical scenarios or for all NTM species given the large spectrum of pathogenicity encountered. Treatment usually consists of at least two antibiotics but the exact regimen will vary depending on the species and there is some variation in recommendations.

Eur Respir Mon 2011. 52, 115–129. Printed in UK – all rights reserved. Copyright ERS 2011. European Respiratory Monograph; ISSN: 1025-448x. DOI: 10.1183/1025448x.10003810

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ontuberculous mycobacteria (NTM) comprise ,140 species, many of which have been reported to cause disease in humans. Based on their rate of growth in subculture, NTM have traditionally been divided into slowly and rapidly growing species (table 1) [1–3]. Also referred to as environmental mycobacteria, NTM have been isolated from natural and drinking water supplies, as well as soil [4–7]. The presumed source of infection is exposure to these environmental reservoirs because human-to-human transmission has not been documented. When inhaled by susceptible individuals, such as those with chronic obstructive lung disease or bronchiectasis, infection with NTM can lead to a chronic, progressive and sometimes fatal lung disease.

Table 1. Examples of slowly growing and rapidly growing nontuberculous mycobacteria that have been reported to cause lung disease Slowly growing mycobacteria

Mycobacterium Mycobacterium Mycobacterium Mycobacterium Mycobacterium Mycobacterium Mycobacterium Mycobacterium Mycobacterium Mycobacterium Mycobacterium Mycobacterium Mycobacterium

arupense asiaticum avium branderi celatum chimaera flavescens florentinum heckeshornense intermedium interjectum intracellulare kansasii

Mycobacterium Mycobacterium Mycobacterium Mycobacterium Mycobacterium Mycobacterium Mycobacterium Mycobacterium Mycobacterium Mycobacterium Mycobacterium Mycobacterium

kubicae lentiflavum malmoense palustre saskatchewanse scrofulaceum shimodei simiae szulgai triplex terrae xenopi

Rapidly growing mycobacteria

Mycobacterium abscessus Mycobacterium alvei Mycobacterium boenickei Mycobacterium bollettii Mycobacterium brumae Mycobacterium chelonae Mycobacterium confluentis Mycobacteium elephantis Mycobacterium goodii Mycobacterium holsaticum

Mycobacterium fortuitum Mycobacterium mageritense Mycobacterium massiliense Mycobacterium mucogenicum Mycobacterium peregrinum Mycobacterium phocaicum Mycobacterium septicum Mycobacterium smegmatis Mycobaterium thermoresistible

NTM INFECTIONS

Despite their frequent isolation in the environment and human specimens, NTM were not widely recognised as a cause of human disease until the late 1950s. Since that time, the number of new species of NTM has grown dramatically [3] and the rate of disease related to NTM has also increased, overtaking the rate of tuberculosis (TB) in some areas [8]. Diagnosis and treatment of NTM lung disease remains challenging for clinicians and depending on the extent of disease and species involved, a cure may be difficult to achieve. When considering treatment, clinicians must weight the potential benefits of therapy against the cost and potential sideeffects of current regimens.

Epidemiology Incidence and prevalence The epidemiology of NTM disease has been difficult to determine because reporting is not mandatory in most countries and differentiation between infection and disease is often difficult. Although the incidence and prevalence of NTM infections have varied significantly across studies, recent studies have reported high rates of NTM pulmonary disease, particularly in older populations [9–12]. Among 933 patients with at least 1 NTM isolate in Oregon (USA), 527 (56%) met the American Thoracic Society (ATS) microbiological definition for disease giving an annualised prevalence of 5.6 cases per 100,000 for pulmonary disease [9]. The prevalence was significantly higher in females (6.4 cases per 100,000) than males (4.7 cases per 100,000) and was highest in persons aged .50 years (15.5 cases per 100,000). In another report from Oregon, the overall 2-year prevalence of NTM pulmonary disease was 8.6 cases per 100,000 and increased to 20.4 cases per 100,000 in those aged o50 years [10]. The annualised prevalence of NTM lung disease within four integrated healthcare delivery systems in the USA ranged from 1.4 to 6.6 per 100,000 [11]. Among persons aged o60 years, annual prevalence was 26.7 per 100,000.

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Studies from Canada [8], Australia [12], Taiwan [13], the Netherlands [14] and the USA [11, 15] have reported increases in the incidence or prevalence of NTM. MARRAS et al. [8] reported an increase in the number of pulmonary NTM isolates in Ontario (Canada) from 9.1 per 100,000 in 1997 to 14.1 per 100,000 in 2003. Difficulty in eradicating NTM infections resulted in a prevalence higher than that of tuberculosis [16]. More recently, two studies from the USA reported increases in NTM pulmonary disease [11, 15]. In a study examining the prevalence of NTM lung disease in four integrated healthcare delivery systems, there was a 2.6% and 2.9% increase per year in females and males, respectively [11]. Pulmonary NTM hospitalisations increased significantly among both males and females between 1998 and 2005 in a study involving 11 states in the USA [15]. Annual prevalence increased among males and females in Florida (3.2% and 6.5%, respectively) and among females in New York (4.6% per year) with no significant changes in California.

Earlier descriptions of pulmonary NTM disease described a male predilection for disease. However, in three recent studies from the USA, a higher proportion of disease was observed in females than males [9–11]. Over an 8-year period from 1998 to 2005, the overall prevalence rate of hospitalisations for NTM pulmonary disease in the USA was highest in females aged o70 years (9.4 per 100,000) compared with similarly age matched males (7.6 per 100,000) [11]. The reasons for the increase in incidence and prevalence have not been explained although increased awareness of the disease and improved diagnostic techniques could be factors. A true increase in incidence could be related to changes in the host such as an aging population, an increased prevalence of chronic lung disease or an increase in the number of immunocompromised individuals. The observation of a decreased incidence of pulmonary TB and an increased incidence of pulmonary NTM [8] could be explained by cross-immunity between mycobacterial species. Finally, an increase in the prevalence or virulence of organisms in the environment or changes in human behaviour that would lead to increased exposure to organisms could be contributors. In support of the latter, the frequency of skin reactivity to purified protein derivative-B, which used antigens from Mycobacterium intracellulare, increased from 11.2% in 1971–1972 to 16.6% in 1999–2000 [17].

Studies utilising delayed type hypersensitivity reaction to subcutaneously injected mycobacterial antigens have estimated that 11–33.5% of the population in the USA has been exposed to NTM [18–21]. A prospective study using skin testing data from Palm Beach, Florida reported that 32.9% of 447 participants in a population-based random household survey had a positive reaction to Mycobacterium avium sensitin [21]. Predictors of a positive reaction included Black race, birth outside the USA and .6 years cumulative exposure to soil. Using data from the National Health and Nutrition Examination Survey (NHANES), investigators reported similar findings with regards to sensitisation to M. intracellulare [17]. Male sex, non-Hispanic Black race and birth outside the USA were each independently associated with sensitisation. These two studies are interesting in that skin test reactivity to either M. avium or M. intracellulare antigens was associated with factors probably associated with soil exposure. However, at least in the USA, disease seems to be more common in older Caucasian females. Thus, the risk factors for exposure and infection may be different from those associated with disease.

C.L. DALEY

Risk factors for NTM infection and disease

Most NTM are significantly less pathogenic than Mycobacterium tuberculosis and probably require some degree of host impairment to result in disease. Impairment can be caused by immune defects or chronic lung disease of which the latter appears to be most common. NTM disease has been described in association with cystic fibrosis (CF), chronic obstructive pulmonary disease including a1-antitrypsin deficiency, cavitary lung disease, pneumoconiosis, bronchiectasis, prior TB,

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Historically male sex has been considered a risk factor for NTM lung disease and males continue to make up the majority of patients in some areas [14]. However, studies from the USA and South Korea have noted a female predominance. In Oregon, as noted previously, the rate of NTM pulmonary diseases was higher in females than males and females made up 60% of cases. Why the shift to a female predominance remains unclear. However, there is likely a genetic link because many females who develop bronchiectasis and NTM infection share a similar body type characterised by tall slender status with a higher frequency of pectus excavatum, kyphoscoliosis and mitral valve prolapse than females who do not have NTM infection [22–24]. This condition was first described by PRINCE et al. [25] in 1989 and later referred to as the ‘‘Lady Windermere Syndrome’’ after the character in Oscar Wilde’s play Lady Windermere’s Fan, the reference referring to fastidious behaviour in the character [26]. Recently, investigators reported that female patients with pulmonary NTM disease were taller, thinner and weighed less than matched control subjects [22]. To date, extensive evaluation of the immune system of these patients has been unrevealing but mutations in the cystic fibrosis transmembrane conductance regulator gene are common [22, 27].

pulmonary alveolar proteinosis and chronic lung injury due to aspiration from gastro-oesophageal disorders [28–34]. Bronchiectasis is an almost universal finding in females with NTM infection and it is seen in many males with NTM infection. However, NTM infections have been reported to occur in only 1–2% of bronchiectasis patients in two small series from the UK [35, 36]. In contrast, studies have documented a high prevalence of NTM from sputum cultures in patients with CF, with estimates ranging from 3% to 19.5% [37, 38]. Pulmonary disease due to NTM has been described in several other immunocompromised patient populations including transplant recipients [39–42], individuals taking tumour necrosis factor-a inhibitors [43–45], and patients with mutations in interferon (IFN)-c receptor 1, IFN-c receptor 2, interleukin (IL)-12 p40 and the IL-12 receptor [46, 47]. Most of these patients present with disseminated disease. Scientists have hypothesised that in slender, older females, decreased leptin, increased adiponectin and/or decreased oestrogens may account for the increased susceptibility to NTM infections [48]. Additionally, anomalies of fibrillin that lead to the expression of the immunosuppressive cytokine transforming growth factor-b may further increase susceptibility to NTM lung disease [48].

NTM INFECTIONS

Diagnosis and management Chronic pulmonary disease is the most common clinical presentation of NTM disease. In order to diagnose pulmonary NTM infection clinicians must weigh clinical, bacteriologic and radiographic information. Diagnostic criteria have been developed to aid the clinician in the diagnostic evaluation of persons suspected of having pulmonary NTM disease (table 2) [3, 49]. Although the diagnostic criteria provide a useful approach for the evaluation of patients with suspected NTM disease, the approach has yet to be validated and it is impossible for a single set of diagnostic criteria to be appropriate for all patients and species of NTM. Unlike with TB, a single positive sputum culture for NTM is not diagnostic of pulmonary disease. However, when two or more sputum cultures are positive the diagnosis of disease is more likely. For example, 98% of patients with two or more positive sputum cultures for M. avium complex (MAC) had evidence of progressive disease in a study from Japan [50]. Whether this microbiologic criterion holds true for other NTM species is not known but given the wide range of pathogenicity among the various NTM species it is unlikely. Patients who are suspected of having NTM lung disease but do not meet the diagnostic criteria should be followed clinically until the diagnosis is either firmly established or excluded.

Laboratory diagnosis Ultimately, the diagnosis of NTM disease is based on isolation of these organisms from clinical specimens. Both solid and broth media should be used for detection of mycobacteria and a semiquantitative reporting of colony counts is recommended [3]. Most NTM grow within 2–3 weeks Table 2. Microbiological criteria for diagnosis of nontuberculous mycobacteria lung disease Respiratory specimen

Sputum specimen Bronchial wash/lavage Tissue biopsy

Culture and histopathological results ATS recommendations

BTS recommendations

At least two separate positive cultures

Positive cultures from specimens obtained at least 7 days apart Not described Not described

One positive culture Compatible histopathology (granulomatous inflammation) and a positive biopsy culture and/or a positive sputum or bronchial wash/lavage culture

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ATS: American Thoracic Society; BTS: British Thoracic Society. Data from [3, 49].

on subculture and rapidly growing mycobacteria usually grow within 7 days of subculture. Identification of specific species can be based on phenotypic, chemotaxonomic and molecular methods [3]. However, none of these procedures are sufficient to differentiate all NTM.

As noted previously, skin test reactions to mycobacterial antigens are common in people living in endemic areas and, thus, are unable to distinguish NTM infection from disease. Tests that could distinguish infection from disease would be very helpful for clinicians. Measurement of anti-A60 immunoglobulin (Ig)G was reported to have a sensitivtity of 87% and specificity of 97% for detection of Mycobacterium abscessus disease in patients with CF [51]. In Japan, KITADA et al. [52] evaluated the performance of an assay that detects serum IgA antibody to glycopeptidolipid core antigen for the diagnosis of MAC lung disease. The sensitivity and specificity of the assay for detecting MAC lung disease were 84% and 100%, respectively. Antibody levels were higher in patients with nodular bronchiectatic disease compared with fibrocavitary disease and levels correlated with extent of disease by chest computed tomography (CT) scans. In a follow-up study of patients who underwent bronchoscopy, the sensitivity, specificity, positive predictive and negative predictive values were 78.6%, 96.4%, 95.7% and 81.8%, respectively [53]. The sensitivity and specificity of the test for MAC pulmonary disease in patients with rheumatoid arthritis was 43% and 100%, respectively [54]. Although these serologic assays are not widely available, they may eventually find their way into diagnostic algorithms.

C.L. DALEY

The clinical usefulness of drug susceptibility testing in the management of patients with NTM disease remains controversial because in vitro results do not correlate well with clinical outcomes for some mycobacterial species. Unfortunately, there is no single susceptibility method that is recommended for all species of slowly growing mycobacteria. For MAC, a broth-based culture method with both microdilution and macrodilution methods are considered acceptable [3]. Initial isolates, as well as those from patients who fail or relapse, should be tested to clarithromycin. Isolates of Mycobacterium kansasii should be tested to rifampin as resistance to rifampin is associated with treatment failure/relapse [3]. Broth microdilution minimum inhibitory concentration (MIC) determination for susceptibility testing is recommended for rapidly growing mycobacteria.

Slowly growing mycobacteria The slowly growing mycobacteria include organisms with wide ranging pathogenicity such as M. kansasii and Mycobacterium szulgai, which are probably second only to M. tuberculosis in terms of disease producing capability and Mycobacterium gordonae and Mycobacterium terrae, which rarely cause lung disease (table 1). MAC is typically the most common NTM to cause pulmonary disease but the frequency of M. avium versus M. intracellulare has varied between studies. Recommendations for treatment vary between guidelines as highlighted in table 3 [3, 49, 55].

Mycobacterium avium complex MAC includes the NTM species M. avium, of which there are several subspecies, M. intracellulare, and some that are as yet poorly described species. The traditionally recognised presentation of MAC lung disease has been as apical fibrocavitary lung disease similar to TB, usually in older males who have a history of cigarette smoking and alcohol abuse (fig. 1). MAC lung disease also presents with nodular and interstitial nodular infiltrates frequently involving the right middle lobe or lingula, predominantly in post-menopausal, nonsmoking Caucasian females. This form of disease, termed nodular/bronchiectatic disease, tends to have a much slower progression than cavitary disease. Nodular/bronchiectatic MAC lung disease is radiographically characterised by chest highresolution CT (HRCT) findings that include multiple small centrilobular pulmonary nodules and bronchiectasis (fig. 2).

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Treatment of MAC pulmonary disease involves a two to three drug regimen, which includes ethambutol, a rifamycin (rifampicin or rifabutin) and a macrolide (clarithromycin or azithromycin).

Mycobacterium xenopi

Mycobacterium malmoense

Mycobacterium kansasii

ATS: American Thoracic Society; BTS: British Thoracic Society; MAC: Mycobacterium avium complex. #: treatment should continue for 12 months beyond the date of culture conversion. Data from [3, 49, 55].

ATS: a fluoroquinolone could be substituted. BTS: addition of clarithromycin may be best in terms of efficacy but would be likely to increase risk of side-effects. Rifampicin Ethambutol¡ macrolide 12+

24

Rifampicin Ethambutol 12+

24

Rifampicin Ethambutol 12+

9

ATS: three times weekly therapy is recommended for those with non-cavitary disease. In cavitary disease, an aminoglycoside is recommended for the first 2 months of therapy. BTS: in patients with compromised immune defences continue treatment for 15–24 months or until the sputum has been negative for 12 months. ATS: specific combinations of these drugs are not described. 24 Rifampicin Ethambutol 12+ MAC

Rifampicin Ethambutol Macrolide Rifampicin Ethambutol Isoniazid Rifampicin Ethambutol Isoniazid¡ fluoroquinolone or macrolide Rifampicin Ethambutol Macrolide Isoniazid¡aminoglycoside

Drugs Drugs

Duration months#

Duration months

Comments BTS recommendations ATS recommendations Organism

Table 3. Recommendations for the treatment of select slowly growing nontuberculous mycobacteria

NTM INFECTIONS

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Unfortunately, treatment outcomes have varied significantly between studies [3, 56]. In a randomised controlled clinical trial conducted by the British Thoracic Society (BTS) comparing clarithromcyin versus ciprofloxacin in combination with rifampicin and ethambutol for treatment of pulmonary MAC, the clarithromycin-containing arm was associated with a higher all-cause mortality (48% versus 30%) but lower rates of failure and relapse (13% versus 23%) compared with the ciprofloxacincontaining arm [55]. In a previous BTS study, rifampicin and ethambutol were associated with a failure and relapse rate of 41% compared to 16% in the comparator arm, which contained isoniazid. Because the macrolides are the only antimicrobial agents for which there is a correlation between in vitro susceptibility and clinical response and the high rate of poor outcomes reported with rifampicin and ethambutol, the ATS recommends inclusion of a macrolide in all patients. [57–62]. Therapy three times a week is recommended for patients with noncavitary disease [3]: this recommendation is based on a study that demonstrated poor bacteriological responses in patients who were treated three times a week and had evidence of cavitary disease [63]. Intermittent therapy with ethambutol may be associated with a lower rate of optic neuritis [64]. In patients with extensive radiographic disease, cavitary disease, marolide resistance disease or treatment failure, an injectable aminoglycoside (amikacin or streptomycin) should be considered. A randomised trial from Japan reported that patients who received streptomycin three times a week for the initial 3 months of therapy along with three other drugs had a faster sputum conversion rate compared with those that were in the placebo arm [65]. However, long-term relapse rates were not different between arms. Macrolide-resistant MAC lung disease is associated with a poor prognosis [66]. The two major risk factors for macrolideresistant MAC disease are macrolide monotherapy or treatment with macrolide and inadequate companion medications. The

treatment strategy associated with the most success for macrolideresistant MAC lung disease includes the use of a multidrug regimen including a parenteral aminoglycoside (streptomycin or amikacin) and surgical resection (debulking) [66]. Clofazimine, in combination with ethambutol and a macrolide, has been used successfully to treat pulmonary MAC infections and, thus, may be a possible alternative drug for macrolide-resistant disease [67].

a)

Mycobacterium kansasii

According to the ATS, the recommended regimen for treating M. kansasii pulmonary disease includes daily rifampicin (600 mg? day-1), isoniazid (300 mg?day-1) and ethfoambutol (15 mg?kg-1?day-1), all administered for 12 months beyond the date of culture conversion [3]. However, the BTS recommends rifampicin and ethambutol therapy for a total of 9 months [49]. Substitution of clarithromycin for isoniazid has been associated with good short-term treatment results with daily [69] and intermittent therapy [70].

b) C.L. DALEY

M. kansasii is one of the most common causes of NTM lung disease in the USA, as well as some parts of Europe and Asia. While most patients with M. kansasii lung disease have upper lobe fibro-cavitary abnormalities similar to TB (fig. 3), essentially any pattern of radiographic abnormality can occur, particularly in HIV-infected patients [68].

Figure 1. A 59-year-old male smoker with cavitary Mycobacterium avium complex lung disease. The patient, who presented with cough, fatigue and weight loss, was found to have acid-fast bacilli on smear microscopy. Previous treatment with rifampicin and clarithromcyin resulted in macrolide resistance. a) Chest radiograph showing cavitary consolidation in right upper lobe, volume loss, pleural thickening and scattered nodular opacities. b) Computed tomography slice showing large cavity in right upper lung with adjacent consolidation and bilateral severe emphysema.

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Patients whose M. kansasii isolates have become resistant to rifampicin as a result of previous therapy have been treated successfully with a regimen that consists of highdose daily isoniazid (900 mg), highdose ethambutol (25 mg?kg-1?day-1) and sulfamethoxazole (1.0 g three times per day) combined with several months of streptomycin or amikacin [71]. The excellent in vitro activity of clarithromycin and moxifloxacin against M. kansasii suggests that multidrug regimens containing these agents are likely to be even more effective for treatment of a patient with rifampicin-resistant M. kansasii disease.

Mycobacterium malmoense Mycobacterium malmoense is considered the second most serious pathogen after MAC in northern Europe, although the clinical relevance of M. malmoense isolates has varied between studies. For example, in the Netherlands [72, 73], 70–80% of isolates are reported to be clinically relevant whereas in the USA, M. malmoense is seldom considered clinically significant. Patients with M. malmoense lung disease frequently have pre-existing obstructive lung disease and present with radiograph findings similar to other cavitary NTM lung disease pathogens.

NTM INFECTIONS

Figure 2. A 65-year-old nonsmoking female with history of cough, fatigue and sinopulmonary infections for several years. Her sputum was culture positive for Mycobacterium avium complex. Chest computed tomography slice showing right middle lobe and lingular bronchiectasis with atelectasis and consolidation. Note the centrilobular nodules in the dependent areas of the lower lobes bilaterally.

In a recent report, clarithromycin, rifampicin and ethambutol were compared with a regimen consisting of ciprofloxacin, rifampicin and ethambutol [55]. Overall, a more favourable response to therapy was reported with the macrolide-containing regimen, although overall mortality was not different between the two regimens. Although the optimal management of M. malmoense has yet to be determined [55, 73, 74] a two to four drug regimen is recommended that would include, at a minimum, ethambutol and rifampicin [73].

Mycobacterium xenopi

Figure 3. A 58-year-old female with Mycobacterium kansasii lung

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disease. The patient presented with cough, fever and weight loss. She was treated as a tuberculosis suspect initially but her sputum specimen grew M. kansasii. Chest computed tomography slice demonstrating a large right upper lobe cavity.

Mycobacterium xenopi is a common cause of NTM lung disease in Canada, the UK, and some parts of Europe [75]. Radiographic findings with M. xenopi pulmonary disease are variable but most often include upper lobe cavitary changes compatible with TB. M. xenopi isolates were reported to have favourable in vitro MICs to isoniazid, rifampin and ethambutol in a study from the Netherlands [75]; however, studies have failed to find a correlation between in vitro drug susceptibility results and treatment outcomes [76]. To date, the optimal treatment regimen has not yet been determined. In a multicentre, randomised trial comparing a regimen of

clarithromycin, rifampin and ethambutol with ciprofloxacin, rifampin and ethambutol [55], there was no difference in the treatment success, failure or relapse rates between groups. All-cause mortality was relatively high and somewhat higher in the ciprofloxacin arm, but death directly related to M. xenopi was low. Even with variable treatment regimens, antimicrobial treatment cured 58% of patients who met ATS criteria for M. xenopi lung disease in a retrospective study from the Netherlands [75]. Currently, the BTS recommends ethambutol and rifampicin for 24 months of therapy whereas the ATS recommends the addition of a macrolide and isoniazid and possibly an aminoglycoside depending on the severity of disease.

Rapidly growing mycobacteria Because many rapidly growing mycobacteria are not pathogenic in humans it is important to identify organisms within this group to the species level since this could affect both treatment and prognosis (table 1).

M. abscessus is one of the most common NTM infections in the USA and accounts for 65–80% of lung infections due to rapidly growing mycobacteria [29, 77]. Recent studies have demonstrated that M. abscessus consists of three species, M. abscessus (sensu stricto) Mycobacterium massiliense and Mycobacterium bolettii [78, 79]. In the USA, most patients with pulmonary disease due to M. abscessus complex are nonsmoking, Caucasian females with a median age of ,60 years [29, 80]. Similarly, in South Korea the median age of patients with pulmonary disease is 55 years and almost all of the patients are nonsmoking females [81]. However, in the Netherlands, over half of the patients are male many of whom have predisposing lung disease [82]. The chest radiograph usually shows multi-lobar, reticulonodular or mixed reticulonodular-alveolar opacities [29]. HRCT findings include the presence of cylindrical bronchiectasis with multiple small nodules, similar to MAC lung disease (fig. 4) [29, 83, 84]. Cavitation has been reported in 10–44% of patients [29, 80, 81].

Figure 4. A 70-year-old nonsmoking female with several year history of cough, fatigue and weight loss. The patient had a history of severe gastro-oesophageal reflux and recurrent pneumonias. Her sputum cultures were consistently positive for Mycobacterium abscessus. Chest computed tomography slice showing diffuse bronchiectasis with scattered centrilobular and subcentimeter nodules. There is an area of airspace of opacity in the posterior left lower lobe.

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Unfortunately, M. abscessus has demonstrated in vitro activity to only a few antimicrobial agents. The ATS recommends therapy with 2–4 months of intravenous antibiotics such as imipenem or cefoxitin plus amikacin given daily or three times per week [3]. Oral agents that have demonstrated in vitro activity should be included in the treatment regimen. Unfortunately, macrolides are the only oral agents typically active in vitro against M. abscessus. Studies have demonstrated the presence of an erythromycin ribosomal methylase gene, erm(41), in M. abscessus, which could result in the development of macrolide resistance and possibly affect treatment responses [77, 85]. Other drugs that sometimes demonstrate in vitro activity

C.L. DALEY

Mycobacterium abscessus complex

include linezolid and tigecycline, however, both drugs are associated with frequent adverse effects [86, 87]. Because of the high levels of in vitro resistance, cure is difficult to achieve in patients with lung disease due to M. abscessus. In South Korea, JEON et al. [81] reported the outcomes of 65 patients with pulmonary disease who were treated with a standardised regimen. Patients were hospitalised and treated with intravenous cefoxitin and twice daily amikacin plus oral clarithromycin, ciprofloxacin and doxycycline. After 1 month the intravenous drugs were stopped and the oral medications continued for a total of 24 months and at least12 months beyond the date of culture conversion [81]. 83% of the patients reported improvement in symptoms and 74% had radiographic improvement as documented by HRCT. Sputum conversion and maintenance of negative sputum cultures for .12 months was achieved in 38 (53%) patients. However, drugrelated adverse events were common. Neutropenia and thrombocytopenia associated with cefoxitin developed in 33 (51%) and four (6%) patients, respectively. Drug-induced hepatoxicity occurred in 10 (15%) patients. Cefoxitin had to be stopped, and in some cases switched to imipenem, in the majority of patients.

NTM INFECTIONS

In a recent report from Denver, CO, USA, the outcomes of 107 patients treated for pulmonary M. abscessus disease were reported [80]. Treatment regimens varied but followed current ATS recommendations. Cough, sputum production and fatigue remained stable, improved or resolved in 80%, 69% and 59% of patients, respectively. Treatment outcomes were disappointing: 20 (29%) out of 69 patients remained culture positive, 16 (23%) patients converted but relapsed, 33 (48%) patients converted to negative and did not relapse and 17 patients (16%) died during the study period. As noted previously, speciation of the rapidly growing mycobacteria may be important because outcomes may vary based on the species of NTM. KOH et al. [77] reported significant differences in the clinical, radiographic and microbiologic outcomes in patients treated for M. abscessus versus M. massiliense. Sputum conversion and maintenance of negative cultures occurred in 88% of patients with M. massiliense compared with 25% of patients with M. abscessus, despite receiving a similar treatment regimen. When isolates of M. abscessus were incubated with clarithromycin, all became resistant within 7 days and the MIC continued to increase at day 14. In contrast, none of the M. massiliense isolates acquired resistance upon exposure to clarithromycin. The erm(41) gene was present in all of the M. abscessus isolates but was partially deleted in the M. massiliense isolates. A combination of surgical resection and chemotherapy may increase the chance of cure in patients who have focal lung disease and who can tolerate resection. Among 14 (22%) patients with pulmonary M. abscessus infection in South Korea who underwent surgical resection, negative sputum culture conversion was achieved within a median of 1.5 months and was maintained in 88% of those with pre-operative culture-positive sputum. Similarly, in a study from the USA, patients who had surgical resection plus medical therapy were more likely to convert their cultures to negative and not relapse compared with medical therapy alone (65% versus 39%; p50.041) [80]. Moreover, significantly more patients who underwent surgery converted sputum cultures to negative and remained negative for at least 1 year when compared with those who received medical therapy alone (57% versus 28%; p50.022).

Mycobacterium chelonae and Mycobacterium fortuitum Although Mycobacterium chelonae and Mycobacterium fortuitum are less likely to cause lung disease than M. abscessus the clinical and radiographic presentations are similar [29, 88]. Of 26 patients in South Korea who grew M. fortuitum from two or more sputum specimens, 25 were not treated and none showed evidence of progressive disease over a median of 12.5 months of followup [88].

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Isolates of M. chelonae are usually susceptible to the macrolides, linezolid, tobramycin and imipenem and uniformly resistant to cefoxitin [89–91]. Other active drugs may include amikacin,

clofazimine, doxycycline and fluoroquinolones. The ATS recommends that treatment of M. chelonae infections should consist of at least two drugs to which in vitro drug susceptibility has been demonstrated. Unlike M. abscessus and M. fortuitum, M. chelonae does not appear to possess a copy of erm(41) [85]. In contrast to M. abscessus and M. chelonae, M. fortuitum demonstrates broader in vitro susceptibility to both oral and intravenous antimicrobial drugs including the newer macrolides, fluoroquinolones, tetracycline derivatives, sulfonamides and intravenous drugs imipenem and cefoxitin [3]. Although most isolates of M. fortuitum are susceptible in vitro to the macrolides, they should be used with caution because of the presence of erm(41) [92]. As with M. chelonae, M. fortuitum lung disease should be treated with at least two drugs to which in vitro susceptibility has been demonstrated [3].

Surgical therapy for NTM lung disease

The benefits must be weighed against the possible complications of surgery. In seven surgical series reported during the macrolide era, the rate of complications varied from 0% to 44% averaging approximately 25% [94–100]. In the largest study to date in Colorado (USA), MITCHELL et al. [99] reported the outcomes of 236 patients who underwent lung resection for NTM pulmonary disease over a 23-year period. Minor complications were reported in 18.5% of the patients with 31 (11.7%) suffering from serious complications. Bronchopleural fistula occurred in 11 (4.2%) cases. No operative mortality was reported in six case series and postoperative mortality ranged from 0% to 11%. In the study from Colorado, seven (2.6%) patients died as a result of the procedure; however, the mortality rate was only 0.6% for the last 162 patients that were operated on from 2001 to 2006. Many of these latter patients underwent video-assisted thoracoscopic surgery. Because case volume may be associated with outcomes, surgery should be performed by thoracic surgeons with extensive experience in performing this type of surgery [99].

C.L. DALEY

Patients who have failed a standard therapeutic regimen, particularly those who harbour resistant organisms, may benefit from surgical resection of the most affected areas. In 12 published series involving a total of 602 patients (range 8–236), the post-operative sputum culture conversion rate ranged from 82% to 100% with a mean conversion rate of 94% [93]. Long-term relapse was not reported in all studies but ranged from 0% to 13%.

Conclusion NTM represent a broad array of organisms with varying prevalence and pathogenicity. Pulmonary infections due to NTM appear to be increasing and the epidemiology is shifting toward a female predominance in some areas. Clinicians must consider clinical, radiographic and bacteriologic information when diagnosing NTM pulmonary infection. Although diagnostic criteria exist, these have yet to be prospectively validated. Consideration of the species of NTM is an increasingly important element of diagnosis and may impact the outcomes of therapy. Treatment regimens vary by NTM species as do treatment outcomes. Future areas of research should focus on the epidemiology of NTM infections, transmission of infection, risks for disease progression, development of new diagnostics and ultimately development of new drugs and treatment regimens. Until we have a better understanding of the transmission and pathogenesis of these difficult to treat infections, it will be difficult to formulate a rationale plan for prevention of infection.

Statement of interest 125

None declared.

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Chapter 9

Ciliary dyskinesias: primary ciliary dyskinesia in adults L.J. Lobo*, M.A. Zariwala# and P.G. Noone*

PCD IN ADULTS

Summary Primary ciliary dyskinesia (PCD) is a genetic disorder of cilia structure and function, chronic infections of the respiratory tract, fertility problems and disorders of organ laterality. Establishing a definitive diagnosis can be challenging, requiring a compatible phenotype and detection of ciliary functional and ultra-structural defects, along with newer screening tools such as nasal nitric oxide and genetics testing. 10 known PCDcausing mutations within two genes are now available in a clinical panel, and in the future, comprehensive genetic testing may serve to identify young infants with PCD to improve the long-term outlook for patients with the disease. Therapy includes regular pulmonary function testing and monitoring of sputum flora to allow a targeted approach to treatment. Referral to an academic centre with expertise in bronchiectasis and/or PCD is prudent to ensure access to the most recent diagnostic testing and therapies. With increased understanding of the disease it is likely that we will expand the definitions of classic and non-classic PCD, as well as its relationship to less common ciliopathies. Keywords: Bronchiectasis, cilia, dynein, mucociliary clearance, nitric oxide, primary ciliary dyskinesia

C

*Division of Pulmonary Diseases, and # Dept of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC, USA. Correspondence: P.G. Noone, CB 7020, Pulmonary Division, University of North Carolina School of Medicine, Chapel Hill NC 275997020, USA, Email [email protected]

Eur Respir Mon 2011. 52, 130–149. Printed in UK – all rights reserved. Copyright ERS 2011. European Respiratory Monograph; ISSN: 1025-448x. DOI: 10.1183/1025448x.10003910

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iliary dyskinesia refers to a syndrome of oto-sino-pulmonary disease with other accompanying phenotypic features. It is often referred to as primary ciliary dyskinesia (PCD) and sometimes referred to as immotile cilia syndrome (ICS) or Kartagener syndrome, or occasionally the motile ciliopathies [1–3]. PCD is currently the preferred term [4]. Although secondary ciliary dyskinesia may be seen in diseases associated with acute and chronic airway inflammation and infection, this chapter will focus primarily on the genetically transmitted form of the disease, that is PCD, rather than nongenetic, generally secondary forms of the syndrome [5]. Since the hallmarks of the disease are chronic lung disease with bronchiectasis, a brief discussion of the major respiratory features (bronchiectasis) is included, as well as a brief review of airway host

defence. This will allow a better understanding of the role of cilia in health and disease. This chapter will focus on disease in adults, as an excellent review of PCD in children was recently published [6].

The major clinical characteristics of PCD are chronic ear, sinus and lower airways symptoms and signs from birth because of the failure of one of the major airway defence mechanisms, that of MCC. By adulthood, bronchiectasis is invariable and is characterised by an abnormal and permanent dilation of bronchi. It is the consequence of inflammation and destruction of the structural components of the bronchial walls, usually in the walls of the medium-sized airways, often at the level of segmental and sub-segmental bronchi. Most experts accept that a ‘‘vicious cycle’’ of infection and inflammation is created by the basic defect in airway host defence. This generates airway damage and further impairment of airway clearance, eventually with chronic colonisation/infection with a variety of microorganisms, leading to further infection and inflammation and eventually destruction of conducting airways and even alveolar surfaces. In its most severe form, bronchiectasis may lead to respiratory failure and death. For the clinician faced with a patient with bronchiectasis, the diagnostic algorithm involves sifting through the various causes of the disease, with a predominant cause often elusive; thus, it may be labelled either as idiopathic or, with an appropriate history, as post-infectious bronchiectasis [9]. However, a careful clinical history, together with focused tests, may find an underlying cause such as CF or PCD, which is almost always helpful from genetic, prognostic, therapeutic and healthcare system standpoints.

L.J. LOBO ET AL.

PCD is a rare, usually autosomal recessive disease characterised by oto-sino-pulmonary disease, including bronchiectasis, organ laterality defects and male infertility. First described early in the 20th century, its disease origins as a defect in ciliary structure and function were described in Sweden in the 1970s [7, 8]. The last decade or so has seen resurgence in interest in PCD, specifically a new focus has emerged from several groups worldwide on more precisely defining the major aspects of the disease phenotype, including elucidating the molecular basis for the ciliary abnormalities. Such data will help clinicians establish a diagnosis of PCD (which can be difficult in many circumstances), which in turn, will hopefully allow more targeted therapeutic approaches. Cystic fibrosis (CF) has long been recognised as a prototype genetic disease associated with severe pulmonary disease and bronchiectasis, with intense research activity devoted to CF pathogenesis and treatment over the last several decades. PCD offers a similar disease model to CF, albeit with a different basic aetiology, offering complementary insights into significant human disease associated with dysregulation of the mucociliary clearance (MCC) apparatus in the respiratory tract.

Thus, structural and functional abnormalities of motile cilia and human flagellated cells (sperm) explain the complex PCD phenotype involving various organ systems. The motile cilia in the respiratory tract are vital components of the mucociliary apparatus used in airway clearance and the flagellated structures are important in the male and female reproductive systems. Left–right asymmetric organ defects may also be part of the phenotype, for example, situs inversus totalis, commonly known as Kartagener syndrome [10].

Normal cilia structure and function In addition to humoral, cellular and innate immune systems, the respiratory tract has developed complex local physical defences to protect the airways from the myriad of inhaled pathogens, allergens and other inhaled noxious particles. One such mechanism is the mucociliary escalator, which mechanically eliminates bacteria and particulates that deposit on the epithelial surface of the respiratory tract.

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Cilia are hair-like attachments found on the epithelial surfaces (,200 per cell) of various organs and are anchored on by a basal body to the apical cytoplasm and extend from the cell surface into the extracellular space. Each cilium is composed of approximately 250 proteins organised into longitudinal microtubules, which make up the basic axonemal structure [11]. Based on the

arrangement of the microtubules, cilia are classified into motile cilia, Inner dynein arm primary cilia and nodal cilia [12]. Motile cilia are the cilia found in the apical surfaces of the upper and Microtubule A lower respiratory tract, the ependymal cells lining the ventricles of the central nervous system, the Microtubule B oviducts of the female reproductive tract and the flagellum of the male Radial spoke sperm. Motile cilia are organised into nine microtubule pair doubCentral complex lets, surrounding a central pair creating a distinctive 9+2 arrangement (fig. 1) [3]. The central pair is linked to the surrounding pair Figure 1. Diagram of a cross-section of the basic ciliary structure. doublet through an array of radial spoke proteins and the surrounding pair doublets are linked to one another via nexin linked proteins. Through ATP-containing dynein arms on the peripheral microtubules, the microtubules slide by one another to produce ciliary motion [13]. The protein links between the microtubules limit the degree of sliding and allow the cilium to bend. Dyneins can be sub-divided into axonemal and cytoplasmic dyneins. Axonemal dyneins move cilia and flagella, as described previously, while cytoplasmic dyneins are involved in the organisation of spindle poles during mitosis [14]. Axonemal dyneins form two structures, the inner and outer arms, and are attached to the microtubules of the nine outer doublets throughout the length of the axoneme, thus they are central to the process of the bending of the cilium or sperm tail. Through coordinated and synchronised bending, wave like movements occur at ,16 Hz, which function to propel mucus and adherent particles/bacteria on the surface of the airway. Integral to the normal function of cilia is normal airway periciliary fluid layer composition and function. One of the main pathogenetic mechanisms in CF is thought to be dysregulation of this fluid layer, which bathes cilia with a thin mucus layer on top [15]. It can be readily seen, therefore, that two discrete abnormalities of MCC, one involving the cilia themselves, the other involving the fluid that bathes the cilia, may result in a broadly similar airway phenotype (bronchiectasis).

PCD IN ADULTS

Outer dynein arm

Finally, nodal cilia occur during embryonic development. In contrast to the 9+2 structure of motile cilia, they have a 9+0 configuration. They have a very interesting rotational movement, resulting in leftward flow of extracellular fluid, which is important for cell signalling during the development of normal human left–right asymmetry (situs solitus) [12]. Defects in the nodal cilia may cause errors with left–right body orientation; for example, dextrocardia, situs inversus totalis and situs ambiguous [16–18]. This explains the association of organ laterality defects in PCD, as well as other rare genetic diseases such as polycystic kidney disease, Senior–Loken syndrome, Alstrom syndrome, Bardet–Biedl syndrome and retinitis pigmentosa [19].

Clinical manifestations The clinical signs and symptoms of PCD are shown in table 1.

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The clinical phenotype that occurs with defective ciliary structure and function is fairly predictable. Cells lining the nasopharanx, middle ear, paranasal sinuses, the lower respiratory tract and the reproductive tract contain cilia and are generally affected in PCD when the disease is fully expressed. In contrast to CF, pancreatic function is preserved, and hepatobiliary disease is usually not a feature. In general, the clinical course of the disease is milder, with absence of the systemic problems associated with CF such as nutritional issues and diabetes. Although there are few data

By system affected

By age of presentation

Middle ear Chronic otitis media with tube placement Conductive hearing loss Nose and paranasal sinuses Neonatal rhinosinusitis Chronic nasal congestion and mucopurlent rhinitis Chronic pansinusitis Nasal polyposis Lung Neonatal respiratory distress Chronic cough (lifelong) Recurrent pneumonia Bronchiectasis Genitourinary tract Male and female fertility problem or history of in vitro fertilisation Laterality defects Situs inversus totalis Heterotaxy (¡ congenital cardiovascular abnormalities) Central nervous system Hydrocephalus (rare) Eye Retinitis pigmentosa

Family history Communities or ethnicities with consanguinity Close (usually first degree) relatives with clinical symptoms Antenatal Heterotaxy on prenatal ultrasound Newborn period Continuous rhinorrhoea Respiratory distress or neonatal pneumonia Childhood Chronic productive cough Atypical asthma unresponsive to therapy Idiopathic bronchiectasis Chronic rhinosinusitis Recurrent otitis media with effusion Adolescence and adult life Same as for childhood Subfertility and ectopic pregnancies in females Infertility in males with immotile sperm Sputum colonisation with smooth/mucoid pseudomonas, other Gram-negative organisms, or nontuberculous mycobacteria

on life expectancy in PCD, it is believed from clinical experience, and some cross-sectional and longitudinal studies, that PCD carries a more favourable prognosis than CF [20, 21]. Nonetheless, the disease may be quite severe and some patients develop respiratory failure requiring consideration for lung transplant [21]. As with CF, a clue to the diagnosis is a family history of PCD, particularly in populations with high levels of consanguinity [22]. For example, there is a reported 1 in 2,200 prevalence of PCD in the Asian population of Britain [23]. The prevalence of PCD in the general population is unknown, although estimates based on mass radiology studies in differing countries (Scandinavia and Japan) suggest a range of ,1:16,000 to 1:40,000 depending on the techniques and calculations involved, and taking into account the likelihood that its prevalence is almost certainly underestimated, even in these focused studies [6].

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Table 1. Clinical signs and symptoms of primary ciliary dyskinesia

Oto-sino-pulmonary disease

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At birth, newborns with PCD often present with a clinical syndrome of neonatal respiratory distress, indicating the importance of ciliary function in clearing the fetal lung [24, 25]. It is useful to consider PCD with this clinical history, whether in later childhood or adulthood (although adult recall of neonatal events may not be reliable). Early childhood atelectasis, pneumonia, hypoxaemia or respiratory failure can be seen [4, 26]. Frequently, these problems may be attributed to other aetiologies (for example wet lung, aspiration or pneumonia), and PCD maybe overlooked for some time. This is borne out by data showing the mean age at diagnosis in children with PCD was .4 years even when persistent pulmonary symptoms occurred, such as chronic cough and persistent rhinitis [27]. Children with wheezing may also be labelled as having ‘‘atypical’’ asthma that is unresponsive to appropriate therapy [28]. Frequently, infants and young children have recurrent upper respiratory tract symptoms, including chronic rhinosinusitis and chronic otitis media [27]. Nasal polyps and conductive hearing loss from the recurrent infections and inflammation is common [29]. Most expert paediatricians discourage placement of drainage

tubes (‘‘grommets’’), as these frequently lead to otorrhoea, worsening of the tympanic scarring and hearing loss over the long term [6, 25]. Although adult nutritional issues are generally not a feature of PCD, infants with PCD may have significant issues with severe gastro-oesophageal reflux, feeding and ability to obtain adequate nutrition and tend to be on the lower end of the growth curve [30]. In later childhood and early adulthood, the impaired MCC in the lower respiratory tract leads to recurrent episodes of bronchitis and pneumonias, which eventually leads to bronchiectasis of the middle and lower lobes [31, 32]. In all age groups, chronic cough is a predominant feature of the disease (often reported by family members), both in response to the chronic inflammation and as a compensatory mechanism for defective ciliary function and MCC [33]. Adults may develop clubbing as a marker of long standing pulmonary disease. By the time patients present to adult clinics, many adults frequently have a history of lobectomy in early life, prior to the diagnosis being established. Since this procedure cannot usually correct what is, after all, a general problem in the lung, it can rarely be recommended [21]. Typically the disease manifests itself as intermittent exacerbations of infectious symptoms, but always with a baseline level of chronic symptoms (as is usual for most patients with bronchiectasis, whatever the cause) [34]. At all stages of the disease the focus should be on minimising symptoms, improving quality of life and slowing declines in lung function (see later). Another unusual, but recently reported complication of chronic airway diseases in older patients with PCD is that of lithoptysis, that is, expectoration of stone-like masses from the airways [35]. The hypothesis is that calcite stone formation is a bio-mineralisation response to the chronic airway inflammation and retention of infected airway secretions in some patients with PCD.

PCD IN ADULTS

Airway microbiology/imaging It is not infrequent that adults present to bronchiectasis clinics having rarely had sputum cultures [21]. However, monitoring of the flora of the airway is important, since older adults often harbour problematic organisms that may require specific treatment [21]. Based on monitoring protocols developed for CF and small studies in PCD, it is recommend that airway cultures be performed every 3 to 6 months [20, 21]. Initially, cultures of airway secretions (sputum cultures) grow Haemophilus influenzae, Streptococcus pneumoniae and Staphylococcus aureus. Once bronchiectasis is evident on chest imaging (high-resolution computed tomography (HRCT)), smooth and mucoid Pseudomonas aeruginosa and other opportunistic pathogens such as nontuberculous mycobacteria (NTM) may be present. In cross-sectional studies series, all adults .30 years of age had evidence of bronchiectasis, with an increasing prevalence of these organisms [21, 36].

Pulmonary function testing Most patients demonstrate progressive obstructive defects with advancing age. Although there are few longitudinal data, cross-sectional studies suggest that the disease is milder than CF in terms of the progression of loss of lung function [21, 36]. Nonetheless, it is important in order to guide treatment, to obtain baseline and serial measures of lung function and assess disease severity and progression, as some patients will develop severe or end-stage lung disease [21]. Ongoing studies, involving larger numbers of patients in multiple centres, will better define longitudinal markers and the natural history of the disease.

Radiology

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With more abundant and specialised imaging, bronchiectasis is being observed more frequently in general. Thus, PCD may be considered in patients with HRCT-proven bronchiectasis. The computed tomography scan characteristics of bronchiectatic airways are well described [37]. However, HRCT features alone do not usually allow a confident distinction between cases of idiopathic versus post-infectious bronchiectasis versus known causes or associations of bronchiectasis, although there are certain patterns of disease distribution that support a diagnosis of PCD, for example, a predilection for the middle and lower lobes has been reported in patients

with PCD, in contrast to the upper lobe distribution of cylindrical bronchiectasis in patients with CF [38]. Some authors suggest that absence of bronchiectasis on a HRCT scan may have a role in excluding the diagnosis of PCD, at least in adults [31].

Reproductive tract abnormalities Infertility in both males and females is also a prominent feature. 98–99% of males with PCD have impaired spermatozoa motility secondary to defective sperm flagella [39]. Data are scattered in females, but a consistent feature is that of normal or delayed fertility in some, while other females show impaired fertility with an increased risk of ectopic pregnancy, presumably because of impaired ciliary function in the oviduct [40].

Organ laterality and other anatomic defects

Rare associations of PCD PCD is occasionally seen with rare diseases linked to abnormalities in primary cilia or sensory cilia, for example in the kidney, retina and embryonic node, which may lead to a wide spectrum of clinical features. An example is PCD with retinitis pigmentosa. Mutations in the X-linked retinitis pigmentosa GTPase regulator gene (RPGR) gene have been identified in a few cases of PCD cosegregating with retinitis pigmentosa [41, 42]. Ciliary dysfunction in both respiratory epithelium and the photoreceptors of the retina seems to be the common factor [42]. Hydrocephalus may be seen in mice with PCD, but its association in humans is less clear, the problem may be secondary to the impaired cilia that line the ventricular ependymal cells of the central nervous system, which helps cerebrospinal fluid flow through the sylvian aqueduct [43–45].

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During the embryonic period, thoraco-abdominal orientation is determined via the unidirectional, rotating beat of nodal cilia. With abnormal nodal ciliary structure and function, thoracoabdominal organ orientation is random. This leads to situs inversus with reversal of the thoracic and abdominal organs in ,50% of patients with PCD [16, 21]. Occasionally, laterality defects are not ‘‘pure’’, that is, situs ambiguous/heterotaxy may be present. This is the phenomenon of left– right asymmetry within specific organ systems, leading to either sole or randomly combined anatomical deformities of the heart, liver and spleen. A recent series found that at least 6% of patients with PCD have heterotaxy, including complex congenital heart defects [17]. Defects in the outer dynein arm (ODA) may be a more common cilia abnormality in patients with laterality defects than that of the inner dynein arm (IDA) or central apparatus [17].

Diagnostic approaches Overview

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Since the first reports of abnormal ciliary structure as the cause of PCD, the diagnosis of PCD has usually been established by obtaining nasal samples of airway cilia for examination under light and electron microscopy. With appropriate techniques, ciliary motion (absent or dyskinetic in PCD) may be defined and ciliary ultra-structure examined for abnormalities, the classic being absent or short/ stubby dynein arms. However, these tests are quite dependent on technical factors and local expertise, and thus it can sometimes be a challenge to definitively diagnose PCD. Recently, however, the diagnostic work-up for PCD has evolved to encompass other methodologies, for example, measures of nasal nitric oxide (NO), more sophisticated analyses of ciliary structure and function and genetic testing (see later). Often, the resources needed to make a definitive diagnosis are only available in specialised centres. Nonetheless, a history yielding the symptomatic clues above should prompt consideration of the diagnosis and, if necessary, referral to the growing number of centres with an interest and expertise in the diagnosis and treatment of PCD and related diseases. An algorithm of currently available tests is presented to help the clinician work through the process (fig. 2). It goes without saying that prior to consideration of the diagnosis of PCD, and embarking on a detailed

Clinical suspicion of PCD

CT chest scan with bronchiectasis (middle/lower lobe predominantly and/or heterotaxy)

No PCD unlikely# Rule out cystic fibrosis with a sweat chloride test and/or CFTR gene mutation analysis

Yes Other aetiologies of bronchiectasis ruled out

No

Rule out primary immunodeficiencies with Ig levels (IgG, IgA and IgM) and serum electrophoresis and consider vaccine response Rule out connective tissue disorders with RF, ANA and ANCA

Yes

Nasal NO level reduced (usually 5– 20% of normal)

Consider ABPA, asthma, allergic rhinitis, gastrooesophageal reflux disease and α1-antitrypsin deficiency No

PCD unlikely#

PCD IN ADULTS

Yes Cilia examination via nasal biopsy Yes Ciliary beat frequency and pattern

Normal

PCD not ruled out¶

Abnormal Electron microscopy with ultra-structural defect in cilia

Normal

Normal beat frequency, pattern and ultra-structure makes PCD unlikely, but could be non-classic disease

Yes

PCD likely

Consider genetic testing for DNAI1 and DNAH5 using clinical panel at select institutions (accounts for ~40%) and there are select genetic tests and gene analysis available at specific academic centres

Figure 2. Diagnosis algorithm for primary ciliary dyskinesia (PCD) in adults. For clinical signs refer to table 1. CT: computed tomography; NO: nitric oxide; CFTR: cystic fibrosis transmembrane conductance regulator; Ig: immunoglobulin; RF: rheumatoid factor; ANA: antinuclear antibodies; ANCA: antineutrophil cytoplasmic antibodies; ABPA: allergic bronchopulmonary aspergillosis. #: if clinical suspicion is still high for PCD other, more specific tests may be undertaken; ": normal ciliary beat frequency and pattern does not completely rule out PCD.

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work-up, other diseases can be considered and ruled out as appropriate [9]. As there are wide variations in PCD presentation and phenotype there may be overlap with CF, immunological deficiencies, allergic bronchopulmonary aspergillosis (ABPA) and other causes of bronchiectasis. Other chapters in this Monograph address these diseases in considerable detail.

Screening tests versus diagnostic tests Tests of ciliary function can be divided into those that are indirect and may be used to screen patients (e.g. the nasal saccharin test) and those that definitively assess function and structure (ciliary beat frequency (CBF)/pattern tests and electron microscopy studies). Newer screening/ diagnostic tests currently undergoing study include nasal NO, which may reflect ciliary structure function indirectly, immunofluorescent analysis of ciliary proteins, high-speed video-microscopy to quantitate ciliary motion and clinically available panels of genetic tests known to be associated with ciliary structural abnormalities.

MCC: the saccharin test The saccharin test is cheap and can be readily performed in the clinical setting as a screening test. However, it is subject to an array of technical factors that render it less reliable than other methodologies. A 1–2 mm particle of saccharin is placed on the inferior nasal turbinate 1 cm from the anterior end (if too far anterior cilia actually beat forwards from the nose). The difficult part is that the patient must sit quietly with the head bent forward without sniffing, sneezing, coughing, eating or drinking. The time it takes for the patient to taste the saccharin is a rough measure of nasal MCC. Generally, tasting saccharin in ,30 minutes is normal. Patients with rhinosinusitis commonly taste it within 60 minutes. If it is not tasted within 60 minutes, PCD can be considered. The test is not suitable for small children who will not sit still for 60 minutes, patients with a poor sense of taste and patients with a cold within the past 6 weeks [46].

NO is present in high concentrations in the upper respiratory tract and is produced by the paranasal sinus epithelium [47]. NO is produced enzymatically from L-arginine by several isoforms of NO synthase. NO appears to contribute to local host defence, modulate ciliary motility and serve as an aerocrine mediator in helping to maintain adequate ventilation–perfusion matching in the lung [48]. Abnormal values of nasal NO have been reported in various sinus and lung diseases; for example, acute and chronic sinusitis, CF and nasal polyposis [48]. Quite fortuitously, low nasal NO levels were first reported in PCD in the early 1990s by a Scandinavian group researching exhaled NO in a variety of normal and diseased states [49]. The observation has been replicated on several occasions and, although not fully understood at a mechanistic level, it seems to be a robust index of classic PCD [21, 50]. In individuals with PCD, levels of exhaled NO are extremely low (,10% of normal values) even when compared with patients with CF and other sinus disorders, where nasal NO may be low, although not usually in the PCD range [51, 52]. Interestingly, in one study, carriers (nonsmoking parents of patients with PCD) had intermediate levels of nasal NO [21]. Confirmation of the diagnosis of PCD requires further diagnostic tests. Nevertheless, the highly reproducible nature of low nasal NO levels make it a valuable screening tool [53].

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NO levels

Ciliary function

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Transnasal brushings or nasal scrape samples may be obtained fairly readily via direct visualisation of the inferior turbinate, without local anaesthesia or sedation [54]. Ciliary beat patterns and frequency can be seen under direct visualisation using a microscope, and classed as qualitatively normal, dyskinetic or immotile [21, 55]. For more quantitative measures, CBF can be measured and the ciliary waveform can be analysed using high-speed digital video imaging to differentiate between abnormal beating cilia and the normal beat patterns [56]. A cilium can be viewed in slow motion or frame by frame, with 40 to 50 frames per ciliary beat cycle [57]. Normal cilia beat forward and backwards within the same plane, with no sideways recovery sweep. Recent advances in computer image processing software may help standardise measures of waveform and direction of multiple cilia, as a measure of the effectiveness of ciliary transport [58, 59]. This software may

also efficiently compute ciliary activity with accuracy and reproducibility. CBF and beat pattern abnormalities have been associated with specific ultra-structural defects such as isolated outer arm defects, isolated inner arm or radial defects or transposition defects [58]. If patients have both a normal CBF and a normal beat pattern, then classic PCD can reasonably be excluded. However, if one or the other is abnormal, further studies are necessary. As with any studies of cilia structure and function, it is critical to exclude ongoing inflammation as a cause of secondary ciliary dysfunction leading to false positives [5].

PCD IN ADULTS

Ciliary structure In patients with an appropriate phenotype, suspicion for PCD for other reasons (for example, respiratory symptoms and a sibling with disease) or positive screening tests (saccharin test, nasal NO or CBF abnormalities), the axonemal structure of the respiratory cilia should be studied using transmission electron microscopy (TEM) [8]. Inflammatory influences can be avoided by sampling the patient in a stable state, post-antibiotics or, if in vitro testing, by culturing the epithelial cells in an inflammation-free environment. The yield may be higher in patients with sino-pulmonary symptoms rather than isolated upper or lower respiratory tract symptoms [60]. There are a number of structural phenotypes associated with PCD [61]. Most cases of PCD are due to a lack of ODA, or a combined lack of both IDA and ODA [21]. Less common defects include IDA defects alone or defects in combination with radial spoke defects, or central microtubule pair defects such as transposition or central microtubular agenesis [62–64]. In a proportion of patients with PCD, no structural defects were defined using existing TEM techniques [53, 60]. This, despite a strong phenotype, defined ciliary functional abnormalities and demonstrated genetic defects, underscoring the notion that the disease is almost certainly under-diagnosed, due to the hitherto reliance on TEM as the ‘‘gold standard’’ for diagnosis of the disease. As seen later, advances in molecular techniques will probably allow a broader definition of PCD (classic and non-classic PCD, akin to the situation with CF), leading to more efficient diagnosis with subsequent beneficial downstream effects for earlier diagnosis, treatment and improved long-term clinical outcomes.

Immunofluorescent stains Immunofluorescent analysis using antibodies directed against the main axonemal components has recently been used to facilitate identification of structural abnormalities of cilia, and is used in diagnosis in some centres in Europe [65, 66]. PCD patients with ODA defects have absence of DNAH5 staining from the entire axoneme and accumulation of DNAH5 at the microtubuleorganising centre as compared with normal individuals with normal DNAH5 staining along the ciliary axoneme [66]. Recent work has also shown that antibody-based techniques can diagnose not only ODA but also IDA abnormalities caused by KTU mutations in PCD [67]. In the future, it may be possible to develop a panel of antibodies directed towards multiple ciliary proteins that may enable the screening of respiratory epithelial samples.

Genetic testing Overview

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As the molecular underpinnings and the genetics of PCD become more defined, genetic testing may overcome some of the drawbacks of the currently available diagnostic tests. Given the complexity of ciliary structure and the genetic heterogeneity of PCD, finding gene mutations causative for PCD has been challenging. Fortunately, non-human models have helped in the process of discovery. Since the basic structure of cilia is highly conserved across species, an example being a simple green alga, Chlamydomonas reinhardtii, extensive information has been gleaned regarding the structure, function and genetics of human cilia, specifically identifying candidate proteins and genes from mutant Chlamydomonas that are critical for normal ciliary function (e.g. slow swimmers with ODA defects and mutant c-heavy chain dynein) [68]. Initial mutations

Table 2. Primary ciliary dyskinesia-causing genes in humans showing extensive locus heterogeneity Human gene

DNAI1 DNAI2 DNAH5 DNAH11 TXNDC3 KTU/PF13 LRRC50 CCDC39 CCDC40 RSPH4A RSPH9

Chromosomal location

Axonemal component

Ultra-structure of patients with mutations

[Ref.]

9p13.3 7q25 5p15.2 7p21 7p15.2 14q21.3 16q24.1 3q26.33 17q25.3 6q22.1 6p21.1

ODA IC ODA IC ODA HC ODA HC ODA IC/LC Cytoplasmic# Cytoplasmic# Ciliary Axoneme Ciliary axoneme RS RS

ODA defects ODA defects ODA defects Normal ultra-structure ODA defects ODA+IDA defects ODA+IDA defects Axonemal disorganisation Axonemal disorganisation Transposition defect CP defects and normal ultra-structure

[69–73] [74, 75] [76–78] [79–83] [84] [67] [85, 86] [87, 88] [87, 89] [90] [90]

found using the candidate gene approach include mutations in DNAI1, homologous to the Chlamydomomas genes IC78. This was discovered in PCD patients with ODA defects and functional ciliary abnormalities [69, 70]. Since then, there have been several more PCD-causing gene mutations published, using a variety of approaches (table 2). Homozygosity mapping in large families that may or may not be consanguineous, but have multiple affected and unaffected siblings can be successfully used to identify disease-causing genes. This method utilises the marker analysis to look for the shared region of the genome from affected and unaffected individuals, to identify the chromosomal locus/loci shared between the affected siblings. Genes within the shared locus/loci are candidates, which can be further aided by the candidate gene approach to prioritise the genes to be tested from the shared locus. OMRAN et al. [91] successfully used this method to localise the shared locus in affected individuals from a large consanguineous family and identified mutations in the DNAH5 gene. Genome-wide linkage analysis, another approach to find disease-causing mutations, using 31 multiplex families with PCD failed to identify disease-causing genes [92]. The main limitation of genome-wide linkage analysis is the extensive genetic and ultra-structural heterogeneity in PCD that limits the comparison of data across the families to get meaningful log of odds (LOD) genetic linkage scores that helps indicate the possible disease-causing loci. Other methodologies include the comparative computational analysis approach, which identifies candidate genes using the DNA information collected from various sequencing projects from various distinct species. It assumes that higher-level organisms independently lost certain genetic information during evolution once the information coding for the specific processes was obsolete. Using subtraction analysis it is possible to find candidate genes necessary for cilia formation and function by comparing the genome of a ciliated eukaryote with eukaryotes not dependent on cilia [93, 94]. A comprehensive discussion of the molecular basis of PCD is beyond the scope of this chapter; the reader is referred to KNOWLES et al. [4].

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DNAI1: dynein, axonemal, intermediate chain 1 gene; DNAI2 : dynein, axonemal, intermediate chain 2 gene; DNAH5: dynein, axonemal, heavy chain 5 gene; DNAH11: dynein, axonemal, heavy chain 11 gene; TXNDC3 : thioredoxin domain containing 3 (spermatozoa) gene; KTU/PF13 : Kintoun; LRRC50 : leucine-rich repeat containing 50; CCDC: coiled-coil domain containing; RSPH4A: radial spoke head 4 homologue A gene; RSPH9 : radial spoke head 9 homologue; ODA: outer dynein arm; IC: intermediate chain; HC: heavy chain; LC: light chain; RS: radial spokes; IDA: inner dynein arm; CP: central pair. #: cytoplasmic protein required for the dynein arms assembly.

DNAI1 and DNAH5 are associated with ODA defects in PCD

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Mutations in DNAI1 and DNAH5 [69, 70, 71–73, 76–78] that encode dynein axonemal intermediate chain 1 and heavy chain 5, respectively, have been well documented in several studies as causative for PCD. DNAI1 accounts for ,2–10% of patients with PCD, although if one ‘‘selected’’ the phenotype to include patients with ODA defects alone, this increases to ,4–14%.

The commonest mutation (founder mutation) in DNAI1 is IVS+2_3insT, accounting for .50% of mutations. DNAH5 is a heavy chain dynein and mutations in the gene were initially found in a large inbred family of Arab descent. Subsequent studies show mutations in DNAH5 to be present in ,15–28% of patients with PCD. Together therefore, DNAI1 and DNAH5 account for ,20–40% of patients with classic disease with ODA defects. Despite extensive allelic heterogeneity, four exons in DNAI1 and five exons in DNAH5 represent mutation clusters, which became the basis of development of the clinical genetic testing for PCD.

PCD IN ADULTS

Miscellaneous other mutations associated with PCD Mutations have been identified in other genes in patients with PCD, specifically, DNAH11, DNAI2, KTU, RSPH9, RSPH4A, TXNDC3 and LRRC50, CCDC39 and CCDC40 (table 2) [26, 67, 74, 79–81, 84, 85, 88–90]. Some genotype–phenotype associations have been defined amidst the plethora of mutations found, primarily at the ultra-structural level (rather than at the clinical level). Mutations in DNAH5, DNAI1 and DNAI2 are exclusively seen in patients with ODA defects, whereas mutations in KTU and LRRC50 are exclusively seen in patients with combined ODA+IDA defects [4]. The genetics of the DNAH11 (which encodes dynein axonemal heavy chain 11) mutation are quite interesting as it was found in a patient with proven CF and situs inversus. It was not clear if this patient had PCD/Kartagener syndrome, or isolated situs inversus, as there is an obvious phenotypic overlap between the CF and PCD. However, the patient had abnormal ciliary beat pattern as seen in PCD, normal ciliary ultra-structure, but with a mutation in the DNAH11 gene that was assumed to be linked to the situs inversus [95, 96]. Subsequently, mutations in DNAH11 were unequivocally shown to be PCD causing in a large German kindred and more recently two patients with PCD were found to harbour mutations in DNAH11 [80, 81]. All of the patients with DNAH11 mutations presented with normal dynein arms. This phenotype highlights the difficulty in diagnosis in those patients with a strong clinical phenotype, but with normal cilia on TEM analysis. Mutations in DNAI2 that encode for a dynein axonemal intermediate chain 2 have been identified in 4% of PCD patients with ODA defects [79]. In contrast to the above proteins and genes, which encode for dynein proteins, KTU is a cytoplasmic protein, required for the assembly of the dynein complex [67]. First noted to be mutated in Mekada fish with laterality defects, and subsequently Chlamydomonas, it was then found to be mutated in PCD patients with both IDA and ODA defects (logical since it is required for normal ODA and IDA assembly and transportation). Mutations in KTU are seen in ,12% of PCD patients with combined ODA and IDA defects. RSPH9, which encodes for the radial spoke head protein 9, was identified as being a PCD-causing gene using homozygosity mapping in two Arab Bedouin families. Subsequently, an identical homozygous 3-bp inframe deletion mutation was identified in both families. Interestingly, the ultra-structure analysis of patients from one family depicted 9+2 or 9+0 microtubular configuration, and from the other family normal ciliary ultra-structure was seen [90]. Using homozygosity mapping in three inbred Pakistani families, RSPH4A was identified as a PCD-causing gene that encode another radial spoke head protein 4A. Ultra-structural analysis showed transposition defects with the absence of a central pair and 9+0 or 8+1 configuration. TXNDC3 (encoding thioreduxin domain-containing protein 3) is a component of the sperm flagella ODA, and a nonsense mutation on one allele and a splice mutation on the other allele were found in one PCD patient [84]. Large genomic deletions, as well as point mutations involving LRRC50 (leucine-rich repeat containing 50), are responsible for a distinct PCD variant that is characterised by a combined defect involving assembly of the ODA and IDA. Functional analyses shows that LRRC50 deficiency disrupts assembly of distally and proximally DNAH5 and DNAI2 containing ODA complexes, as well as DNALI1-containing IDA complexes, resulting in immotile cilia [85]. Multiple other candidate genes have been tested in patients and families with PCD, and were found to be negative.

Other genetic associations

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X-linked retinitis pigmentosa, sensory hearing deficits and PCD have been associated via mutations in the RPGR, essential for photoreceptor maintenance and viability [41]. In addition, a

single family was reported with a novel syndrome that is caused by oral-facial-digital type 1 gene (OFD1) mutations, and characterised by X-linked recessive mental retardation, macrocephaly and PCD [45].

Animal models for PCD have been reported to occur in nature, although they have rarely been studied in depth [97]. Similarly animals with a PCD phenotype have been constructed using molecular techniques, mainly in mice [4]. Other than the Mdnah5 deficient mouse and the Dpcd/ poll knock out mouse, the causative gene in the other models are unknown. The Mdnah5 deficient mice were created via transgenic insertional mutagenesis that leads to a frame shift mutation. The mice have the classic PCD phenotype and the ultra-structural analysis reveals absent ODA [98, 99]. The Dpcd/poll knock out mice present a phenotype of sinusitis, situs inversus, hydrocephalus, male infertility and ciliary IDA defects [43]. Recently, a murine mutation of the evolutionarily conserved adenylate kinase 7 (Ak7) gene resulted in animals presenting with pathologic signs characteristic of PCD, including ultra-structural ciliary defects and decreased CBF in respiratory epithelium [100]. The mutation is associated with hydrocephalus, abnormal spermatogenesis, mucus accumulation in paranasal passages and a dramatic respiratory pathology upon allergen challenge. Ak7 appears to be a marker for cilia with 9+2 microtubular organisation. Mutations of the human equivalent may underlie a subset of genetically uncharacterised PCD, although no human mutations have been identified as yet. Finally, a novel method of developing a mouse model with a PCD phenotype was recently published [101]. A transgenic mouse lacking an ODA was developed by deleting Dnaic1, a mouse intermediate chain dynein. Importantly, the mice did not develop many of the problems that usually result in an early death for the animals, such as hydrocephalus or other severe developmental defects. Thus, the survival of the animals allowed the investigators to show that the animals did experience problems consistent with defective MCC, at least in the upper airway (severe rhinosinusitis). Objective measures of MCC were also consistent with defective ciliary function in the nasal passage, though interestingly not in the lower airway, possibly reflecting differing turnover of ciliated epithelium in various regions of the respiratory tract (upper versus lower). This animal model may allow studies that attempt to dissect out the relative importance of the various components of the MCC apparatus in different airway regions.

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Future directions

Summary: an algorithm for testing As there is no easy, single diagnostic test to diagnose PCD, it is recommended that the diagnosis be based on multiple contributing pieces of data (fig. 2). A typical clinical presentation to suggest additional testing for PCD includes recurrent respiratory tract infections (either upper or lower, or both), neonatal respiratory distress, childhood ear infections, adult bronchiectasis in the absence of a diagnosis and male/female fertility problems. Additional features to provoke further tests include organ laterality defects, and complex congenital heart or other organ defects and retinitis pigmentosa. Ciliary dyskinesia, sperm immotility or identification of specific defects of axonemal structures on electron microscopy are also suggestive of the diagnosis. The reader should bear in mind that patients with PCD with atypical histories may have no demonstrable ciliary ultrastructural defects on standard TEM. Nasal NO, if available, helps exclude the disease if normal or very high and, if very low, strongly suggests the diagnosis. Recently, clinically available genetic testing, a rapidly evolving field, may assist in an increasing number of patients with PCD.

Therapeutic approaches Overview

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The goal for the management of PCD is to prevent exacerbations and complications as much as possible, and to slow the progression of disease. As the disease is generally not as severe as CF, and

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the diagnosis may be delayed, adults with the disease may not fully appreciate or understand the nature and/or severity of the disease. Thus, education as to the diagnosis, prognosis and therapeutic avenues need to be discussed thoroughly with the patients once the diagnosis is secure (usually on several occasions). Although there are few literature-based studies in PCD, there are enough studies in CF and non-CF bronchiectasis to allow significant extrapolation (although not total, see later) into patients with PCD, to at least frame a plan of treatment depending on disease severity, sputum microbiology and patient circumstances. Medical therapy has been shown to slow the deterioration in lung function [20, 102]. ELLERMAN and BISGAARD [20] reported longitudinal lung function in 24 patients diagnosed before and after the age of 18 years. They observed worse lung function in patients diagnosed in adulthood, but did not find further deterioration in lung function in either group once the diagnosis was established and routine care initiated. This suggests that aggressive treatment could prevent further lung damage. It should be noted, however, that other larger patient cohorts followed for a longer time period suggest that PCD may be a serious threat to lung function as early as pre-school, with a high degree of variation in the loss of lung function once diagnosed [103]. There was no link to either age or level of lung function at diagnosis and early detection did not slow the rate of decline in lung function. These data support the genetic and phenotypic heterogeneity of PCD. Despite this, regular clinical surveillance is strongly recommended to establish trends of disease progression, and to detect exacerbations early to attempt to prevent irreversible lung damage. This should include at least lung function testing, sputum or throat cultures to assess airway microbiology and annual chest radiographs [104]. Pulmonary function in PCD patients appears to decline slower compared with patients with CF and the majority of patients with PCD seem to have a normal to near normal life span [21]. However, there are patients that develop progressive bronchiectasis, leading to severe lung disease and respiratory failure.

Specific therapies There are no therapies to date that have been shown to correct ciliary dysfunction in PCD patients. Some pilot or single case reports suggest benefit for some of the underlying pathogenetic pathways in PCD, but none are yet available on a general basis, or proven in randomised controlled studies (although patients will often inquire as to their availability) [33, 105, 106]. Thus, therapies to enhance airway clearance, as well as to suppress or kill bacteria are the cornerstones to PCD care.

Airway clearance As with CF, routine airway clearance with cardiovascular exercise, percussion vests, chest physical therapy and various valve/positive expiratory pressure devices should be performed on a daily basis. The aims of respiratory physiotherapy include mobilising and aiding expectoration of broncho-pulmonary secretions, improving efficiency of ventilation, maintaining or improving exercise tolerance, improving knowledge and understanding and reducing breathlessness and chest pain. There are no data in either CF or PCD to support any one method of airway clearance over another, and in adults a good practice is to facilitate a consultation with a chest physiotherapist for an education ‘‘class’’, and to determine what modality of airway clearance and what devices the patient prefers. As with any chronic lung disease, exercise is highly recommended for cardiovascular fitness and specifically for airway clearance. Even though a chronic cough is a major complaint, it should not be suppressed as it is a compensatory mechanism for mucus clearance with dysfunctional cilia [33].

Antibiotics

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Antibiotics are the mainstay of treatment for bacterial infections of the airways associated with PCD. The microbiological flora of the airways is broadly similar to that of CF, although with a delayed appearance of P. aeruginosa. Antibiotic therapy should be based on regular sampling of

Modulation of airway secretions In the CF population, nebulised hypertonic saline (7% hypertonic saline) is beneficial by modulating the liquid content of the periciliary fluid layer, thereby thinning thick secretions and triggering a cough reflex [109, 110]. However, in PCD, its utility is less clear as it stimulates cough to help clear secretions but its role in thinning secretions is not known [111]. A small study of 24 patients with non-CF bronchiectasis showed that hypertonic saline resulted in greater expectorated sputum weight and a greater reduction in sputum viscosity compared with the active cycle of breathing technique alone [112]. Thus, it may be considered in the PCD population as it can augment mucus clearance with little to no risk, other than time. Other aerosolised hypertonic agents such as dry powder mannitol are currently being investigated and may be promising in the future [113]. Deoxyribonuclease (dornase alfa), an enzyme that hydrolyses eukaryotic DNA released from decaying neutrophils to diminish mucus viscosity and enhances clearance, is beneficial in CF patients, but its use by extrapolation into PCD patients remains unproven and may even be detrimental to lung function [114, 115].

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airway secretions for Gram-positive, Gram-negative and acid fast pathogens to build a pattern of the main pathogens in any given patient’s airways [21, 107]. In adults, sputum is usually easy to acquire and bronchoscopy is not usually necessary to gather specimens. When PCD patients have symptoms of a respiratory tract infection, they require treatment with antibiotics based on airway cultures and sensitivities. H. influenzae, S. aureus, and S. pneumoniae are commonly isolated from the airways of PCD patients. There are no randomised placebo-controlled studies evaluating the efficacy of antibiotics in exacerbations in adults or children although numerous studies indicate that antibiotics can improve symptoms and hasten recovery. Antibiotics are recommended for exacerbations that present with an acute deterioration (usually over several days) with worsening local symptoms (cough, increased sputum volume or change of viscosity, increased sputum purulence with or without increased wheeze, breathlessness and haemoptysis) and/or a decrease in lung function based on lung function testing. Expert consensus is that 2 weeks of therapy is reasonable. The choice of antibiotics may be initially empirical, based on the likely microbial agent or guided via previous sputum cultures in an individual (hence the recommendation to gather serial samples). The recommended route of antibiotics needs further study to address the optimal regimen, but most clinicians use oral antibiotics for milder exacerbations and combined antipseudomonal intravenous drugs for more significant deteriorations. Previous studies suggest that the combination of intravenous and inhaled antibiotics might have greater efficacy than intravenous therapy alone [34]. In patients chronically colonised with P. aeruginosa, the addition of nebulised tobramycin to high-dose oral ciprofloxacin for 14 days led to a greater reduction in microbial load at day 14 although there was no clinical benefit [108]. Attempts at early eradication of newly acquired bacteria are recommended as in CF, although there are no data that show that such an approach prevents the progression of lung disease. Long-term antibiotics or nebulised antibiotics (tobramycin, colomycin or aztreonam) may be used in patients with chronic or frequent exacerbations. Some patient do well on ‘‘rotating’’ cycles of oral antibiotics, although there are no data to support such an approach and there is a general concern about inciting microbial resistance.

Other airway treatments

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Bronchodilators are not particularly effective in PCD or CF unless a coexisting asthmatic component exists [116]. PCD patients may be initially misdiagnosed as asthmatics unresponsive to conventional therapy, including b-agonists and inhaled corticosteroids. b-Adrenoceptor agonists have been shown to augment CBF in functional cilia but there is little data in the dyskinetic cilia seen in the PCD population [117]. Anti-inflammatory strategies such as alternate-day prednisolone have not been shown to be effective in CF; there are no studies in PCD [118]. Inhaled steroids may or may not be of benefit in individual patients with PCD; a recent Cochrane

review concluded no benefit in non-CF bronchiectasis overall [119]. As with other inflammatory diseases of the lung, the macrolide antibiotics may exert long-term benefits for the modulation of airway inflammation and thus disease expression [106, 120].

Miscellaneous lung treatments

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L-Arginine

might hypothetically have a therapeutic role in PCD patients, in augmenting the production of airway NO, theoretically enhancing CBF (although the exact role of NO in this process is unknown). However, in the small studies performed, L-arginine did not normalise nasal NO levels and no improvement in lung function was observed [121]. Uridine-5-triphosphate (UTP), or its analogues, is also a potential therapy for CF and similar diseases [122]. UTP stimulates chloride ion secretion and mucin release in goblet cells, therefore increasing airway fluid hydration and enhancing cough clearance in healthy individuals. A small acute clinical trial of nebulised UTP in PCD demonstrated enhanced airway clearance during cough, but no long-term benefits in pulmonary function have been shown [33]. Localised surgery may be considered in situations that resemble that of CF or idiopathic bronchiectasis, where occasionally very localised lung disease is considered to be problematic in causing severe systemic symptoms, frequent exacerbations and/or life threatening haemoptysis [123, 124]. Patients with such localised disease, haemoptysis or refractory pulmonary infections, have undergone surgical resection of the bronchiectatic lung but the long-term effects are unknown [124]. If PCD does progress to endstage lung disease, lung transplantation must be considered. PCD patients have undergone successful heart-lung, double lung or living donor lobar lung transplant [125]. In patients with situs inversus, the anatomic disorientation adds an extra challenge when considering the anastomotic sites but is not a contraindication. The long-term survival appears similar to other lung transplant recipients.

Treatment of extrapulmonary disease in adults As PCD affects other aspects of the respiratory tract other than the lungs, treatment of those areas must be considered. Chronic rhinitis and sinusitis may cause significant morbidity in patients with PCD. As of now, no treatments have been shown to be unequivocally effective, although most patients are treated with intranasal corticosteroids, sinus lavage procedures and antibiotics. Antibiotics should be used sparingly for sinus symptoms as resistance occurs quickly and antibiotics should be reserved for more pressing pulmonary symptoms. If sinus symptoms persist despite aggressive medical management or are severe, endoscopic sinus surgery can be used to promote drainage and better delivery of topical medications [126]. Male infertility due to sperm immotility can be overcome by assisted fertilisation techniques such as intracytoplasmic sperm injections [127]. Females, who are infertile secondary to fallopian tube dysfunction, can have direct ovum harvesting from the ovaries and can get in vitro fertilisation.

Statement of interest P.G. Noone is principal investigator on an industry sponsored study (multicentre) looking at the effects of inhaled mannitol in non-cystic fibrosis bronchiectasis (Pharmaxis). He is also principal investigator on an industry sponsored study (multicentre) looking at the effects of inhaled aztreonam in non-cystic fibrosis bronchiectasis (Gilead).

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94. Fliegauf M, Omran H. Novel tools to unravel molecular mechanisms in cilia-related disorders. Trends Genet 2006; 22: 241–245. 95. Supp DM, Witte DP, Potter SS, et al. Mutation of an axonemal dynein affects left-right asymmetry in inversus viscerum mice. Nature 1997; 389: 963–966. 96. Supp DM, Brueckner M, Potter SS. Handed asymmetry in the mouse: understanding how things go right (or left) by studying how they go wrong. Sem Cell Develop Biol 1998; 9: 77–87. 97. Cavrenne R, De Busscher V, Bolen G, et al. Primary ciliary dyskinesia and situs inversus in a young dog. Vet Rec 2008; 163: 54–55. 98. Ibanez-Tallon I, Gorokhave S, Heintz N. Loss of function of axonemal dynein Mdnah5 causes primary ciliary dyskinesia and hydrocephalus. Hum Mol Genet 2002; 11: 715–721. 99. Tan SY, Rosenthal J, Zhao XQ, et al. Heterotaxy and complex structural heart defects in a mutant mouse model of primary ciliary dyskinesia. J Clin Invest 2007; 117: 3742–3752. 100. Fernandez-Gonzalez A, Kourembanas S, Wyatt TA, et al. Mutation of murine adenylate kinase 7 underlies a primary ciliary dyskinesia phenotype. Am J Respir Cell Mol Biol 2009; 40: 305–313. 101. Ostrowski LE, Yin W, Rogers TD, et al. Conditional deletion of dnaic1 in a murine model of primary ciliary dyskinesia causes chronic rhinosinusitis. Am J Respir Cell Mol Biol 2010; 43: 55–63. 102. Hellinckx J, Demedts M, de Boeck K. Primary ciliary dyskinesia: evolution of pulmonary function. Eur J Pediatr 1998; 157: 422–426. 103. Marthin JK, Petersen N, Skovgaard LT, et al. Lung function in patients with primary ciliary dyskinesia: a crosssectional and 3-decade longitudinal study. Am J Respir Crit Care Med 2010; 181: 1262–1268. 104. Gibson RL, Burns JL, Ramsey BW. Pathophysiology and management of pulmonary infections in cystic fibrosis. Am J Respir Crit Care Med 2003; 168: 918–951. 105. Loukides S, Kharitonov S, Wodehouse T, et al. Effect of arginine on mucociliary function in primary ciliary dyskinesia. Lancet 1998; 352: 371–372. 106. Yoshioka D, Sakamoto N, Ishimatsu Y, et al. Primary ciliary dyskinesia that responded to long-term, low-dose clarithromycin. Intern Med 2010; 49: 1437–1440. 107. Cystic Fibrosis Foundation. Patient Registry. Annual Report to the Center Directors. Cystic Fibrosis Foundation Annual, Bethesda, MD, USA. www.cff.org/UploadedFiles/research/ClinicalResearch/Patient-Registry-Report2009.pdf 108. el Din MA, Palmer LB, el Tayeb MN, et al. Nebulizer therapy with antibiotics in chronic suppurative lung disease. J Aerosol Med 1994; 7: 345–350. 109. Donaldson SH, Bennett WD, Zeman KL, et al. Mucus clearance and lung function in cystic fibrosis with hypertonic saline. N Engl J Med 2006; 354: 241–250. 110. Robinson M, Regnis JA, Bailey DL, et al. Effect of hypertonic saline, amiloride, and cough on mucociliary clearance in patients with cystic fibrosis. Am J Respir Crit Care Med 1996; 153: 1503–1509. 111. Bennett WD, Olivier KN, Zeman KL, et al. Effect of uridine 5-triphosphate plus amiloride on mucociliary clearance in adult cystic fibrosis. Am J Respir Crit Care Med 1996; 153: 1796–1801. 112. Kellett F, Redfern J, Niven RM. Evaluation of nebulised hypertonic saline (7%) as an adjunct to physiotherapy in patients with stable bronchiectasis. Respir Med 2005; 99: 27–31. 113. Wills P, Greenstone M. Inhaled hyperosmolar agents for bronchiectasis. Cochrane Database Syst Rev 2006; 2: CD002996. 114. Fuchs HJ, Borowitz DS, Christiansen DH, et al. Effect of aerosolized recombinant human DNase on exacerbations on respiratory symptoms and on pulmonary function in patients with cystic fibrosis. N Engl J Med 1994; 331: 637–642. 115. Donnell AE, Barker AE, Ilowite JS, et al. Treatment of idiopathic bronchiectasis with aerosolized recombinant human DNase. Chest 1998; 113: 1329–1334. 116. Phillips GE, Thomas S, Heather S, et al. Airway response of children with primary ciliary dyskinesia to exercise and b2-agonist challenge. Eur Respir J 1998; 11: 1389–1391. 117. Bennett WD. Effect of b-adrenergic agonists on mucociliary clearance. J Allergy Clin Immunol 2002; 110: Suppl. 6, S291–S297. 118. Balfour-Lynn IM, Lees B, Hall P, et al. Multicenter randomized controlled trial of withdrawal of inhaled corticosteroids in cystic fibrosis. Am J Respir Crit Care Med 2006; 173: 1356–1362. 119. Kapur N, Bell S, Kolbe J, et al. Inhaled steroids for bronchiectasis. Cochrane Database Syst Rev 2009; 1: CD000996. 120. Saiman L, Marshall BC, Mayer-Hamblett N, et al. Azithromycin in patients with cystic fibrosis chronically infected with Pseudomonas aeruginosa: a randomized controlled trial. JAMA 2003; 290: 1749–1756. 121. Grasemann H, Gartig SS, Wiesemann HG, et al. Effect of L-arginine infusion on airway NO in cystic fibrosis and primary ciliary dyskinesia syndrome. Eur Respir J 1999; 13: 114–118. 122. Deterding RR, Lavange LM, Engels JM, et al. Phase 2 randomized safety and efficacy trial of nebulized denufosol tetrasodium in cystic fibrosis. Am J Respir Crit Care Med 2007; 176: 362–369. 123. Balkanli K, Genc O, Dakak M, et al. Surgical management of bronchiectasis: analysis and short-term results in 238 patients. Eur J Cardiothorac Surg 2003; 24: 699–702.

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124. Smit HJ, Schreurs AJ, Van den Bosch JM, et al. Is resection of bronchiectasis beneficial in patients with primary ciliary dyskinesia? Chest 1996; 109: 1541–1544. 125. Rabago G, Copeland JG III, Rosapepe F, et al. Heart-lung transplantation in situs inversus. Ann Thorac Surg 1996; 62: 296–298. 126. Parsons DS, Greene BA. A treatment for primary ciliary dyskinesia: efficacy of functional endoscopic sinus surgery. Laryngoscope 1993; 103: 1269–1272. 127. Gerber PA, Kruse R, Hirchenhain J, et al. Pregnancy after laser-assisted selection of viable spermatozoa before intracytoplasmatic sperm injection in a couple with male primary cilia dyskinesia. Fertil Steril 2008; 89: 9–12.

Chapter 10

Channelopathies in bronchiectasis I. Sermet-Gaudelus*,#,",+, A. Edelman*,# and I. Fajac*,+,1

CHANNELOPATHIES

Summary Channelopathies are diseases caused by dysfunction of ion channel subunits. They result in impaired mucociliary clearance and may therefore lead to bronchiectasis. The main channelopathy associated with bronchiectasis is cystic fibrosis (CF), an autosomal recessive disease caused by mutations in the CFTR gene, which encodes the chloride CFTR channel. Bronchiectasis can be associated to channelopathies in following cases: 1) patients with already known typical CF; 2) patients with bronchiectasis who, on investigation, are found to have a single-organ manifestation of CF; 3) patients with only one or none mutation of CFTR with abnormal sweat test or nasal potential difference (PD) where CFTR mutations play the role of a modifier deleterious gene; and 4) patients with only one or no mutation of CFTR with normal sweat test or nasal PD, who may still have an undefined channelopathy. In these last two cases, it may be that, CFTR mutation combined with another ion transport abnormality, in a situation of transheterozygosity, creates the conditions for abnormal airway surface liquid (ASL) hydration regulation and defective mucociliary clearance. Keywords: Airway surface liquid, bicarbonate, calciumdependent chloride channel, cystic fibrosis, cystic fibrosis transmembrane conductance regulator, epithelial sodium channel

*Universite´ Paris Descartes, # INSERM Unite´ 845, " Service de PneumoPe´diatrie, Hoˆpital Necker-Enfants Malades, + Assistance Publique Hopitaux de Paris, and 1 Service de Physiologie-Explorations Fonctionnelles, Hoˆpital Cochin, Paris, France. Correspondence: I. Sermet-Gaudelus, Service de PneumoPe´diatrie, Universite´ Paris Descartes, Hoˆpital Necker-Enfants Malades, 149 rue de Se`vres, 75015, Paris, France, Email [email protected]

Eur Respir Mon 2011. 52, 150–162. Printed in UK – all rights reserved. Copyright ERS 2011. European Respiratory Monograph; ISSN: 1025-448x. DOI: 10.1183/1025448x.10004010

B

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ronchiectasis is defined as an abnormal dilation of proximal medium-sized bronchi due to weakening or destruction of the muscular and elastic components of the bronchial walls [1]. It is caused by a vicious cycle of transmural infection and inflammation, resulting in retained secretions that damage the airways and impair mucociliary clearance. Bronchiectasis can appear as either a local obstructive process or a diffuse disease involving both lungs. In the latter case, a systemic condition must be sought. These can include autoimmune disease, a1-antitrypsin deficiency, connective tissue disorders, immunodeficiency states, allergic

bronchopulmonary aspergillosis and primary ciliary dyskinesia. Channelopathies, defined as diseases caused by dysfunctioning ion channel subunits, are another possibility. Channels are pore-forming proteins that provide pathways for the controlled movement of ions into or out of cells, and are hence important in regulating mucociliary clearance [2]. The present chapter focuses on the role of channelopathies as causative factors for the development of bronchiectasis.

The link between ion transport and mucus transport in the airways Two opposing transport systems tailored to controlling the volume of liquid on the epithelial surface

When ASL volume is depleted, normal airway epithelium exerts dynamic regulation by switching its status from net NaCl absorption to net secretion (fig. 2) [3, 4]. This requires the accumulation of Clwithin the cell through the action of the Na+/K+/2Cl- cotransporter located in the basolateral membrane. Cl- then exits the cell across the apical Cl- channels, at the Luminal Basolateral same time as apical Na+ absorption Cl slows and Na+ moves paracelluK+ Na+, K+-ATPase larly to maintain electroneutrality. Na+ + Na Adenosine triphosphate (ATP), reENaC leased on to the airway surface, is the main sensor for this regulation K+ Cl[5]. Its actions are mediated by two + PKA CFTR purinergic receptor subtypes, the Clpertussis-toxin-insensitive G-proCltein (Gq)-coupled ATP/uridine triphosphate (UTP)-sensing P2Y2 Figure 1. Cellular models of electrolyte secretion: absorption P2Y receptor and the stimulatory pathway. In airway epithelial cells, under resting conditions, Na+ is G-protein (Gs)-coupled A2B adenotaken up by a luminal epithelial sodium channel (ENaC); Cl- is sine receptor. Activation of the A2B transported via the paracellular shunt and probably via cystic fibrosis purinoreceptor raises cell cyclic adetransmembrane conductance regulator (CFTR) Cl- channels. Na+ is pumped out of the cell by the basolateral sodium–potassium nosine monosphosphate (cAMP), adenosine triphosphatase (Na+,K+-ATPase), whereas Cl- and K+ which, in turn, activates the CFTR leave the cell via Cl- and K+ channels, respectively. PKA: protein sufficiently to provide CFTR-depenkinase A. -: inhibition; +: stimulation. dent Cl- secretion and negative ENaC

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Under resting conditions, airway surface epithelia display net salt and fluid absorption (fig. 1), driven by active apical Na+ absorption through the amiloride-sensitive epithelial sodium channel (ENaC), passively accompanied by Cl-, in part, via a transcellular pathway, mainly the cystic fibrosis transmembrane conductance regulator (CFTR), and, in larger part, via the paracellular pathway [3]. This absorptive pattern occurs due to basolateral sodium–potassium adenosine triphosphatase (Na+,K+-ATPase), which generates an electrochemical gradient favourable for apical Na+ absorption. ASL remains isotonic under basal conditions because of the airway epithelium’s permeability to water (due to the relative leakiness of the tight junctions) and the isoosmotic conditions of ion transport.

I. SERMET-GAUDELUS ET AL.

The thin film of liquid covering airway surfaces, called airway surface liquid (ASL), is partitioned into two compartments, the mucus layer, which entraps particles and pathogens and has lubricant activity, and the periciliary liquid (PCL) layer, which facilitates ciliary beating and separates the mucus layer from the mucins tethered to the cell surface [3]. Normal airway surface epithelia can regulate ASL volume by setting the height of the PCL to approximately the height of the extended cilia (,7 mM) [3]. The coordination of sodium and chloride ion transport regulates ASL homeostasis to provide efficient mucus transport.

Apical Na H2O ENaC

K+

Na+ -

-

regulation by the CFTR. Higher ATP concentrations then activate the P2Y2 receptor, promoting, on the one hand, the inhibition of Na+ absorp+ + + Na , K -ATPase Na tion and, on the other, Cl- secretion, Na+/K+/2Cl- cotransporter mediated by another apical channel, the calcium-activated chloride chanK+ nel (CaCC). KV7.1 Basolateral

+

CFTR

Cl HCO3Adenosine ATP UTP CaCC Cl-

PKA cAMP Ca2+

2ClK+

K+ KCa3.1

Na+ H2O

CHANNELOPATHIES

Figure 2. Cellular models of electrolyte secretion: secretory pathway. In airway cells, under conditions triggering secretion, Clis taken up from the basolateral (blood) side by the Na+/K+/2Clcotransporter. K+ recycles through basolateral K+ channels. This leads to basolateral membrane hyperpolarisation, which, in turn, electrically drives Cl- to the luminal side of the epithelium and stimulates Cl- secretion through the cystic fibrosis transmembrane conductance regulator (CFTR) and/or calcium-activated chloride channels (CaCCs). Activation of the A2B adenosine receptor results in raised cell cyclic adenosine monosphosphate (cAMP) levels, which, in turn, activate the CFTR sufficiently to provide CFTRdependent regulation of the epithelial sodium channel (ENaC) and Cl- secretion, together with activation of the cAMP-dependent potassium channel (KV7.1). Higher ATP concentrations activate the P2Y2 P2Y receptor, inhibiting Na+ absorption and activating both CFTR-dependent and CFTR-independent Cl- secretion, the latter mediated by the release of cytoplasmic Ca2+, which, in turn, activates the CaCC and the calcium-activated potassium channel (KCa3.1). Na+ is pumped out of the cell by sodium–potassium adenosine triphosphatase (Na+,K+-ATPase). Na+ is secreted via the paracellular shunt following the electrical driving force generated by the negative transepithelial voltage in the lumen. ATP: adenosine triphosphate; UTP: uridine triphosphate; PKA: protein kinase A. q: increased; -: inhibition.

Finally, when ASL volumes are depleted, the epithelium rehydrates airway surfaces by: 1) inhibiting absorption (in the surface epithelium); and 2) activating secretion (in the submucosal glands).

Ion transporters involved in mucociliary clearance Consistent with these fundamental observations, most of the channelopathies identified as possible causes of the impaired clearance of bronchial tree secretions appear to involve Cl-, Na+ and bicarbonate transport.

Cl- transporters Cystic fibrosis transmembrane conductance regulator

The CFTR is a member of the ATP-binding cassette transporter superfamily, principally expressed in the apical membrane of epithelia. It plays a fundamental role in transepithelial fluid and electrolyte transport because it functions as an anion channel and a regulator of ion transporters in epithelial cells. The CFTR is a cAMP- and ATP-regulated Cl- channel that permits Cl- to be released from the cell [6]. Recent data also suggest that the CFTR pore may switch dynamically from a conformation permeable to Cl- to a conformation permeable to large anions, such as glutathione and HCO3-, and may, therefore, be involved in pH regulation of the ASL and mucus [7].

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Apart from its secretory function, the CFTR has the regulatory function of other epithelial channels. The CFTR inhibits ENaC activity and, therefore, conveys reduction in Na+ resorption [8]. The CFTR upregulates an outwardly rectifying chloride channel (ORCC) following its activation by protein kinase A (PKA) [9]. The CFTR can also interact via its extreme C-terminal amino acid sequence with PDZ-domain-containing proteins, which are important organisers for receptors, ion transporters and regulatory elements present in airway epithelium [10]. For example, reciprocal activation between the CFTR and the solute carrier (SLC) 26 transporter (SLC26T) family of HCO3-/Cl- exchangers has been shown to depend upon PDZ domain interaction and binding of the sulfate transporter and anti-s factor antagonist (STAS) domain of SLC26T family proteins to the CFTR regulatory (R) domain [11].

Calcium-activated chloride channels Airway epithelial cells display Ca2+-dependent Cl- secretion through CaCCs in response to mucosal nucleosides. The mechanism relies on the stimulation by ATP or UTP of the Gq-coupled P2Y2 purinergic receptors, which increases inositol 1,4,5-trisphosphate (IP3) production and subsequently cytosolic Ca2+ release [12]. Transmembrane protein 16A (TMEM16A), which generates Ca2+-activated Cl- currents with similar biophysical and pharmacological properties to those in native epithelial tissues, is a very likely candidate for these CaCCs [13].

Chloride channel-2 Chloride channel (ClC)-2 is a member of the pH- and voltage-activated chloride channel family and is present on the apical membranes of airway epithelial cells [14]. Activation of ClC-2 is hypothesised to provide a parallel pathway for Cl- secretion [15].

Indirect activation of Cl- secretion by K+ channels

Na+ transporters The ENaC is a heteromultimer composed of distinct but homologous a-, b- and c-subunits known to be activated by selective endoproteolysis [17]. As pointed out above, it provides the main pathway for apical Na+ absorption at the apical membrane [3, 4]. The ENaC and the CFTR physically associate in mammalian cells [18], an interaction that may impede ENaC proteolytic cleavage and inhibit stimulation of the channel open probability [19].

HCO3- transport

I. SERMET-GAUDELUS ET AL.

Activation of K+ channels at the basolateral side of the epithelium causes hyperpolarisation of the basolateral membrane, which electrically drives Cl- to the luminal side of the epithelium and stimulates Cl- secretion through the CFTR and/or CaCCs. At least two different populations of K+ channel are located on the basolateral side of airway epithelial cells that are activated by an increase in either intracellular cAMP (cAMP-dependent potassium channel (KV7.1)) or Ca2+ (calciumactivated potassium channel (KCa3.1)) [16].

HCO3- plays a critical role in determining the viscosity of mucins and mucus by decondensing mucin granules. Intracellularly, mucins are condensed in granules by high concentrations of Ca2+ and H+ that shield the repulsive forces of the anionic sites of mucin glycoproteins. As granules are secreted, Ca2+ and H+ have to dissociate quickly from the mucin to unshield the negative sites, so that Na+ can replace Ca2+ to allow mucin network hydration, swelling and dispersion. HCO3- is critical for sequestration of Ca2+ and H+ and maintenance of a low concentration of these free cations in solution, which, in turn, favour their disassociation from mucins [20, 21]. Moreover, a normal pH is necessary for effective mucociliary clearance, as assessed by several observations. For example, a reduction in extracellular pH of 0.5 reduces mucociliary beat frequency by 22% in bronchi and 16% in bronchioles [22]. As stated above, the CFTR clearly plays a role in HCO3- transport. Cell membrane ion transporters besides CFTR may also be involved in ASL and/or gland fluid pH regulation [23]. These include the following.

The basolaterally located isoform sodium bicarbonate cotransporter (NBC) 1 permits the basal influx of HCO3- followed by efflux through the apical CFTR [24].

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Na+/HCO3- cotransporters

Cl-/HCO3- exchangers Based on an analogy to SLC26A3 function in HCO3- secretion by the pancreatic duct epithelium, WHEAT et al. [25] proposed a model for HCO3- transport in the airway epithelium: Cl-/HCO3exchange activity, governed by SLC26A3 in the apical membrane, might secrete HCO3- into the ASL, with Cl- recycling through the CFTR. However, experiments in polarised airway epithelial cells failed to confirm this hypothesis [26]. The role of Cl-/HCO3- exchangers in ASL pH regulation at the apical membrane therefore remains speculative.

Investigation of ion transport in airway epithelium Transepithelial potential difference (PDte) results from ion movements across both the basolateral and apical membrane and leakiness of tight junctions. Its assessment has been applied in vivo to both nasal and bronchial mucosa [27]. Nasal potential difference (PD)-based outcomes include the stable maximum baseline (basal PD) and the successive net voltage changes after perfusion of the mucosa with: 1) amiloride (an ENaC inhibitor), to assess Na+ transport (Damiloride); 2) lowchloride solution, to drive Cl- secretion (Dlow-chloride); and 3) isoproterenol in low-chloride solution (Disoproterenol), to stimulate the cAMP-dependent Cl- conductance related to the CFTR (fig. 3). The sum of Dlow-chloride and Disoproterenol serves as an index of CFTR function [28].

CHANNELOPATHIES

This PDte can also be measured in Ussing chambers, using either epithelial biopsy specimens or airway epithelial cells in culture. This system measures transepithelial ion transport by evaluating PDte in volts [29], by either applying a PD and measuring the resulting change in current (technique of voltage clamping) or short-circuiting the tissue, i.e. clamping PDte at 0 V and measuring the amount of current required.

Channelopathies: cystic fibrosis Pathophysiology Cystic fibrosis (CF) is one of the principal channelopathies resulting in abnormal mucus clearance. It is an autosomal recessive disease caused by mutations in the CFTR gene (CFTR), which encodes the CFTR Cl- channel [30]. In CF, defects in the mechanisms governing both Na+ absorption and Cl- secretion severely disrupt ASL volume regulation on airway surfaces. Specifically, they accelerate the basal rate of net epithelial Na+ absorption in CF airway epithelia, causing isotonic volume hyperabsorption that reflects the absence of the tonic inhibitory effect of CFTR on ENaC activity [31, 32]. The mechanism linking the missing CFTR and increased Na+ absorption in CF airway epithelia may be the failure to protect ENaC from proteolytic cleavage and consequent activation [33]. CF airway epithelia also lack the capacity to enhance Cl- transport [34]. Therefore, whereas non-CF epithelium can rehydrate when ASL volumes are depleted, by activating secretion and inhibiting absorption, CF epithelium cannot switch from net absorption to net secretion [31]. This inability may be due to its dependence on ATP signalling alone, in contrast to the dual signalling (ATP and adenosine) systems that control ASL volume in normal epithelia [35]. In this model, ATP can inhibit ENaC and activate CaCC, via the P2Y2 receptor, but the A2B pathway is blocked because the CFTR is not functional. Under resting conditions, the P2Y2 pathway may be sufficient to produce an ASL volume consistent with mucus transport. It may, however, be overwhelmed in a context of respiratory infections, e.g. virus infections, which are frequent in early life. These infections and their effect on this system might, therefore, be the initiating event of CF disease [31].

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The resultant reduction in ASL volume is shared by the two layers: 1) the water content of the mucus layer is reduced, producing a highly viscoelastic adhesive material; and 2) the water content of the periciliary environment is depleted, causing the collapse of this layer and a loss of its

PD mV

-40 -30

Basal PD

-20

Δamiloride

Δlow chloride

-10

Δisoproterenol

Δlow-chloride−Δisoproterenol

-50

0 b)

-50 -40 Basal PD

Ringer+amiloride

-30 Δamiloride

Low chloride +amiloride

-20

Low chloride +amiloride +isoproterenol

-10 Ringer 0

Time seconds

Figure 3. Nasal potential difference (PD) trace showing the response to perfusion of various solutions in a) a healthy control and b) a cystic fibrosis (CF) patient. Baseline nasal PD (basal PD) is measured after perfusion of nasal epithelium with saline solution. Nasal PD changes (D) were recorded after perfusion with the following solutions: 100 mM amiloride in saline solution (Damiloride), 100 mM amiloride in low-chloride solution (Dlow-chloride), and 100 mM amiloride plus 10 mM isoproterenol in low-chloride solution (Disoproterenol). The sum of Dlow-chloride and Disoproterenol (Dlow-chloride–Disoproterenol) serves as an index of transepithelial cystic fibrosis transmembrane conductance regulator (CFTR)dependent Cl- transport because it reflects the cyclic adenosine monosphosphate (cAMP) activation of nasal mucosal Cl- permeability. In CF patients: 1) basal PD is more negative than in healthy controls because of increased Na+ transport (high depolarisation following amiloride perfusion); and 2) there is no response following low-chloride perfusion and isoproterenol administration, showing the absence of Cl- permeability.

I. SERMET-GAUDELUS ET AL.

A recent hypothesis suggests that a defect in HCO3- secretion plays a critical role in the pathophysiology of CF [39]. As pointed out above, the level of monovalent cations in ASL in CF patients is normal and constant, whereas it is the concentration of HCO3- that is notably subnormal, because of reduced secretion due to the CFTR defect [40]. Several studies have shown a relatively acidic ASL [41] and an intrinsic acidification defect in fluid gland secretion in CF [42]. This reduced HCO3- level is associated with increased mucus viscosity due to reduced Ca2+ chelation, necessary for rapid mucin swelling and dispersion [21]. Importantly, the extent of these defects correlates with the level of HCO3-, which suggests a relationship between disease severity and the degree of impairment in HCO3- secretion [43, 44].

a)

PD mV

lubricant activity. The combination of the PCL and ASL defects causes the mucus to adhere to the airway [36]. Evidence of adhesion is available from early pathological studies of CF airways, which reveal bronchiolar mucous plugs within 48 hours of birth [37], and from radioparticle deposition studies that show the inability of the cough manoeuvre to clear mucus adhering to airway surfaces [38].

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One consequence of mucus stasis is the formation of thick mucous plaques and plugs, in which microorganisms are embedded. Several features of this thickened adherent CF mucus promote persistent biofilm growth [45]. First, the increased concentration of mucins limits bacterial motility, increases their binding to mucin epitopes and feeds them. Thus bacteria deposited in CF mucus may proliferate densely in the area of droplet deposition. Secondly, the concentrated mucin gel also limits the effectiveness of secondary defence mechanisms that might normally resolve a bacterial infection, such as neutrophil migration or diffusion of antimicrobial substances. Finally, cellular oxygen is consumed at high rates in CF airway epithelium to fuel this increased Na+ transport, thereby creating hypoxic zones in adherent mucous plaques near the cell surface that link the special CF low-oxygen environment and infection [46]. Pseudomonas aeruginosa, specifically, adapts to the hypoxic zones by producing alginate and forming biofilm, thus setting the stage for chronic infection. The persistence of chronic bacterial infection of the airway

lumen then stimulates airway defences and induces a chronic hyperinflammatory response, mainly via the nuclear factor (NF)-kB-mediated pathway [47]. Taken together, these findings indicate that the combination of abnormal Na+ and Cl- transport in CF leads to ASL volume regulation failure, mucus stasis, bacterial infection and inflammation. These, in turn, result in inhibition of mucociliary and cough clearance, and, as a final consequence, induction of bronchiectasis.

Clinical description

CHANNELOPATHIES

The diagnosis of CF is based on an abnormal sweat test result (sweat Cl- level of .60 mM) and the finding of two CF-causing mutations in the CFTR and/or an abnormal PDte [30]. In the latter case, the response to amiloride is increased because of lack of inhibition of Na+ resorption, and Clsecretion is absent in the presence of low-chloride solution and isoproterenol. CF clinical presentation can be divided into two types: 1) classic disease, readily diagnosed based on clinical and laboratory data; and 2) less-severe disease that manifests later in life and yields ambiguous genetic testing results [48]. In the first case, CF is a life-limiting multisystemic disorder that affects the Cl- transport system in exocrine tissues. The hallmark is a classic triad of symptoms, most often from infancy or childhood: progressive obstructive lung disease with sputum infected by Staphylococcus aureus or P. aeruginosa, exocrine pancreatic insufficiency, and a high sweat Cl- level. In males, this triad is associated with congenital absence of the vas deferens, leading to sterility. Other specific clinical phenotypes include CF-related liver disease, meconium ileus, CF-related diabetes, pansinusitis and nasal polyposis. Mortality occurs mainly due to progression of lung disease and respiratory insufficiency [49]. In children, bronchiectasis is a marker of respiratory disease severity, because it is associated with increased morbidity and accelerated decline in pulmonary function [50]. It can appear as early as 3 months in CF children [38]. In a cohort of 125 Australian children (from birth to 6 years) diagnosed with CF after newborn screening, 22% showed evidence of bronchiectasis, and the prevalence increased with age [51]. In the paediatric (but not adult) population, the presence and severity of bronchiectasis is significantly related to respiratory infection with P. aeruginosa [52], and, more specifically, mucoid P. aeruginosa [53]. In the second case, advances in basic CF science have broadened the clinical spectrum of CF and highlighted less-severe, so-called CFTR-related, presentations. Most of these patients carry one CF-causing mutation and one or two mutations retaining residual CFTR function [54]. It is not clear whether CFTR-related bronchiectasis, in such cases, is a single-organ manifestation of CF or a condition in which CFTR mutations play the role of a modifier deleterious gene, acting with an environmental contribution.

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Several studies [55–66] have investigated the frequency of CFTR mutations in patients with disseminated bronchiectasis (table 1). The prevalence of CFTR mutations in this population is controversial. Four studies [55–58] found no evidence of an increased prevalence of CFTR abnormalities compared with the general population. Other series [57–64] observed very few patients finally diagnosed with CF on the basis of carriage of two CF-causing mutations and/or elevated sweat Cl- levels (approximately 7% of all of the patients enrolled in those studies). Most patients had at least one non-CF-causing mutation, including mutations classified as ‘‘associated with CFTR-related disorder’’ [54]. Some of these mutations were associated with normal or borderline sweat Cl- levels (substitution of aspartic acid 1152 with histidine (Asp1152His or D1152H), cytosine to thymidine substitution 10 kb downstream of nucleotide 3849 (3849+10 kbC.T), 5T allele of polythymidine tract in intron 8 (IVS8-5T) and Arg117His). It should be pointed out that many of the sequence variations identified are not recognised as CFTR mutations, and still less as CF-disease-causing mutations, mainly because of the lack of established or substantiated knowledge of their pathogenic potential. In these cases, CFTR functional evaluation in epithelium might help in identifying patients with CFTR-related disease [28, 66]. A cohort of patients with bronchiectasis and a sweat Cl- level of ,60 mM were investigated [66];

Table 1. Studies showing an increased prevalence of cystic fibrosis transmembrane conductance regulator (CFTR) mutation in patients with bronchiectasis of unknown origin First author [ref.]

Subjects n

P IGNATTI [60]

16

G IRODON [61] B OMBIERI [62] H UBERT [63] C ASALS [64] Z IEDALSKI [65] B IENVENU [66]

32 23 601 55 50 122

Controls n

Healthy 66; COPD 33; nonobstructive RD 36; atopic 85 0 Healthy 33 0 Local historical cohort 0 Healthy 26; obligate heterozygotes 38; typical CF 92

CFTR mutations Two

One

0

4; IVS8-5T: 9

5 0 45 0 3 15

6 11 43 14; IVS8-5T: 4 18 22

COPD: chronic obstructive pulmonary disease; RD: respiratory disease; IVS8-5T: 5T allele of polythymidine tract in intron 8; CF: cystic fibrosis.

Other channelopathies The epithelial sodium channel There are two principal, and rare, human clinical disorders that occur due to ENaC mutations [67]. The first is Liddle’s syndrome, caused by gain-of-function mutations leading to enhanced Na+ resorption in the renal tubule, and characterised by volume-expanded low-renin hypertension and apparently no respiratory disease [68]. The other is pseudohypoaldosteronism (PHA) type I, due to loss-of-function mutations [69]. In addition to kidney impairment, characterised by renal salt wasting, hyperkalaemia and metabolic acidosis, such children also show defective Na+ transport in the sweat gland, which leads to elevated sweat Cl- and Na+ concentrations. Moreover, children with PHA-I frequently exhibit respiratory tract diseases that involve increased mucociliary clearance and decreased mucus viscosity [69].

I. SERMET-GAUDELUS ET AL.

15 patients carried two CFTR mutations and exhibited abnormal ion transport in the nasal mucosa (i.e. increased Na+ transport and decreased Cl- secretion). They were finally diagnosed with a CFTR-related disorder. In the same series, 22 patients carried only one mutation but displayed abnormal ion transport in the nasal mucosa, intermediate between the normal and the CF range. This led to the hypothesis that an as yet unidentified other factor, genetic or environmental, may trigger the pathogenic role of a unique CFTR mutation. Among the possibilities, abnormalities in ion tranporters other than CFTR should be considered.

157

Recently, ENaCs have been shown to play a critical role in the physiology of mouse airways. Transgenic mice with airway-specific overexpression of the ENaC (b-subunit) develop CF-like lung disease with mucous obstruction and poor bacterial clearance. The airway surfaces of these mice absorb three times more Na+, causing ASL volume depletion, increased mucus concentration, delayed mucus transport and increased mucus adhesion to airway surfaces [70]. These events cause spontaneous and severe lung disease that shares features with CF, including mucous obstruction, goblet cell metaplasia, neutrophilic inflammation and poor bacterial clearance. This outstanding proof-of-concept study demonstrates that increasing airway Na+ absorption creates all of the conditions for the onset of bronchiectasis and initiates a CF-like lung disease [71]. Further support for this mechanism comes from the following two observations: 1) modulation of ENaC activity in CF patients may potentiate disease severity, as suggested by studies showing an enhanced response to amiloride solution in patients with poor respiratory function [72] or chronic P. aeruginosa colonisation [66]; and 2) Na+ transport is significantly higher in bronchiectatic patients, even in those with no or only one CFTR mutation, compared with control subjects [66].

The role of ENaCs in non-CFTR-related bronchiectasis has been investigated in a few studies. SHERIDAN et al. [73] studied 20 patients with diffuse bronchiectasis and elevated sweat Clconcentrations but without two CFTR mutations and identified four patients with five missense mutations and one splicing mutation in ENaC genes. Moreover, among 55 patients with idiopathic bronchiectasis who did not have two mutations in the CFTR coding regions, 10 were identified with an ENaC mutation [74, 75]. This was higher than the expected frequency, and, as these variants had not been previously described, they are unlikely to be common polymorphisms. Moreover, six patients showed evidence of abnormal ion transport, in either sweat glands or nasal epithelium. Hence, although these variants were each found in a heterozygous state, they might be expected to result in abnormal ENaC function. This hypothesis is further supported by recent evidence of ENaC mutations leading to proved channel dysfunction and associated with atypical CF [76].

Other Cl- channels The model of ASL homeostasis suggests that dysfunction of other Cl- channels may alter ASL homeostasis. ClC-2 mutations have been identified in people with idiopathic generalised seizures, but they are not associated with a history of lung disease [77]. Moreover, the ClC-2 knockout mouse undergoes normal lung development, possibly because it has multiple alternative Cl- channel conductance pathways. A ClC-2 abnormality may, therefore, not be related to any human lung disease [15].

CHANNELOPATHIES

To date, no human disease has been linked to a defect in Ca2+-dependent Cl- channels. However, mice that do not express TMEM16A, the best candidate for CaCCs, show greatly reduced mucociliary clearance [13]. Therefore, the role of this channel in human bronchiectasis requires further investigation.

Indirect inactivation of Cl- transport A defect in basal K+ channels may affect the driving force necessary for Cl- to migrate to the apical membrane, as shown by the strong reduction in Cl- transport in nasal, tracheal and bronchial cells carrying mutations of KV7.1 and KCa3.1 [16, 78]. However, no lung disease has been reported among patients with these channelopathies [16]. Alternatively, defective interaction between an ion transporter and a mutated protein modulating its function may impair the channel function, as demonstrated for CFTR and SLC26A3. The interaction between these two proteins leads to their reciprocal functional activation [11]. When SLC26A3 displays a mutation identified in humans, i.e. responsible for congenital Cl- diarrhoea, its interaction with the CFTR is altered, and CFTR activation suppressed [11, 79]. Therefore, mutations in proteins that interact with the CFTR, and specifically other SLC26T members, may affect the CFTR and induce a CF-like phenotype.

Bicarbonate There is evidence that defective HCO3- secretion is associated with abnormal mucus hydration and impaired mucociliary clearance [20]. The amount of mucus discharged is significantly reduced when HCO3- secretion is impeded in the intestines [80] and uterine cervix [43]; a similar mechanism might be anticipated in airways. Extracellular acidification also favours inflammation, by inducing neutrophil activation [81] and delaying neutrophil apoptosis [82]. CF is clearly associated with a defect in ASL pH regulation. Defects in HCO3- transporters other than the CFTR can be envisioned, but require further investigation.

Transheterozygosity 158

After extensive genetic screening, 33–50% of patients with diffuse bronchiectasis are characterised as heterozygous for the CFTR [66]. As the theoretical frequency of this heterozygosity in the

general population is 3.3%, this highly elevated frequency suggests that heterozygosity for the CFTR may have pathogenic consequences. It may predispose to the development and severity of bronchiectasis by potentiating other genetic factors affecting airway physiology or add to deleterious environmental factors. Further support for this hypothesis comes from evidence of an abnormal nasal electrophysiological phenotype in patients with bronchiectasis carrying one CFTR mutation, intermediate between control subjects or patients with no CFTR mutations, on the one hand, and patients with two CFTR mutations on the other [66]. However, the absence of any increased prevalence of bronchiectasis in obligate heterozygotes [83], although they display abnormal Cl- transport [84], suggests that carrying a single CFTR mutation is not solely responsible for development of the disease. A total of 55 patients with diffuse idiopathic bronchiectasis were studied and an unexpectedly high proportion (5%) of heterozygosity was found for both CFTR and ENaC mutations [75]. As the expected frequency of such transheterozygosity in the general population is 0.3%, the finding of so high a prevalence of mutations of both ion transporters suggests that it is clinically relevant. Slight defects in both channels, which separately would not be sufficient to alter ASL homeostasis, are likely to combine their deleterious effects and lead to deficient ENaC/CFTR interaction. Along this line, we speculate that transheterozygosity of a single CFTR mutation and a mutation in another ion channel might create the conditions for abnormal ASL hydration regulation and defective mucociliary clearance.

It is likely that the true incidence of cases of ion-transport-related bronchiectasis among all bronchiectasis is underestimated, given the lack of specific symptoms. Although much is now known about the CFTR, the study of other channelopathies is only just beginning. Except for typical CF and CFTR-related syndrome, it is difficult to demonstrate a causal relationship between bronchiectasis and ion transport defects. The continuum of ion transport dysfunction from normal to disease phenotype makes it difficult to define a clear-cut level for the involvement of ion transport defect in the physiopathology of bronchiectasis [85]. Therefore, in order to ascertain the role of channelopathies in the genesis of bronchiectasis, mutations in a given channel and the related ion transport function should be systematically investigated in bronchiectatic patients. Such studies may point to interesting therapeutic pathways aimed at normalising the first cause of the pathogenic cascade resulting in bronchiectasis.

I. SERMET-GAUDELUS ET AL.

Conclusion

Statement of interest None declared.

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Chapter 11

Bronchiectasis associated with inflammatory bowel disease Ph. Camus* and T.V. Colby#

The two major inflammatory bowel diseases (IBD), ulcerative colitis and Crohn’s disease (CD), can involve the respiratory system in several ways. The most typical pattern of involvement is in the form of airway inflammation and narrowing, which may involve specific areas of the tracheobronchial tree from the trachea to the bronchioles or which can be diffuse. Marked inflammation, which can be granulomatous in CD, causes, at times, marked airway obstruction. This pattern of involvement is amenable to different forms of inhaled and oral corticosteroid therapy. Drugs used to treat IBD are though to have no responsibility in causing the syndrome. This is in contrast to parenchymal lung disease in IBD. Colectomy may trigger the onset of airway involvement and will not improve or cure established airway inflammation in IBD. Keywords: Airway inflammation, bronchiectasis, bronchiolitis obliterans-organising pneumonia, granulomatous inflammation, inflammatory bowel disease

*Dept of Pulmonary Disease and Intensive Care, University Medical Center Le Bocage and Medical School, Universite´ de Bourgogne, Dijon, France. # Dept of Pathology, Mayo Clinic, Scottsdale, AZ, USA. Correspondence: Ph. Camus, Dept of Pulmonary Disease and Intensive Care, University Medical Center Le Bocage and Medical School, Universite´ de Bourgogne, POB 77908- F-21079, Dijon, France, Email [email protected]

Ph. CAMUS AND T.V. COLBY

Summary

Eur Respir Mon 2011. 52, 163–177. Printed in UK – all rights reserved. Copyright ERS 2011. European Respiratory Monograph; ISSN: 1025-448x. DOI: 10.1183/1025448x.10004110

P

163

atients with either of the two major inflammatory bowel diseases (IBD), ulcerative colitis (UC) and Crohn’s disease (CD), may develop a host of unusual, well-defined thoracic manifestations (table 1) [1–6]. Among these manifestations, a distinctive pattern of airway inflammation and scarring involving the major and minor airways (depending on the patient) has emerged clinically, endoscopically and pathologically as a consistent and increasingly recognised form of respiratory involvement in IBD. The severity ranges from the asymptomatic state to copious and disabling bronchorrhea or acute asphyxia. In addition, IBD is also associated with interstitial lung disease (ILD) with a variegated pattern on high-resolution computed tomography (HRCT), sterile necrobiotic neutrophilic nodules and pleuropericardial involvement. It is important to appreciate that therapy with several IBD-modifying drugs can also produce diffuse ILD,

164

UC,CD

UC,CD

+

UC,CD

CD..UC UC.CD UC..CD

+ ++ Uncommon Does not apply +++ ++ ++ ++ ++ ++ ++ + Does not apply Does not apply +

Moderate Weak Unknown

UC.CD CD..UC

+++ +++ Very rare

Moderate

Weak

Low Weak No

Strong Strong

+ ++

UC,CD

No

NSIP Pulmonary infiltrates with eosinophilia BOOP ILD with granulomas Desquamative interstitial pneumonia Localised mass or masses and nodules Granulomatous inflammation Localised BOOP Necrobiotic nodules" Therapy-related lung disease Drug-induced pneumonitis Opportunistic infections Pleural surface Serositis Effusion Pericardial surface Pericarditis Pericardial effusion or tamponade

Unknown

UC.CD

Drugs

Moderate

Moderate

Strong with CD Moderate Moderate

Strong Strong with CD Unknown

Moderate Low

Strong

IBD

Evidence base for association with

+++ ++ ++

++

+

+++

+++

UC versus CD

Main bronchi Small/peripheral airways# Infiltrative lung disease Diffuse ILD

Trachea

Airway inflammation/ deformity/scarring Glottis, larynx, subglottic region

Onset post-colectomy

Frequency/ incidence

Table 1. Airway involvement in inflammatory bowel disease

INFLAMMATORY BOWEL DISEASE

Malignancy, infection, autoimmune

Malignancy, infection, autoimmune

Bacterial infection

TB, sarcoidosis

Malignancy

BOOP of other causes TB, sarcoidosis, HSP

ILD due to drugs/other causes, metastatic lymphangitic spread ILD due to drugs/other causes PIE due to drugs/other causes

ANCA related (granulomatosis with polyangiitis (Wegener’s)), TB, sarcoidosis Herpes virus, polychondritis, TB, maligancy, papilloma Classic chronic bronchitis/smoking bronchiectasis Other causes of acute/chronic bronchiolitis

Main competing diagnosis

The evidence that IBD is causally associated with airway inflammation is based on: 1) the steady flow of consistent clinical descriptions of an association worldwide since the 1960s; 2) the common embryologic ancestry of the bronchi and bowel suggests coinvolvement in the same disease process; 3) the frequent reports of airway involvement occurring post-colectomy in individuals with UC with no history of lung disease [1, 7]; 4) the impressive response of airway inflammation to inhaled or oral corticosteroid therapy at least in patients with mild or moderate disease [1, 8–11]; and 5) epidemiologic studies showing greater prevalence of bronchitis in IBD patients overall [12]. Taken together, these findings suggest a true causal association of IBD with airway inflammation [12, 13]. In approximately 75% of IBD patients who develop airway involvement, the onset of respiratory symptoms is weeks to years after the development of clinically confirmed IBD. Post-colectomy patients are not immune to the development of airway involvement (which may be very severe) and colectomy may even be a risk factor for onset and progression of severe airway involvement in UC [1, 7]. Less often, IBD-related airway involvement pre-dates the onset of the IBD (raising difficult diagnostic issues), develops concomitantly with the inaugural flare of the IBD, or parallels flare ups of the IBD [1, 6]. Contrasting with ILD (the other major pattern of respiratory involvement in IBD), many IBD patients who develop airway involvement do so at a time when they are no longer exposed to IBDmodifying drugs, either because the IBD is quiescent or because of their post-colectomy status.

Ph. CAMUS AND T.V. COLBY

This chapter will focus on airway inflammation in IBD which can occur in both UC and CD, with greater incidence in the former. Although some overlap exists, the inflammation associated with each condition has distinct clinical and pathologic features. Notably, granulomatous inflammation is observed in the airways and/or lung parenchyma in CD, while non-granulomatous inflammation is seen in UC.

Airway involvement in IBD is generally inflammatory in nature and therefore typically amenable to therapy with inhaled or oral corticosteroids, may

165

+ Very rare at present

UC,CD – + ++

Other thoracic manifestations Venous/pulmonary thromboembolism Fistulas1 Low serum albumin and pulmonary oedema

Onset post-colectomy Frequency/ incidence

UC: ulcerative colitis; CD: Crohn’s disease; IBD: inflammatory bowel disease; BOOP: bronchiolitis obliterans-organising pneumonia; ILD: interstitial lung disease; NSIP: nonspecific interstitial pneumonia; ANCA: antineutrophil cytoplasmic antibody; TB: tuberculosis; PIE: pulmonary infiltrates and eosinophilia; HSP: hypersensitivity pneumonitis. –: no/never; +: unusual; ++: occasional; +++: common among IBD-related respiratory manifestations (overall incidence is low with 177 instances in 155 patients in 2007 [6]; .: greater, ..: far greater; ,: lower; ,: equal to. #: .7th generation; ": also named pulmonary Pyoderma gangrenosum; 1: colobronchial or oesotracheal.

No

Drugs

IBD

Strong; odds ratio,3 Strong

Main competing diagnosis Evidence base for association with UC versus CD

Table 1. Continued.

involvement of the pleural space or cardiac hypersensitivity reactions. Several drugs used to treat IBD, such as anti-tumour necrosis factor (TNF)-a therapy, put patients at risk of developing opportunistic pulmonary infections, including pulmonary tuberculosis and should be considered in the list of differential diagnoses.

localise from the glottis to the smallest airways depending on patient and stage of the disease, may be localised or diffuse in the airways, or may lead to a reduction in airway patency which, when involving the upper airway (in particular larynx, vocal cords or glottis), carries the risk of rapidly progressive life-threatening airway obstruction [1, 14, 15]. The diagnosis of any respiratory manifestation in IBD is one of exclusion and the main competing diagnoses are listed in table 1. Differential diagnoses include other systemic conditions capable of involving the central airways, such as sarcoidosis, relapsing polychondritis, tracheal amyloidosis or papillomatosis, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (granulomatosis with polyangiitis (Wegener’s)), idiopathic subglottic stenosis, chronic bronchitis, bronchiectasis or suppurative airway disease of other causes [16, 17]. One must also consider drug-induced disease, since the IBD-modifying drugs sulfasalazine and mesalazine can produce adverse reactions in the lung or heart [18]. Similarly, therapy with corticosteroid drugs and anti-TNF-antibody therapy increases the risk of developing opportunistic pulmonary infections including tuberculosis. Therefore, IBD patients who present with ILD, purulent necrobiotic nodules, acute bronchiolitis and granulomatous airway inflammation need to be carefully investigated to exclude infection and drug induced changes [3, 19, 20].

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Literature milestones The first report on airways disease in UC by LOPEZ BOTET and ROSALEM ARCHER [21] described the essentials of a unique disease, subsequently identified in many patients in several studies. The authors reported the occurrence of aggressive ulcerous bronchitis and bronchiectasis (confirmed on contrast bronchography), associated with profuse bronchorrhoea and haempotysis, in a 38year-old female 10 years after colectomy for UC. Prednisolone treatment improved her symptoms temporarily before she developed refractory airways disease, amyloidosis and eventually died. The authors suggested that the two manifestations reflected one single disease, and that the inflammatory process may have shifted to the airways. In 1976, KRAFT et al. [22] drew attention to the potential association of IBD and disabling airway disease. In their seminal paper they described six adult IBD patients; five UC and one with regional enteritis (CD). All of the patients were nonsmokers who developed chronic, otherwise unexplained, bronchorrhea 3–13 years after the onset of their IBD. In two patients, the airway disease developed following total proctocolectomy. There was a correlation of bowel and respiratory symptoms in four patients. Five patients had an obstructive pattern of pulmonary dysfunction. Bronchiectasis was evidenced using contrast bronchography in four patients. Oral corticosteroid therapy used to treat the underlying IBD was not reported to notably influence the course of airway involvement. HIGENBOTTAM et al. [8] described 10 nonsmoking patients with UC who presented with a chronic productive cough, which was not felt to be due to sulfasalazine treatment. Bronchial epithelial biopsies from four patients revealed basal reserve cell hyperplasia, basement membrane thickening and submucosal inflammation. Treatment with inhaled corticosteroid (beclomethasone diproprionate) relieved the cough in seven patients. These investigators highlighted the possibility that airway involvement in UC might be explained by the common embryologic ancestry of the bronchial and intestinal epithelium, representing a new extra-intestinal manifestation (EIM) of the disease.

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These observations were expanded in a study by CAMUS et al. [1] of 33 IBD patients (UC n527, CD n56) of whom 20 presented with airway involvement. Three out of these 20 patients presented with severe upper airway inflammation narrowing and tortuosity, 15 with central airway inflammation or suppurative airways disease (trachea or major bronchi), of whom six had documented bronchiectasis, and two with small airway involvement or bronchiolitis. In the three patients with central airway involvement and upper airway obstruction, airway endoscopy showed friable, velvety airway inflammation with cobble stoning and haemorrhage. Airway patency was reduced to 20% of normal in one case and appearance of the mucosa in the airway was reminiscent of that in the colon. In the 15 patients who presented with large airway inflammation,

airway endoscopy also showed severe inflammation with glittering erythema and oedema severely narrowing the airway lumen with effacement of bronchial cartilaginous rings. The bronchoalveolar lavage (BAL) showed increased neutrophil counts, which diminished in responders once corticosteroid therapy was administered in parallel with the resolution of the airways symptoms of cough and sputum. Pulmonary function (notably forced expiratory volume in 1 second) also improved dramatically by o50%, even in patients with bronchiectasis. Six further patients presented with febrile pulmonary infiltrates corresponding pathologically to bronchiolitis obliterans-organising pneumonia (BOOP), a disease of the transitional zone of the lung that is traditionally considered an ILD. However in IBD, BOOP was notable for prominent ulcerative or suppurative involvement of the distal bronchioles, raising the question of the dominant site of involvement in IBD-related BOOP. Inhaled corticosteroids were effective in controlling the symptoms of cough, sputum and airway pathology in those patients with chronic bronchitis (in ,60%), but were less efficacious in doing so in patients with bronchial suppuration, bronchiectasis or chronic bronchiolitis (,30%) in whom oral corticosteroid were effective. A literature review indicated that upper airway involvement accounted for 11.1% of the reported cases, large and small airway involvement 83.3% and 5.6%, respectively, and that about half the cases of IBD-associated airway involvement had developed post-coletomy.

CASEY et al. [24] reviewed their experience with 11 lung biopsies from CD patients who presented with diffuse or localised pulmonary opacities. Workup for an infection was negative in all 11 cases. The major pathologic features in four patients were chronic bronchiolitis with non-necrotising, non-coalescent granulomatous bronchocentric inflammation. Two further patients had acute bronchiolitis associated with a neutrophil-rich bronchopneumonia with suppuration and vague granulomatous features resembling that seen in UC. The remaining five patents were diagnosed with ILD or organising pneumonia. In 2007, BLACK et al. [6] reviewed the literature on 171 instances of respiratory pathology (99 with airway involvement) in 155 IBD patients. Large airway involvement was found to be the most common pattern of involvement, accounting for 67% of the cases overall, with bronchiectasis being the most frequently reported pattern. Involvement of the upper airway (glottis and larynx) and small airway accounted for 15% and 17% of the cases, respectively.

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GARG et al. [23] described the HRCT features of airway inflammation in seven patients with UC (five post-colectomy) who presented with cough and recurrent respiratory infections. Fibreoptic bronchoscopy in six patients showed diffuse mucosal erythema and oedema that were most severe in the proximal airways. Sinus imaging showed mucosal thickening in six patients, a feature that has not been described previously. HRCT features included bronchiectasis in six patients, peripheral airway involvement in four patients and a rigid and stenotic trachea in three patients.

Several other notable papers have consistently described similar, if not identical, cases and/or reviewed earlier literature. Taken together, these studies further confirm a true association of IBD and large or small airway involvement, and the beneficial effect of corticosteroid therapy in many cases [3, 9, 11, 20, 25–30].

Epidemiology: risk factors Clinically apparent airway involvement is uncommon in IBD. KRAFT et al. [22] calculated a prevalence rate of 0.21% in their IBD clinic. In a recent study of 165 patients with bronchiectasis detected on computed tomography scans, an underlying cause was identified in 122 (74%) patients; five patients had a history of IBD (up to 10 years earlier in one case), two were postcolectomy and in one patient the diagnosis was made during a flare up of IBD [17].

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Figures for prevalence may be higher if subclinical airway involvement is defined by subnormal pulmonary physiology (a common occurrence in IBD, particularly during flare ups) [31–34], increased exhaled nitric oxide [35], minimal changes of uncertain significance on imaging [36] or changes in induced sputum cytology [35, 37, 38]. However, although subclinical changes in BAL

cell profile have been found in IBD [35, 39], there is no current evidence to suggest a link between these subtle changes and the likelihood of developing overt airway or parenchymal lung involvement at a later time. Females outnumber males with an approximate ratio of 1.8–2.1 [1, 6]. Colectomy has been suspected to be a risk factor for the development of IBD (mainly UC)-related airway involvement [1, 8, 22]. Recently, KELLY et al. [7] confirmed this in 10 patients with IBD (CD n55) and bronchiectasis. Eight of these patients had developed respiratory symptoms from within a few weeks to decades after colectomy. One may question whether IBD-associated airway involvement is linked to colectomy per se, or occurs as a result of IBD-modifying drug withdrawal post-colectomy. However, the long time delay of several decades in some patients tends to support the notion that airway involvement in IBD is an EIM of the disease, rather than a complication of drugs or a result of drug withdrawal. Furthermore, colectomy in patients with IBD and airway involvement may lead to deterioration of the respiratory condition and should not be proposed in an attempt to cure the airway involvement [1]. A high rate (52%) of EIM other than in the lung was noted in IBD patients with airway involvement. Smoking is unlikely to play a causal role as most patients with the association are nonsmokers or reformed smokers [1, 40].

Clinical presentations

INFLAMMATORY BOWEL DISEASE

Upper airway obstruction: glottic and subglottic This presentation is unusual and it is the most worrisome pattern of involvement in IBD as this may cause rapidly progressive, severe airway compromise and acute asphyxia. IBD-related upper airway obstruction has been described in both UC and CD, often in association with active IBD, having a similar clinical presentation in both conditions (figs. 1 and 2). Early onset of symptoms of sore throat and hoarseness can be mistaken as upper respiratory tract infection [1, 6, 14, 15]. These annunciating symptoms may not receive appropriate attention. Following this a continuous resonant deep-toned barking cough may develop, sometimes with hoarseness due to vocal cord oedema or dysmotility, stridor and blood-tinged sputum [1, 14, 41, 42]. The overall amount of sputum is usually insignificant, except if patients have associated tracheal or large airway involvement, which is frequent. In a few patients, flow reduction [43] is noted on the inspiratory and expiratory limb of the flow–volume loop [26], indicating fixed as opposed to variable airway obstruction. Inexplicably, upper airway inflammation can accelerate and progress rapidly, producing severe airway compromise within a few hours or days [1, 6, 14, 15, 28], at times requiring mechanical ventilation [15]. Unequivocal airway stenosis can be visualised on computed tomography [44, 45]. On endoscopy, there is marked erythema of the vocal cords, glottis or subglottic region with oedema, a velvety friable oedematous mucosal swelling, whitish or reddish nodules, distorted anatomy and pus. In some cases, the 5-mm fibreoptic bronchoscope could not be passed through and beyond the stenotic area without causing further compromise [1, 14], or progression of the scope in the trachea required repeat bending to reach the more distal trachea [1]. Macroscopically, appearance of the airway walls is reminiscent of that in the colon in UC [25, 40]. Beyond the stenotic area, there is marked inflammation and bulging of tracheal walls. The extent of involvement varies depending on the patient, being limited to the upper trachea in some and in others extending upstream beyond the tracheal bifurcation to involve the main stem bronchi, also in the form of diffuse inflammation or erythematous or haemorrhagic nodular deformity, distorting and reducing airway patency [25]. Imaging studies using HRCT planar reconstruction or magnetic resonance imaging demonstrate marked thickening of the airway wall and a correlative reduction in airway calibre [1, 43, 45, 46]. Pathologically, bulging of the airway wall corresponds to dense lymphoplasmacytic and oedematous mucosal infiltrate with, sometimes, lymphocytes, neutrophils or rare eosinophils permeating the mucosa up to the epithelium which is also infiltrated (fig. 2a and b). The overlying airway mucosa may show squamous metaplasia or may be ulcerated [1, 47]. When present, noncaseating granulomas suggest a diagnosis of CD as opposed to UC.

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The pattern of upper airway obstruction in UC requires expeditious and emergent management to restore airway patency via interventional endoscopy using debridement, laser, argon plasma

a)

g)

e)

c)

f)

h)

Figure 1. Chest and endocopic imaging in inflammatory bowel disease-related airway involvement. Upper airway inflammation and stenosis is best evidenced using a) computed tomography (CT) reconstruction or magnetic resonance imaging and b) fibreoptic bronchoscopy. Inflammation may localise in the glottic or subglottic area, often involving the trachea (b) and proximal airways which show b) cobble stoning and c) inflammation and pus. d) Radiographically, minimal changes are present in early disease in the form of bibasilar bronchial tramlines. On CT examination there is a combination of e) airway wall thickening, f) glove-finger shadows reflecting airway filling by inspissated secretions or g) a tree-in-bud appearance reflecting small airway inflammation. h) Late changes are in the form of bronchiectasis. Often changes on endoscopy and imaging will improve with inhaled alone or inhaled and oral corticosteroid therapy.

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d)

b)

coagulation, electrocautery, dilation, stent placement (if the lesion does not occupy the glottic and subglottic space and does not involve the vocal cords) and topical injections of corticosteroids and/ or mitomycin C [28, 45, 46]. Inhaled, nebulised and parenteral corticosteroids and infliximab have also been used and this has met with success in a few cases [45]. Breathing a helium–oxygen mixture (heliox) is indicated during the acute phase of the disease. Prudent dilatation of the airway using calibrated bougies can be considered to restore airway patency. However, this was complicated by mediastinitis in one case [43]. Overall, the response to combined treatment is encouraging.

Central airway involvement: trachea and main stem bronchi

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This is the most common and most disabling pattern of airway involvement in IBD with 67 cases reported overall (figs. 1 and 2) [1, 6, 22, 23, 40, 47–52]. Age at onset of the airway disorder is, on average, 43 years. Two-thirds of the patients were females [6]. Three main patterns were described: 1) chronic bronchitis with cough and moderate sputum, 2) suppurative airway disease with abundant bronchorrhea, and 3) chronic bronchiectasis [1, 6]. It is unclear whether there is a

a)

b)

c)

d)

e)

f)

g)

INFLAMMATORY BOWEL DISEASE

Figure 2. Airway pathology in inflammatory bowel disease (IBD)-related airway involvement. a, b) Tracheobronchial inflammation is in the form of a dense and florid mixed submucosal lymphoplasmacytic infiltrate within the airway wall, sometimes markedly reducing airway patency. The mucosa can be ulcerated (a) and the inflammatory infiltrate (including neutrophils and a few eosinophils) can be seen permeating and homing toward the airway mucosa (b). Bronchial glands may be damaged or destroyed (not shown). Inflammation may also involve c) the more distal airways or bronchioles (diameter of the airway lumen ,1.8 x 1 mm) down to d) the smallest airways (showing at least six bronchioles involved) in the form of acute and chronic exquisitely bronchoor bronchiolocentric inflammation, while the vasculature is spared and uninvolved. Occasionally, there is e) purulent bronchiolitis (can also be seen in IBD-associated bronchiolitis obliterans-organising pneumonia) and/or f) purulent bronchiolar and tissue necrosis. g) In a few cases constrictive bronchiolitis and chronic obstruction to airflow develop as a late manifestation.

continuum from chronic bronchitis to suppurative airways disease or bronchiectasis in a given patient. However, the clinical impression is that some patients do progress from simple chronic bronchitis to bronchiectasis in the absence of, and sometimes in spite of, corticosteroid therapy for a few months or years. Cough and sputum are typically unexplained other than by the background history of IBD. The condition essentially occurs in adulthood in nonsmoking IBD individuals with no history of lung of airway disease. Typically, IBD-related large airway disease manifests with the insidious or rapid development of cough productive of variable amounts of clear, purulent or blood-stained sputum. Copious bronchorrhea (.100 mL and o500 mL) has been reported in a few cases [1, 53]. Some patients experienced parallel flare ups of bowel and bronchial symptoms, further reinforcing the notion of a true association [1, 8, 49]. In several instances abundant bronchorrhea and severe airway involvement developed a few days to a few weeks after total colectomy as though aggressive inflammation had ‘‘shifted’’ away from the bowel to the airways [7, 48, 51]; although inexplicably, airway involvement can occur much later [1, 7, 21].

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Pulmonary function testing usually reveals a moderate-to-severe obstructive or mixed obstructive and restrictive spirometric profile [1]. There is little change in airflow upon inhalation of a bronchodilator drug. Bronchial responsiveness to methacholine is usually normal [1], and this contrasts with the background of pronounced inflammation noted on pathology. The figures often improve dramatically following inhaled and/or oral corticosteroid therapy [1, 11].

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Findings on endoscopy may be near normal in patients with early or mild symptoms such as cough, or may show diffuse erythema. Bronchial biopsy specimens at this stage may evidence submucosal inflammation [1, 3]. Neutrophils are increased in the BAL [1] and on follow-up these cells diminish in number and percentage in patients who respond to inhaled corticosteroids in terms of improvement in cough and sputum [1]. In general, in patients with IBD-related airway involvement, changes are evident endoscopically [1, 9, 23] in the form of erythema, oedema, velvety bulging of the tracheal or bronchial walls and whitish or reddish cobble stoning [46, 47]. The changes may be predominant in the trachea or they may extend in the form of sparkling oedema in main stem bronchi and more distally. At times, reduced airway patency prevents full inspection of the bronchial tree [1, 60]. Pathologically, the underlying IBD seems to repeat the abnormalities found in the bowel [1, 3]. A dense submucosal collection of plasma cells and lymphocytes deeply infiltrates the airway wall [1, 3, 11]. The epithelium undergoes squamous metaplasia and/or is ulcerated. Neutrophils and rare eosinophils may be interspersed in the cellular infiltrate and epithelium. Subepithelial airway glands beneath the mucosa may be destroyed by the infiltrate and inflammatory cells may extend around the ducts of the bronchial glands and into the glands themselves [1]. IBD-related bronchiectasis differs Table 2. Airway involvement in inflammatory bowel disease clinically and pathologically from typical Site of involvement bronchiectasis. The former is positively Larynx/glottis/subglottic area 2 (2.2) influenced by corticosteroid therapy and, Tracheal¡subglottic inflammation/stenosis 15 (16.6) pathologically, the latter shows a less Bronchiectasis 44 (48.9) dense and conspicuous cellular infiltrate Chronic bronchitis 13 (14.4) and with more germinal centres (follicuSuppurative airways disease 5 (5.6) lar bronchiectasis). The inflammatory Bronchiolitis/granulomatous bronchiolitis 10 (11.1) Diffuse panbronchiolitis 1 (1.1) infiltrate in IBD-related airway involvePure constrictive bronchiolitis 2 (2.2) ment may extend to more distal airways ILD with a bronchiolitis component 21 which, if available for examination, for such as BOOP example on a lung resection specimen [1, 3], Data are presented as n (%) or n. ILD: interstitial lung show a similar pattern of inflammation disease; BOOP: bronchiolitis obliterans-organising pneuand stenosis down to the bronchioles monia. Data from [6]. (fig. 2c) [1, 57]. There is histological

Ph. CAMUS AND T.V. COLBY

Although the extent of abnormalities on imaging is generally in proportion to the severity of clinical symptoms, abnormalities on the chest radiograph can be surprisingly small and discreet, being simply in the form of linear basilar opacities or the ‘‘dirty lung’’, despite disabling cough and abundant sputum. Radiographically, early or mild cases show minimal or no changes. More advanced or progressive cases show bibasilar tramlines indicating bronchial wall thickening, especially in cases with suppurative airway disease. Tubular or cystic bronchiectases are seen in yet more advanced cases [52]. On HRCT examination, early cases may evidence non-uniform lung emptying on full expiration thought to reflect peripheral airway obstruction [36, 52]. In more advanced cases, thin-cut sections of airway on HRCT [54] show airway wall thickening [11] and an increased external diameter of the airway compared to the adjoining vessel. In more severe cases, extensive bronchial wall thickening and basilar or widespread dense-tubulated or dichotomously-branched opacities, which are also known as glove-finger shadows, are seen [1, 53]. The latter changes are reminiscent, if not similar to, those in allergic bronchopulmonary aspergillosis and may represent impaction of inspissated mucoid or purulent secretions filling the airway lumen. However, more advanced cases show basilar or more widespread cystic bronchiectasis in addition to the aforementioned changes [1, 53, 55–58]. Subtle changes can be present in distal regions of the lung in the form of small irregular dichotomously branched shadows, the so-called tree-in-bud appearance, more often than not [58] subpleurally in the bibasilar lung [53]. These changes are thought to represent peribronchiolar cellular cuffing and may correlate pathologically with acute, subacute and/or chronic bronchiolitis [3, 20, 59]. HRCT imaging of maxillary and ethmoid sinuses may demonstrate mucosal thickening in up to 60% of patients with UC-related large airway involvement [23].

Table 3. Airway involvement in inflammatory bowel disease (IBD): evidence of relationship High prevalence of co-existing extra-intestinal manifestations including sclerosing cholangitis Absence of a history of airway or lung disease in childhood or adulthood Low incidence of smoking No other cause identified at the origin of the airway inflammation or bronchiectasis, no immune deficiency Onset of airway involvement following the onset of the IBD Parallel flares of airway and bowel manifestations (rare) Onset of airway involvement after (sometimes very shortly or up to several years) proctocolectomy Colectomy tends to aggravate symptoms and extent of involvement in the airways Distinctive pathologic features or airway (trachea to the smallest airways) involvement Similar macroscopic appearance and microscopic features of airway and interstinal inflammation Marked improvement with corticosteroid therapy, unlike classic airways diseases except asthma Relapse of airway symptoms and inflammation with corticosteroid withdrawal Similar embryologic ancestry of airways and bowel

INFLAMMATORY BOWEL DISEASE

similarity between the airways and colonic mucosa in UC-related large airway involvement, particularly with regards neutrophilic infiltration, mucosal ulceration and dense underlying chronic inflammation with plasma cells [3]. There is little correlation between the degree of airway inflammation seen on endoscopy and the amount of expectorated sputum. Stains and cultures yield inconsistent results, being sterile or showing normal flora, with rare Pseudomonas colonisation. Symptoms inconsistently improve following a course of antibiotics except if used in conjunction with inhaled or oral corticosteroids (see later section). In patients who respond to corticosteroid therapy, the airway appearances can return to normal [30]. In a few patients, however, late changes will develop in the form of tracheal stricture or deformity, cicatricial obliteration of one or more bronchial orifices or localised weblike strictures.

Small airway involvement: bronchiolitis There is some confusion surrounding the term ‘bronchiolitis’, according to whether the condition is suspected clinically using HRCT, pulmonary function testing or BAL, or is diagnosed pathologically using transbronchial sampling or surgical biopsies (the latter is rarely indicated). Bronchiolitis is best defined pathologically by inflammatory events centred on small noncartilagenous airways generally measuring ,2 mm (approximately the 7th generation). These airways are situated in the central portion of the secondary pulmonary lobule and, when inflamed, result in centrilobular nodules visible on HRCT. Bronchiolitis may be the predominant finding on a lung biopsy specimen (although it may simply reflect or accompany inflammatory changes in proximal bronchi in bronchiectasis) and/or may extend and transition into more distal alveolar lung in the form of BOOP. Evaluation of bronchiolitis requires careful exclusion of an infectious aetiology. Small airway involvement in IBD has been reported in 17 patients overall [6]. The condition occurs at a younger age (29 years on average) and in both sexes equally, compared to large airway involvement [6]. In approximately a third of the patients, bronchiolits pre-dated the onset of the IBD [6]. Cough and sputum are not always present and the condition may manifest with cough, dyspnoea, or wheeze accompanied by obstructive or restrictive lung function abnormalities [1, 43, 61]. Radiographically, the chest film can be normal or demonstrate small diffuse irregular or nodular opacities [24, 62, 63].

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Although bronchiolitis can be the predominant histopathologic finding in both UC and CD [1, 3, 6, 19, 24, 43, 62–65], the pathological features differ between these conditions. In CD, there is associated non-caseating, non-coalescent bronchiolocentric granulomatous inflammation [24, 66] while in UC, there is dense bronchiolocentric neutrophilic inflammation of the airway wall or suppurative bronchiolitis with neutrophils filling the lumen. Although inflammation has a predilection to involve the bronchioles, inflammation of the neighbouring lung can be present, producing some parenchymal shadowing or consolidation on imaging [62], or focal suppuration

resembling Pyoderma gangrenoum in the skin [19]. Some cases show florid organising pneumonia (BOOP) in addition to acute bronchiolitis [1, 67]. A few cases exhibited a pattern identical to diffuse panbronchiolitis [1], as originally described in Japanese individuals [68], with interstitial foam cells in addition to acute and chronic bronchiolitis [1, 3]. Scarring may follow acute and chronic bronchiolitis in the form of constrictive bronchiolitis characterised by severe obstruction to airflow (fig. 2g) [43]. In such patients, lung transplantation may be an option. The link between UC or CD and small airways involvement is more than tenuous and acute or chronic bronchiolitis should be considered as part of the spectrum of UC-related airway involvement. Some investigators have compared bronchiolitis, as it occurs in UC, to sclerosing cholangitis, another UC-associated EIM.

Management There is sparse and limited evidence to indicate classic IBD-modifying drugs specifically in patients with IBD-related airway involvement as these agents are largely ineffective. Although anecdotal reports described improvement of airway pathology after infliximab [45], IBDmodifying drugs are not recommended as a first-line treatment in this condition. Similarly, no response has followed therapy with azathioprine or cyclophosphamide.

Corticosteroid drugs are the mainstay of treatment of IBD-related airway involvement. The route of administration, dosage, titration and duration of treatment with corticosteroid varies with the patient and is largely empirical. In patients with airway involvement of moderate severity, such as mild chronic bronchitis, inhaled corticosteroids are the treatment of choice. It is customary to start with a high dosage (2,000– 2,500 mg?day-1) [1, 60]. Adjunctive oral corticosteroid therapy may be used but does not seem to be an absolute requirement in early/mild disease. Inhaled corticosteroid therapy often provides convincing improvement and excellent clinical control of the airway disease at this stage. Improvements in pulmonary function (if decreased prior to onset of treatment), imaging, endoscopy and BAL neutrophilia accompany the clinical improvement [1, 11, 30, 46]. Once a satisfactory response to treatment is obtained, inhaled corticosteroids can be slowly tapered every month or so to lower dosages similar to those used to treat asthma (1,200–1,600 mg?day-1). Patient education will permit any recrudescence in symptoms to be self managed by an increased dose of inhaled corticosteroids. The addition of oral corticosteroids (e.g. 25–60 mg oral prednisolone or equivalent depending on sex, weight and severity) is normally indicated when there has been no or very slow clinical improvement after a few weeks of inhaled corticosteroid therapy. Oral steroids are more readily efficacious [40] enabling quicker control of symptoms and are indicated in patients with moderate or severe airway involvement. It seems important to reach the normal clinical state as quickly as possible to ensure the best possible quality of remission. Oral corticosteroids are tapered in a few weeks to the minimal effective dosage and withdrawn if possible. Short (2–6 weeks) bursts of oral corticosteroid may be indicated during relapses, should inhaled corticosteroids not suffice in controlling the disease.

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Colectomy has not shown to be of benefit in the management of IBD-associated airways disease, and bowel surgery should be critically discussed in such patients. Furthermore, a number of instances have described the sudden onset, or clear deterioration, of IBD-associated airway involvement shortly after colectomy.

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Importantly, patients with more advanced or aggressive IBD-related large airway involvement, with or without bronchiectasis, may also benefit from long-term inhaled corticosteroid therapy. Imaging or pathology cannot readily identify which patient will respond to corticosteroid therapy [1], and clinical response may be associated with no change on imaging and little change in physiology [55–56, 69]. Cases with copious bronchorrhea are less likely to improve on inhaled corticosteroids, possibly due to altered pharmacokinetics of ICS in the diseased bronchial tree [1]. In such patients a nebulised corticosteroid is indicated (e.g. 1 mg budesonide b.i.d. to q.i.d.), in addition to more classic oral and inhaled corticosteroids until improvement in symptoms occurs [1].

Dosage and duration of treatment with oral and inhaled steroids are guided by clinical response, pulmonary function, bronchoscopy and follow-up HRCT (weighing up the risk of increased radiation exposure particularly in young people). Although there is no evidence favouring this, we advise patients to: 1) take their drugs accurately, avoiding any drug holiday even though they may feel better; 2) exercise regularly with the hope that inspissated secretions dislodge, enabling inhaled corticosteroids to reach deeper, more distal airways and with the hope of minimising the musculoskeletal adverse effects of corticosteroids regardless of the route of administration; and 3) receive regular chest physiotherapy unless they reach the asymptomatic state. Fine-tuning of all aspects of steroid treatment in IBD-related airways disease is best carried out in close co-operation with the patient, who is often a very astute observer of his/her own illness. It is interesting that paying attention to such small details such as careful explanation of how treatment works, and punctuality in terms of inhalation and exercise often meet with improved compliance and significant clinical improvement, while the nominal dosage of corticosteroids was left unaltered.

INFLAMMATORY BOWEL DISEASE

Additional treatment options include courses of antibiotics since bouts of infection may repeatedly complicate the course of the airways disease, and expectorate actively by positional and voluntary coughing to clear the airways. There is no published or presented experience with azithromycin in IBD-associated airway involvement. Given the benefit of this drug in other forms of inflammatory airways disease, an empirical therapy may be worth trying in selected patients [70]. Two issues are currently unresolved. 1) Although corticosteroid therapy is indicated, the specific effect of inhaled, nebulised, systemic or topical corticosteroids in IBD-related upper airway involvement is unclear and difficult to evaluate separately. 2) Management of patients who present with aggressive airway inflammation and stenosis immediately or later during the course of their disease are a real concern. Corticosteroids may have transient or not perceptible effects and few options are left available, in as much as patients may suffer adverse effects of prolonged corticosteroid therapy. We attempted to deliver higher steroid dosages topically via bronchial instillations of methylprednisolone in saline via the fibrescope three times per week. A typical 40–80 mg dose in normal saline is instilled alternatively in the right and left bronchial tree every 2–3 days. Responders show a decrease in symptoms and some bleaching in the airways consistent with reduced inflammation. The time interval between two instillations can be expanded to 5–6 days in those who respond. Still, some patients’ illness is refractory to any form of therapy, with bronchial inflammation progressing to uncontrollable destruction of the entire tracheobronchial tree, pulmonary function deteriorating and adverse effects of corticosteroid therapy tragically increasing with time. Lung transplantation and novel techniques of airway management need to be discussed in such desperate cases [71, 72].

Statement of interest None declared.

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40. Spira A, Grossman R, Balter M. Large airway disease associated with inflammatory bowel disease. Chest 1998; 113: 1723–1726. 41. Karasalihoglu A, Kutlu K, Yilmaz T. Laryngotracheal obstruction in ulcerative colitis (apropos of a case). Rev Laryngol 1988; 109: 469–471. 42. Kerneis J, Bernard R, Frances JL, et al. Tracheal stenosis and hemorrhagic rectocolitis. Gastroenterol Clin Biol 1993; 17: 63–65. 43. Wilcox P, Miller R, Miller G, et al. Airway involvement in ulcerative colitis. Chest 1987; 92: 18–22. 44. Kinebuchi S, Oohashi K, Takada T, et al. Tracheo-bronchitis associated with Crohn’s disease improved on inhaled corticotherapy. Intern Med 2004; 43: 829–834. 45. Kirkcaldy J, Lim WS, Jones A, et al. Stridor in Crohn’s disease and the use of infliximab. Chest 2006; 130: 579–581. 46. Plataki M, Tzortzaki E, Lambiri I, et al. Severe airway stenosis associated with Crohn’s disease: case report. BMC Pulm Med 2006; 6: 7. 47. Vasishta S, Wood JB, McGinty F. Ulcerative tracheobronchitis years after colectomy for ulcerative colitis. Chest 1994; 106: 1279–1281. 48. Butland RJA, Cole P, Citron KM, et al. Chronic bronchial suppuration and inflammatory bowel disease. Q J Med 1981; 50: 63–75. 49. Moles KW, Varghese G, Hayes JR. Pulmonary involvement in ulcerative colitis. Br J Dis Chest 1988; 82: 79–83. 50. Gionchetti P, Schiavina M, Campieri M, et al. Bronchopulmonary involvement in ulcerative colitis. J Clin Gastroenterol 1990; 12: 647–650. 51. Gabazza EC, Taguchi O, Yamakami T, et al. Bronchopulmonary disease in ulcerative colitis. Intern Med 1992; 31: 1155–1159. 52. Mahadeva R, Flower C, Shneerson J. Bronchiectasis in association with coeliac disease. Thorax 1998; 53: 527–529. 53. Mahadeva R, Walsh G, Flower CD, et al. Clinical and radiological characteristics of lung disease in inflammatory bowel disease. Eur Respir J 2000; 15: 41–48. 54. Kosciuch J, Krenke R, Gorska K, et al. Relationship between airway wall thickness assessed by high-resolution computed tomography and lung function in patients with asthma and chronic obstructive pulmonary disease. J Physiol Pharmacol 2009; 60: Suppl. 5, 71–76. 55. Serrano J, Plaza V, Franquet T, et al. Bronchiolitis associated with ulcerative colitis. Arch Broncopneumol 1996; 32: 151–154. 56. Eaton TE, Lambie N, Wells AU. Bronchiectasis following colectomy for Crohn’s disease. Thorax 1998; 53: 529–531. 57. Ward H, Fisher KL, Waghray R, et al. Constrictive bronchiolitis and ulcerative colitis. Can Respir J 1999; 6: 197–200. 58. Faruqi S, Avery G, Morice AH. Chronic cough associated with Crohn’s disease. Cough 2010; 6: 6. 59. Sugino K, Hebisawa A, Uekusa T, et al. Histopathological bronchial reconstruction of human bronchiolitis obliterans. Pathol Int 2011; 61: 192–201. 60. Kuzniar T, Sleiman C, Brugie`re O, et al. Severe tracheobronchial stenosis in a patient with Crohn’s disease. Eur Respir J 2000; 15: 209–212. 61. Stanescu D, Veriter C. A normal FEV1/VC ratio does not exclude airway obstruction. Respiration 2004; 71: 348–352. 62. Hilling GAL, Robertson DAF, Chalmers AH, et al. Unusual pulmonary complication of ulcerative colitis with a rapid response to corticosteroids: case report. Gut 1994; 35: 847–848. 63. Haralambou G, Teirstein A S, Gil J, et al. Bronchiolitis obliterans in a patient with ulcerative colitis receiving mesalamine. Mt Sinai J Med 2001; 68: 384–388. 64. Desai SJ, Gephardt GN, Stoller JK. Diffuse panbronchiolitis preceding ulcerative colitis. Chest 1989; 95: 1342–1344. 65. Mazer BD, Eigen H, Gelfand EW, et al. Remission of interstitial lung disease following therapy of associated ulcerative colitis. Pediatr Pulmonol 1993; 15: 55–59. 66. Vandenplas O, Casel S, Delos M, et al. Granulomatous bronchiolitis associated with Crohn’s disease. Am J Respir Crit Care Med 1998; 158: 1676–1679. 67. Swinburn CR, Jackson GJ, Cobden I, et al. Bronchiolitis obliterans organising pneumonia in a patient with ulcerative colitis. Thorax 1988; 43: 735–736. 68. Iwata M, Sato A, Colby TV. Diffuse panbronchiolitis. In: Epler GR, ed. Diseases of the Bronchioles. New York, Raven Press, Ltd, 1994: pp. 153–179. 69. Alcazar Navarrete B, Quiles Ruiz-Rico N, Gonzalez Vargas F, et al. Bronchiectasis following colectomy in a patient with ulcerative colitis and factor V Leiden mutation. Arch Broncopneumol 2005; 41: 230–232. 70. Verleden GM, Dupont LJ. Azithromycin therapy for patients with bronchiolitis obliterans syndrome after lung transplantation. Transplantation 2004; 77: 1465–1467.

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Chapter 12

Immunodeficiencies associated with bronchiectasis J.S. Brown*, H. Baxendale#," and R.A. Floto",+

IMMUNODEFICIENCY

Summary Bacterial infection of the lung is a cause of bronchiectasis and also the main clinical problem in patients with bronchiectasis. As a consequence, inherited or acquired immunodeficiencies that allow repetitive lung infection with respiratory pathogens (such as Streptococcus pneumoniae and Haemophilus influenzae) can drive the development and progression of bronchiectasis. The immune defects most strongly associated with bronchiectasis are those resulting in hypogammaglobulinaemia. These include the primary immunodeficiencies, common variable immunodeficiency and X-linked agammaglobulinaemia and the secondary immunodeficiences caused by lymphoproliferative malignancy, allogeneic bone marrow transplantation and chemo/immunotherapy. Identifying hypogammaglobulinaemia is important and allows patients to be given immunoglobulin replacement, reducing exacerbation frequency and probably progression of bronchiectasis. Conditions resulting in T-cell dysfunction (such as chronic HIV infection or immunosuppression), reduced bacterial opsonisation (such as complement deficiencies), failure of phagocyte migration (leukocyte adhesion deficiency) and impaired intracellular killing of bacteria (chronic granulomatous disease) may also predispose to bronchiectasis. In this chapter we describe the main immunodeficiencies associated with bronchiectasis and suggest a staged approach to immunological investigations. Keywords: Antibody, bronchiectasis, haematopoietic stem cell transplant, HIV, immunodeficiency, T-helper cell type 17

B

*Centre for Respiratory Research, Dept of Medicine, Rayne Institute, Royal Free and University College Medical School, # Division of Infection and Immunity, Dept of Immunology, Royal Free and University College Medical School, London, " Cambridge Centre for Lung Infection, Papworth Hospital, and + Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK. Correspondence: J.S. Brown, Centre for Respiratory Research, Dept of Medicine, Rayne Institute, Royal Free and University College Medical School, 5 University Street, London WC1E 6JF, UK, Email [email protected]

Eur Respir Mon 2011. 52, 178–191. Printed in UK – all rights reserved. Copyright ERS 2011. European Respiratory Monograph; ISSN: 1025-448x. DOI: 10.1183/1025448x.10004210

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ronchiectasis is characterised by damage and dilatation of the bronchi allowing chronic colonisation with significant numbers of bacterial pathogens. The initial damage to the bronchi can be caused by infection. It is therefore not surprising that a range of immunodeficiences predisposing to recurrent respiratory tract infections can lead to the development of bronchiectasis.

Immunodeficiencies are defined as primary immunodeficiencies (PIDs), resulting from deleterious genetic mutations, or secondary immunodeficiencies (SIDs), where acquired insults have compromised immune function. The pathogens usually associated with milder bronchiectasis include Streptococcus pneumoniae and Haemophilus influenzae. Both are common nasopharyngeal commensals in adults and children that also cause acute bronchitis and pneumonia. It is probable that impaired host immunity to these pathogens initiates the development of bronchiectasis, which then further compromises mucosal defences permitting infection and sometimes colonisation with environmental bacteria, such as Pseudomonas aeruginosa. Existing data on the causes of bronchiectasis is derived from relatively low numbers of patients, usually from specialist centres, who have been immunologically investigated to a variable extent as discussed by BILTON and JONES [1] in the first chapter of this Monograph. As a consequence, the true proportion of bronchiectasis patients with a definable immunodefiency is unclear (and will certainly increase with modern molecular diagnostic approaches). From published reports, up to 7% of adults and up to a third of children presenting with bronchiectasis will have a PID [2–6]. Reported rates of SID are lower but are likely to increase in line with more frequent use of immunotherapy, solid organ and bone marrow transplantation and improved survival from HIV. In this chapter we will discuss each of the major PIDs and SIDs that have been associated with bronchiectasis (table 1), before drawing some more general conclusions about mucosal immunity to bacterial infection.

Primary immunodeficiencies In most case series the commonest immune disorders associated with bronchiectasis are antibody deficiencies [2–7]. Antibody deficiency can be inherited or acquired and can be caused by a range of specific defects in antibody production, leading to several distinct immunological phenotypes the most important of which are discussed below. S. pneumoniae and H. influenzae are both surrounded by an antigenic polysaccharide capsule which is a major virulence determinant for invasive infection. The close association of antibody deficiencies as causes of bronchiectasis perhaps indicates that antibody-mediated immunity is a non-redundant mechanism for airways immunity to these pathogens. Since antibody deficiency syndromes are responsible for significant numbers of patients with bronchiectasis [3, 5, 6] and require specific management strategies including antibody replacement, it is important that patients presenting with bronchiectasis should be appropriately investigated for these conditions by measuring total serum antibody levels, specific antibody titres and antibody responses to vaccination.

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Antibody deficiency syndromes

Failure of any of the steps involved in antibody production can potentially lead to defective humoral immunity. Gene mutations affecting early pre-B-cell development (such as recombination-activating gene) will usually also impair T-cell production and lead to severe combined immunodeficiencies which almost always present in childhood. In contrast, adults may present de novo (although with a long history) with a block in pre-B-cell to immature B-cell development giving rise to: X-linked agammaglobulinaemia (XLA) (usually caused by mutations in Bruton’s tyrosine kinase (Btk)); defects in class switch recombination and/or somatic hypermutation (which are necessary to generate high-affinity immunoglobulin (Ig)G, IgA and IgE) resulting in hyper IgM syndromes; and defects (which are currently only partially characterised) in generating functional antibody responses leading to common variable immunodeficiency (CVID), IgA or IgM deficiency, IgG subclass deficiency and isolated specific antibody deficiency. The most common of these conditions are discussed below.

Common variable immunodeficiency

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CVID is the most common adult primary immunodeficiency, with an estimated prevalence of 1 in 25,000 in Caucasians [8]. Although many patients with CVID develop bronchiectasis, CVID is

Table 1. Primary and secondary immunodeficiencies associated with bronchiectasis Type of immunodeficiency

Mechanism of immune defect(s)

Patients with bronchiectasis

Bronchiectasis attributable to this immunodeficiency

Primary X-linked agammaglobulinaemia# CVID#

Mutation in Bruton’s tyrosine kinase 85% unknown

f32%

,3% children, very rare in adults 2–10% children, 0.7–2.4% adults

10–15% mutations in TACI, CD28, ICOS Unknown

Unknown

Unknown

Unknown

Specific antibody deficiency" Hyper IgE syndrome

Poor antibody response to polysaccharide antigens Mutations STAT3

Unknown Unknown

Phagocyte defects

Varied

Unusual

TAP deficiency

TAP1 or TAP2 mutations

Most patients

Antibody deficiency Antibody deficiency Unknown

Unknown Unknown Unknown

Rare Rare Rare

Associated with bronchiolitis obliterans

42% of bronchiolitis obliterans patients

Rare

Unknown

Rare

Rare?

Rare

6–16%

Unknown - depends on incidence of HIV

f50% severe COPD

Potentially common

IgG subclass deficiency" IgA deficiency"

Secondary CLL" Multiple myeloma" Other haematological malignancy HSCT"

IMMUNODEFICIENCY

37%

Antibody deficiency Post-infective? Immuosuppressive therapy? Lung transplant Associated with bronchiolitis obliterans Post-infective? Immuosuppressive therapy? Other solid organ transplant Post-infective? Immuosuppressive therapy? HIV infection Recurrent pneumonia

COPD

Associated with LIP Low CD4 count Post-tuberculosis? Impaired mucosal immunity?

Children unknown, 4–48% adults Children unknown, 2% adults Children unknown, 4–11% adults? ,2.5% children, very rare adults ,1–10% children, ,1% adults Rare in children, very rare in adults

CVID: common variable immunodeficiency; Ig: immunoglobulin; TAP: transporter antigen peptide; CLL: chronic lymphocytic leukaemia; HSCT: haematopoietic stem cell transplantation; COPD: chronic obstructive pulmonary disease; TACI: transmembrane regulator, calcium modulator and cyclophilin ligand interactor; ICOS: inducible T-cell surface expressed CD28 co-stimulatory molecule; STAT3: signal transducer and activator of transcription 3; LIP: lymphocytic interstitial pneumonitis. #: treated with intravenous Ig (IVIG) ": consider treatment with IVIG.

relatively rarely identified as the cause of bronchiectasis in most published data for adult patients, varying from 0.7% to 2.4% of cases [3, 4, 6], and in 2–10% of childhood cases [2, 5].

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CVID is characterised by reduced circulating Ig concentrations of one or more isotypes, with IgG levels two standard deviations below normal [9] and poor responses to immunisation. The mean age at diagnosis has two peaks of around 30 years and in younger children [10, 11]. Adult patients with CVID have often had symptoms for many years before diagnosis [11]. Familial CVID

CVID is characterised by reduced serum IgG concentrations, so finding levels of serum IgG below the normal range will identify nearly all potential cases. Patients with low IgG levels (even if just above the bottom of the normal range) should be further evaluated initially by measuring: 1) serum IgA, IgM and IgE; 2) IgG subclasses; and 3) levels of specific antibodies (against for example, tetanus toxin, pneumococcal serotype-specific capsular polysaccharide and H. influenzae capsular polysaccharide B) before and following vaccination if appropriate. More detailed investigations, usually conducted by clinical immunologists, include B-cell and T-cell immunophenotyping and T-cell proliferative responses to common mitogens (to subclassify CVID and exclude T-cell immunodeficiency). Patients will most likely require lifelong Ig replacement therapy. The main complications of therapy are fever, headache and chills, which are managed through pre-medication with anti-histamines and hydrocortisone. Anaphylactic reactions are rare. Ig replacement may be given by intravenous infusion (i.v. Ig (IVIG), 400 mg?kg-1 every 3–4 weeks) or by subcutaneous injection (100 mg?kg-1 weekly). Although there are few data on the long-term consequences of IVIG treatment, IVIG reduces the incidence of respiratory tract infections [18–20] and computed tomography (CT) scores of inflammation associated with bronchiectasis [21], so is likely to prevent or slow the progression of bronchiectasis. Early identification of CVID cases is therefore important, and measuring serum IgG levels in all cases of bronchiectasis is recommended in the recent British Thoracic Society guidelines [22]. CVID patients are often given prophylaxis with continuous low-dose oral antibiotics as well as IVIG therapy. The long-term prognosis of bronchiectasis in CVID patients is not known, but chronic lung disease is a prominent cause of death for CVID patients [23]. Historically, the dose of replacement IVIG given is based on a trough IgG level with the objective of keeping this within the normal range (7– 15 g?L-1). Generally, this results in most patients running a trough IgG at the lower end of the normal range, but recent data suggests that for some patients this is inadequate to keep them free of infection. Individualised Ig therapy using a dose that prevents infection has therefore been advocated to minimise the risk of progressive lung disease [24].

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accounts for 10–20% of cases, generally with an autosomal dominant inheritance pattern (often with partial penetrance); although many of the more recently identified genetic defects associated with CVID have an autosomal recessive inheritance pattern. For the majority of patients the molecular defects causing CVID are not known, but in 10–15% mutations affecting Ig production have been described. These include mutations of the inducible T-cell surface expressed CD28 costimulatory molecule (,1% CVID); the B-cell activating factor receptor (,1% CVID); the CD19 component of the co-receptor for the B-cell antigen receptor (,1% CVID); the transmembrane regulator, calcium modulator and cyclophilin ligand interactor (10–15% CVID); and a B-cell surface receptor involved in B-cell proliferation [8, 12]. These mutations affect quite different parts of the immunological response required for antibody production, suggesting that the molecular causes of CVID are heterogeneous and perhaps explaining why there is a large range of clinical associations with CVID that only affect a proportion of patients [13]. For example, up to 25% of patients with CVID also develop autoimmune and lymphoproliferative complications including granulomatous disease, lymphocytic infiltrations of the lungs or lymphoma [7, 10, 11, 14]. Although these complications have been particularly associated with known genetic polymorphisms, variations in gene dosage and penetrance has frustrated attempts to generate robust clinical phenotype–genotype classifications [15–17]. Most patients seem to be susceptible to respiratory and gastrointestinal infections, although selection bias and small numbers means that the precise incidence of respiratory tract complications in CVID varies between publications. In a large French series encompassing the national experience of patients with CVID, pneumonia occurred in 58% (31% due to S. pneumoniae and 12% due to H. influenzae), bronchitis in 69% and sinusitis in 63% of patients [11]. In total, 37% of patients were diagnosed with bronchiectasis. The pattern of bronchiectasis in CVID tends to be diffuse lower and middle lobe disease associated with chronic upper respiratory tract symptoms, similar to idiopathic bronchiectasis [5–7].

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Long-term management of patients with CVID should also include: 1) optimised treatment of their bronchiectasis focusing on appropriated oral and/or nebulised antibiotic prophylaxis

(as discussed by HAWORTH [25]), anti-inflammatory therapy (described by SMITH et al. [26]) and airway clearance strategies (described by BYE et al. [27]); 2) vigilance for the development of lymphoma, lymphoproliferative lung infiltration and granulomatous disease (although there is no consensus on the type or frequency of screening [28]); 3) a low threshold for investigation of gastrointestinal symptoms or B12/folate deficiency to pick up CVID-associated inflammatory enteropathy and Giardia infection; and 4) specialist management of associated idiopathic thrombocytopenic purpura and other autoimmune disease if present.

X-linked agammaglobulinaemia

IMMUNODEFICIENCY

XLA is a rare disorder of B-cell development characterised by absent serum antibodies and no circulating B-lymphocytes. It is usually caused by inherited mutations in the Btk gene, although clinically similar autosomal recessive diseases have been described due to other mutations affecting B-cells [7]. Patients present with recurrent bacterial and viral infections in early childhood. Similar to CVID, patients with XLA are particularly susceptible to infections caused by encapsulated bacteria such as S. pneumoniae and H. influenzae. As a consequence of recurrent lung infection, lung disease can develop bronchiectasis; in one survey 32% of adult patients with XLA had chronic lung disease, mainly bronchiectasis [29]. The relative risk of developing structural lung damage is, however, reported to be less with XLA compared with CVID [20]. XLA has been associated with up to 3% of cases of childhood bronchiectasis [5] but is only a rare cause in adults. No specific pattern of bronchiectasis in patients with XLA has clearly been described. The long-term prognosis has improved with aggressive treatment with IVIG and antibiotic therapy, although there are few data on the rate of progression of bronchiectasis and chronic lung disease remains a significant cause of death [29].

IgA, IgM and IgG subclass deficiencies In case series of paediatric and adult patients with bronchiectasis, small numbers of patients have selective IgM (,1%), IgA (2%) [3] or IgG subclass deficiency [30–32]. However, the clinical significance of deficiency of IgM or IgA with normal IgG remains unclear. The incidence of isolated IgM deficiency in the normal population is not known and whether IgM can mediate immunity at the mucosal surface has not been clarified. IgA is present at mucosal surfaces including the airway lining fluid [33] and is thought of as an important component of mucosal immunity. IgA deficiency is relatively common [3, 9], with a prevalence of 1 in 600 of the population, but may be more likely to lead to lung damage if combined with IgG subclass deficiencies or specific antibody responses to carbohydrate antigens (see later) [34]. IgG subclass deficiency, especially IgG2, has been associated with bronchiectasis, particularly in children. However, the incidence of IgG subclass deficiency varies widely in patients with bronchiectasis, from 4% to 48% [3, 6, 30, 35, 36] and the significance of subclass deficiency has been questioned as it is relatively common in a normal population [37]. IgG2 deficiency may be associated with reduced natural or vaccine-induced specific antibody to S. pneumoniae or H. influenzae as discussed later. As such, IgG2 deficiency may reflect poor antibody responses to the bacteria that are associated with bronchiectasis and thus represent a risk factor for disease [38]. Overall, at present there is no clear consensus that identification of isolated IgA, IgM or IgG subclass deficiency in a patient with bronchiectasis is necessarily clinically relevant [6].

Specific antibody deficiency

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The high incidence of bronchiectasis in patients with hypogammaglobulinaemia is probably related to lack of antibody-mediated immunity to the encapsulated respiratory pathogens S. pneumoniae and H. influenzae. Antibody responses to S. pneumoniae and H. influenzae that recognise capsular polysaccharides are protective, and hence a number of groups have explored whether selective deficiencies in antibody responses to polysaccharide antigens could also cause bronchiectasis. Antibody responses to polysaccharide antigens are described as being T-independent and generated

through mechanisms that are different from T-dependent antigens [39]. Distinct B-cell subpopulations respond to polysaccharide antigens and patients who have poor responses to capsular polysaccharide vaccines or who lack particular B-cell subpopulations are particularly susceptible to S. pneumoniae pneumonia [40] and perhaps the development of bronchiectasis [41]. Antibody responses to polysaccharide antigens can be tested by evaluating capsule-antigen specific responses after vaccination against S. pneumoniae or H. influenzae, and can be compared with antibody responses to a protein antigen vaccine, such as diptheria or tetanus [42]. Specific antibody deficiency has been identified in 58% of patients with idiopathic bronchiectasis [38], but this was a small study in which the immunological criteria used for specific antibody deficiency has been queried [43]. Other larger series of adult patients with bronchiectasis suggest specific antibody deficiency has an incidence varying from 4% to 11% [3, 41]. In some cases, an impaired specific antibody response was associated with selected IgG subclass deficiencies [36]. However, antibody responses to vaccination with polysaccharide antigens are variable and affected by age. Up to 10% of the normal population may be nonresponders [44, 45]. Hence it is difficult to evaluate the significance of specific antibody deficiency as a cause of bronchiectasis without further studies involving large numbers of bronchiectasis patients and matched controls. Furthermore, naturally acquired immunity to at least S. pneumoniae may actually be partially dependent on antibody responses to protein rather than capsular antigens [46], undermining the reasoning why a specific defect in carbohydrate responses could cause bronchiectasis.

There are many other immunodeficiencies reported to lead to recurrent lung infection, many of which have been associated with bronchectasis. Although often very rare, these diseases are of importance as they indicate which components of the immune response are necessary for preventing recurrent bacterial infections of the lung.

Transporter antigen peptide deficiency syndrome Transporter antigen peptide (TAP) proteins are required for the transfer of peptide antigens from the cytosol into the endoplasmic reticulum where they associate with human leukocyte antigen (HLA)-1 for presentation on cell surfaces. Autosomal recessive mutations in the TAP1 or TAP2 genes result in reduced HLA-1 expression and CD8 lymphocyte numbers, but with an increase in natural killer (NK) and cd T-cells [47, 48]. The majority of subjects with TAP deficiency have recurrent sino-pulmonary infections with common respiratory tract bacterial pathogens and develop bronchiectasis [47, 48]. Only a handful of families with TAP deficiency have been described, and this genetic defect will be responsible for a vanishingly small proportion of cases of bronchiectasis. However, the association of TAP deficiency and other very rare familial T-cell disorders [49, 50] with bronchiectasis demonstrates that there are previously unsuspected mechanisms of immunity to extracellular bacterial pathogens involving CD8 lymphocytes that requires further investigation. In addition, it has been suggested that an excess of NK and cd Tcells might promote bronchiectasis due to a dysregulated inflammatory response in reply to infection with bacterial pathogens [48].

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Other PIDs and bronchiectasis

Disorders of macrophage or neutrophil function

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There are a wide range of inherited disorders affecting neutrophil function such as chronic granulomatous disease (CGD), leukocyte adhesion deficiency and Chediak–Higashi syndrome [51]. Although these disorders are extremely rare, making it difficult to accurately evaluate their clinical associations, neutrophil disorders classically lead to recurrent pneumonia and abscesses but are not necessarily closely associated with bronchiectasis. In relatively large series of adult patients with bronchiectasis, tests of neutrophil function only occasionally identify patients with abnormal responses and even in these patients the relationship of the defect to bronchiectasis is not clear [2, 3, 5]. CGD has been associated with cases of bronchiectasis in some paediatric case

reports or case series but these reports are likely to have been affected by selection bias as they originate from specialist centres [2, 5, 52]. The seemingly weak association of neutrophil defects with bronchiectasis may also reflect the range of pathogens these patients are most susceptible to, which include Staphylococcus aureus, Nocardia, Aspergillus and Candida species but excludes S. pneumoniae and H. influenzae, the pathogens most closely associated with development of bronchiectasis in Ig deficiencies [51]. Primary defects of macrophage function generally affect intracellular killing and lead to increased incidences of infection with intracellular pathogens such as mycobacteria, Histoplasma, Listeria and Salmonella species [51] but again are generally not directly associated with the development of bronchiectasis. What is unclear is the extent to which functional polymorphisms of phagocytic receptors (such as Fc gamma RIIA H/R 131) or pattern recognition receptors (such as Toll-like receptors) may predispose to bronchiectasis through impaired phagocytosis of opsonised/non-opsonised bacteria or aberrant inflammatory responses.

IMMUNODEFICIENCY

Hyper IgE syndrome Hyper IgE syndrome is a rare autosomal dominant inherited syndrome that causes susceptibility to a range of infections as well as bone, dental, vascular and joint abnormalities [53]. Most patients have the classical clinical triad of massively raised IgE levels, recurrent pneumonia and soft tissue abscesses (hence the condition is also called Job’s syndrome). The majority of cases are caused by mutations affecting the signal transducer and activator of transcription 3, an intracellular signalling protein important for regulating cellular responses to cytokines [53, 54]. Patients have both an exaggerated and reduced cytokine response to infection. In particular, patients with hyper IgE syndrome have an impaired T-helper cell type 17 (Th17) CD4 response [55], which seems to be important for mucosal immunity to some respiratory pathogens such as Klebsiella pneumoniae and S. pneumoniae [56, 57], as well as S. aureus [58] and Candida species [59]. Th17 CD4 immune responses assist neutrophil recruitment to sites of infection as well as local mucosal immunity [56, 57]. Pneumonia in patients with hyper IgE syndrome is often complicated by pneumatoceles, but can also lead to bronchiectasis in a significant proportion of patients [53]. Although hyper IgE syndrome is a rare disease that is only occasionally found in cases series of patients with bronchiectasis [2, 5], the identification that the underlying genetic defect of a Th17 response demonstrates the importance of this pathway for immunity to common bacterial pathogens of the lung.

Other PIDs associated with bronchiectasis Patients with inherited disorders of DNA repair such as ataxia telangiectasia are more susceptible to infections as the development of adaptive immunity is impaired. Many of these patients are antibody deficient and have bronchiectasis [60]. Similarly Wiskott–Aldrich syndrome, an X-linked immunodeficiency caused by mutations in the WASP gene leading to low levels of T- and Blymphocytes, NK cells and serum IgM, develop infections with encapsulated organisms and therefore are at risk of bronchiectasis [61]. Both these disorders are rare causes of bronchiectasis in paediatric case series [2, 5]. A major component of immunity to extracellular bacterial pathogens is the complement system, and inherited complement deficiencies such as C2 or mannose-binding lectin (MBL) deficiency are associated with recurrent respiratory infections [62, 63]. However, although MBL deficiency is a relatively common condition affecting up to 25% of the normal population [62] there are only occasional reports linking isolated MBL deficiency with bronchiectasis [5]. MBL deficiency may increase the likelihood of bronchiectasis in patients with CVID [64–66] and is associated with more severe disease in patients with cystic fibrosis (CF) [67], suggesting MBL may help control disease progression in other immunodeficiencies associated with bronchiectasis. Lower levels of L-ficolin, another MBL pathway opsonin, has also been found in patients with bronchiectasis compared with controls [68], although these data need to be replicated. Other complement deficiencies are very rare and there are no data linking them to bronchiectasis.

184

The majority of patients with CF and ciliary dyskinesias will develop bronchiectasis and clearly have impaired physical immune defences of the lung through the effects of the gene defects on

mucociliary clearance. Neither are usually characterised as immunodeficiencies. However, recent data suggest mutations of the CF transmembrane conductance regulator in CF also cause a variety of defects in mucosal innate immunity. These include impaired phagocyte function, reduced efficacy of antibacterial peptides, and failure of bacterial internalisation by epithelial cells, as well as an exaggerated inflammatory response to infection [69, 70]. This constellation of multiple defects in innate immunity could make a significant contribution to the development of bronchiectasis in patients with CF, but this will be difficult to establish conclusively.

Secondary immunodeficiencies Good clinical data on the associations of different secondary immune deficiencies with bronchiectasis are more limited than the available data for PIDs. In general an accurate assessment using the published data of the importance of SIDs as causes of bronchiectasis is not possible. However, recognised causes of SIDs are probably relatively rare causes of bronchiectasis, with the potential exception of children in areas with a high incidence of HIV infection.

Many haematological malignancies result in B-cell and/or T-cell dysfunction and predispose to recurrent lung infection and subsequent development of bronchiectasis. In addition, profound immunodeficiency may occur as a result of treatment for these conditions. Case reports or case series have described bronchiectasis complicating chemotherapy, acute and chronic leukaemias, myeloma and lymphomas [5, 71–73]. In particular, due to the combination of prolonged survival and the high frequency of secondary hypogammaglobulinaemia, multiple myeloma and chronic lymphocytic leukaemia (CLL) seem to be relatively commonly associated with bronchiectasis, although the exact incidence has not been reported [72]. CLL and myeloma patients with proven bronchiectasis and hypogammaglobulinaemia should be assessed for IVIG therapy. Bronchiectasis has also been reported to develop in association with more acute haematological malignancies, perhaps as a consequence of severe lung infections and/or due to the affects of leukaemia or chemotherapy on host immunity [71]. However, there are no precise data on the incidence and rate of progression of bronchiectasis in patients with haematological malignancies.

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Haematological malignancies

Post-transplantation Haematopoietic stem cell transplantation (HSCT) is associated with an increased incidence of respiratory infections and potentially prolonged defects in cellular and humoral immunity in survivors [74]. These factors could predispose to bronchiectasis [75] and, in the authors’ experience, serial CT scans after allograft HSCT can demonstrate rapidly developing bronchiectasis over a period of weeks to months. In addition, up to 10% of HSCT allograft recipients will develop bronchiolitis obliterans (the main pulmonary manifestation of graft versus host disease) which precedes the appearance of diffuse bronchiectasis in ,40% of cases [76, 77]. Hence, although there are no precise prevalence data on bronchiectasis post-HSCT, it is probably a relatively common complication, especially in allograft recipients. Similarly, patients who develop bronchiolitis obliterans after lung transplantion may also have CT evidence of bronchiectasis [78], and there are case reports of bronchiectasis developing after transplantation of other solid organs [79], presumably because of damage caused by intercurrent pneumonias and/or impaired pulmonary immunity due to prolonged immunosuppressive therapy.

HIV

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HIV infection in most patients leads to a progressive T-cell defect characterised by a fall in CD4 Thelper cells. HIV-infected subjects suffer recurrent infections with conventional and opportunistic pulmonary pathogens, including mycobacteria species and S. pneumoniae. With the increasing duration of long-term survival after HIV infection it is therefore perhaps not surprising that up to

16% of HIV-infected children develop bronchiectasis [80, 81]. The incidence of bronchiectasis in HIV-infected adults may also be significant [82, 83]. The aetiology of HIV-related bronchiectasis is not well understood but may include direct effects of HIV infection on T-cell-dependent immunity and local macrophage- and monocyte-dependent pulmonary immunity, secondary effects on humoral responses, as well as direct effects of bronchial wall damage due to intercurrent pneumonia or tuberculosis, and possibly the association of HIV in adults with chronic obstructive pulmonary disease (COPD) [84]. The limited available publications suggest that in children bronchiectasis is more likely in subjects with CD4 counts ,100 mm3, or who have had recurrent pneumonia [80]. Interestingly, there is also a specific association with lymphocytic interstitial pneumonitis (LIP), with up to 40% of HIV-infected children with LIP developing bronchiectasis [80, 85]. Whether this reflects accelerated bronchial wall damage due to the lymphocytic infiltrate or reduced mucosal immunity in LIP is not clear. There are no comparative data on the pattern and progression of bronchiectasis in HIV-positive patients compared with patients with bronchiectasis due to other causes. More studies are required on the prevalence and associations of HIV infection with both adult and paediatric bronchiectasis to allow specific risk groups to be defined and managed aggressively to prevent progressive bronchiectasis. In addition, in areas with significant levels of HIV infection whether patients diagnosed with bronchiectasis warrant a HIV test as part of the diagnostic work-up needs consideration.

IMMUNODEFICIENCY

COPD and asthma There is a high incidence of bronchiectasis in patients with severe asthma and COPD according to CT criteria [86, 87], although the exact incidence is not known and is confounded by both asthma and irreversible airways obstruction being complications of bronchiectasis. Bronchiectasis could be associated with asthma and COPD due to the cycles of recurrent infection and localised bronchial wall inflammation associated with both conditions. The clinical importance of bronchiectasis in patients with airways disease is not clear at present, but as bacterial infections frequently drive exacerbations of COPD, significant bronchiectasis could be clinically highly relevant. Patients with asthma and COPD may have altered mucosal immune responses to microbial pathogens and impaired macrophage function that, along with the marked airway inflammation that characterises both diseases, might contribute towards the development of bronchiectasis [88, 89]. The effects of asthma and COPD on pulmonary immunity need further investigation. Due to the rising incidence of COPD and more extensive use of CT scanning, severe COPD is likely to become an increasingly common association in series of adult patients with bronchiectasis.

Biological therapies Therapies that inhibit tumour necrosis factor-a (such as infliximab) or deplete B-cells (rituximab) are increasingly used to treat rheumatological and other autoimmune conditions. Both therapies are associated with increased risks of infection [90, 91]. These therapies may make management of existing bronchiectasis more challenging and, in our experience, usually require an escalation of antibiotic prophylaxis. Furthermore, they could potentially trigger the development of bronchiectasis by increasing the frequency and/or severity of respiratory infections. Repeated administration of rituximab is often associated with the development of hypogammaglobulinaemia, which in the context of recurrent infection, should be managed by immunoglobulin replacement [92]. The effects of biological therapies are discussed in detail in the chapter by DHASMANA and WILSON [93].

What information do PIDs and SIDs provide about immunity to airways infection? 186

The identification of patients with bronchiectasis due to PIDs provides clear evidence for which aspects of the immune system are required for protection against bacterial infections of the lung.

The close association of bronchiectasis with CF, primary ciliary dyskinesia and antibody deficiency syndromes such as CVID and XLA demonstrate that physical defences and IgG (and perhaps IgA, specific IgG subclasses or anti-polysaccharide antibody responses) are required for the prevention of chronic bacterial infection of the lungs, as discussed in the chapter by LAMBRECHT et al. [94]. Although the mechanisms remain poorly defined, the clinical manifestations of TAP deficiency and hyper IgE syndrome with bronchiectasis suggest there is also an important and previously unsuspected role for CD8 and Th17 CD4 lymphocytes for the prevention of bacterial lung infection. Conversely, despite the prominence of neutrophil and macrophage infiltration in pneumonia and bacterial bronchitis, defects of phagocyte and complement function are only loosely associated with bronchiectasis. Humoral immunity therefore seems to be more important for bacterial clearance from the bronchial tree than phagocytes. This is perhaps a surprising observation as the main mechanism by which antibody assists pulmonary immunity to bacterial infection would have been predicted to be through promoting bacterial phagocytosis. Despite these clues provided by PIDs and SIDs, large gaps remain in our knowledge on the immune mechanisms required to prevent bacterial infections of the lung. Specific important areas of future research include the mechanisms by which antibody promotes clearance of bacteria from the lung, the bacterial target antigens for these antibody responses, and the role of different T-cell subsets for lung immunity.

We recommend a sequential approach to investigation of immune function in patients with bronchiectasis or recurrent infection summarised in table 2. First-line investigations involve measurement of total serum Ig, IgG subclasses and specific antibody levels before and after vaccination (to detect CVID, XLA, IgA/IgM and IgG subclass deficiency) and, where appropriate, test for HIV infection. Further testing can then be initiated (following discussion with a clinical immunologist). Second-line tests include T- and B-cell immunophenotyping (to examine for defects in lymphocyte differentiation), neutrophil superoxide measurements (to look for CGD) and complement (to check for deficiency). A number of third-line tests involving gene sequencing and functional assays (examples shown in table 2) may also be indicated. One important clue to the type of immunodeficiency is the type of infections affecting the patient which can direct

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A strategy for immunological investigation of patients with bronchiectasis

Table 2. Suggested staged immunological investigations of patients with bronchiectasis First-line tests

Second-line tests

Third-line tests

Serum IgG, IgA, IgM, IgE

Immunophenotyping (including B-cell subsets)

Specific gene sequencing (e.g. ICOS, TACI, STAT)

IgG subclasses

Targetted genotyping (MBL, FccRIIa)

TCR Vb usage

Levels of specific antibodies against: Neutrophil superoxide pneumococcal serotype specific capsular polysaccharide, tetanus toxin If low, assess vaccination response

TCR spectratyping

Autoantibody screen

Functional assays (e.g. chemotaxis, cytokine release assays, phagocytosis and bacterial opsonisation assays)

Complement levels

White cell differential count HIV test

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Ig: immunoglobulin; MBL: mannose-binding lectin; ICOS: inducible T-cell surface expressed CD28 costimulatory molecule; TACI: transmembrane regulator, calcium modulator and cyclophilin ligand interactor; STAT: signal transducer and activator of transcription 3; TCR: T-cell receptor.

laboratory investigations: encapsulated bacteria in B-cell immunodeficiencies; fungi, viruses and mycobacteria in T-cell immunodeficiencies and catalase-positive organisms (e.g. Staphylococcus, Aspergillus) in neutrophil disorders.

IMMUNODEFICIENCY

Future directions 26–53% of patients with bronchiectasis have no defined cause [3, 4]. Many of these patients also have upper respiratory tract disease such as sinusitis, suggesting they may have a global defect in preventing chronic bacterial infection of the respiratory tract. Recently, there has been increasing evidence that unsuspected immune defects may underpin many childhood infectious diseases [95] and intensive screening of children with idiopathic bronchiectasis may identify additional PIDs. For example, as untreated patients with primary ciliary dyskinesia and CF almost always develop significant bronchiectasis, other more minor defects in physical defences could be important causes of idiopathic bronchiectasis in adults and children. However, redundancy may limit the role of immunological defects as causes of bronchiectasis. For example, even with significant IgG deficiency the clinical phenotype of bronchiectasis has only partial penetrance and a significant proportion of subjects do not develop chronic lung infection. Hence, in adults, bronchiectasis could be a multifactorial disorder requiring two or more immune defects or a combination of an immune defect with a specific environmental insult in order to develop. The role of many aspects of lung immunity such as mucosal anti-bacterial peptides and proteins have yet to be investigated, and the complexity of the respiratory immune system could make identifying novel immune defects associated with bronchiectasis difficult. Despite this, polymorphisms affecting NK cell function or TAP and HLA associations with bronchiectasis have been described [96–98]. Further genetic studies of large numbers of patients with bronchiectasis are likely to identify additional polymorphisms or mutations affecting different aspects of immune function which could be related to the development of bronchiectasis.

Conclusions Characterisation of patients with bronchiectasis has demonstrated close associations with a wide range of PIDs and SIDs, confirming that effective pulmonary immunity is necessary to prevent chronic bronchial damage due to bacterial infection. PIDs associated with bronchiectasis provide clear evidence for the vital role of physical defences for preventing lung infection, with important supportive roles from antibody and T-cell. SIDs causing bronchiectasis are less well characterised, but the effects of long-term HIV infection, the new biological therapies and perhaps chronic airways disease on pulmonary immunity are likely to be increasingly associated with the development of bronchiectasis. Patient with SID should be monitored for the development of recurrent lung infections and, where appropriate, the development of hypogammaglobulinaemia. Despite intense investigation for all the known causes of bronchiectasis, a large proportion of patients will still have idiopathic disease. An even more detailed immunological assessment of patients with idiopathic bronchiectasis combined with investigations for novel gene defects and polymorphisms will probably reveal a range of minor defects that affect immune function in a significant proportion of these patients. Although the challenge will then be to confirm that these minor immune defects actually contribute to the development of bronchiectasis, we would predict that increasing numbers of immunodeficiencies associated with bronchiectasis will be identified in the future.

Statement of interest

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H. Baxendale has received research grant funding from Biotest and GlaxoSmithKline PLC to explore natural and vaccine related immunity to Streptococcus pneumoniae. Travel to ESI 2010 biannual meeting was funded by Grifols UK, Ltd.

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Nat Med 2006; 12: 1023–1026. 90. Lieberman-Maran L, Orzano IM, Passero MA, et al. Bronchiectasis in rheumatoid arthritis: report of four cases and a review of the literature–implications for management with biologic response modifiers. Semin Arthritis Rheum 2006; 35: 379–387. 91. Cooper N, Arnold DM. The effect of Rituximab on humoral and cell mediated immunity and infection in the treatment of autoimmune disease. Br J Haematol 2010; 149: 3–13. 92. Cooper N, Davies EG, Thrasher AJ. Repeated courses of rituximab for autoimmune cytopenias may precipitate profound hypogammaglobulinaemia requiring replacement intravenous immunoglobulin. Br J Haematol 2009; 146: 120–122. 93. Dhasmana DJ, Wilson R. Bronchiectasis and autoimmune disease. Eur Respir Mon 2011; 52: 192–210. 94. Lambrecht BN, Neyt K, GeurtsvanKessel CH. Pulmonary defence mechanisms and inflammatory pathways in bronchiectasis. Eur Respir Mon 2011; 52: 11–21. 95. Casanova JL, Abel L. Human genetics of infectious diseases: a unified theory. EMBO J 2007; 26: 915–922. 96. Dogru D, Ozbas Gerceker F, Yalcin E, et al. The role of TAP1 and TAP2 gene polymorphism in idiopathic bronchiectasis in children. Pediatr Pulmonol 2007; 42: 237–241. 97. Boyton RJ, Smith J, Ward R, et al. HLA-C and killer cell immunoglobulin-like receptor genes in idiopathic bronchiectasis. Am J Respir Crit Care Med 2006; 173: 327–333. 98. Boyton RJ, Smith J, Jones M, et al. Human leucocyte antigen class II association in idiopathic bronchiectasis, a disease of chronic lung infection, implicates a role for adaptive immunity. Clin Exp Immunol 2008; 152: 95–101.

Chapter 13

Bronchiectasis and autoimmune disease D.J. Dhasmana and R. Wilson

BRONCHIECTASIS AUTOIMMUNITY

Summary The association between bronchiectasis and autoimmune disease is well recognised, and best described with rheumatoid arthritis. The prevalence of bronchiectasis in rheumatoid arthritis varies considerably in studies, with obliterative bronchiolitis a common feature. The prognosis of rheumatoid arthritis with bronchiectasis seems to be worse than either condition alone. The advent of high-resolution computed tomography has increased the sensitivity of detecting bronchiectasis, but this should be assessed for clinical significance. Traction bronchiectasis results from interstitial fibrosis pulling the airway wider, rather than damage weakening the bronchial wall, and is less likely to lead to bronchial suppuration. Bronchial wall damage in bronchiectasis is caused by inflammation, but it is difficult to differentiate damage caused by severe or recurrent infections, predisposed to by immunosuppression related to the autoimmune disease itself or its treatment, from damage caused by the autoimmune process. Increased use of new immunomodulatory or immunosuppressive agents has proved successful in modifying autoimmune disease processes, but has also led to emergence of infective complications that can cause bronchiectasis or exacerbate preexisting disease. Keywords: Autoimmune, bronchiectasis, immunosuppression, rheumatoid arthritis, Sjo¨gren’s syndrome, vasculitis

A

Host Defence Unit, Royal Brompton Hospital, London, UK. Correspondence: R. Wilson, Royal Brompton Hospital, Fulham Road, London, SW3 6NP, UK, Email [email protected]

Eur Respir Mon 2011. 52, 192–210. Printed in UK – all rights reserved. Copyright ERS 2011. European Respiratory Monograph; ISSN: 1025-448x. DOI: 10.1183/1025448x.10004310

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n association between bronchiectasis and autoimmune disease has long been recognised. The main autoimmune diseases in which bronchiectasis has been described are discussed in this chapter with emphasis on rheumatoid arthritis, for which there is best evidence of a true association. When information is available we discuss estimated prevalence, pathogenesis, clinical features and management where this differs from that in usual bronchiectasis and prognosis. In addition, we have discussed screening and risk stratification in the context of immunosuppression following the use of biological agents such as anti-tumour necrosis factor (TNF) in autoimmune disease.

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Mixed selected and unselected Various, including large prospective and cross-sectional Mixed selected and unselected Mixed selected and unselected Mixed selected and unselected Various selected Primary bronchiectasis not described Mixed selected and unselected

,10–46%

,2–21%

7–23%

f59%

f50% 12%

Rare

Sjo¨rgen’s

SLE

Largest body of data, variable conclusions but largest evidence base of autoimmune diseases Most data based on incidental findings on HRCT, patients often asymptomatic Frequent finding of bronchiectasis on HRCT but poor correlation with symptoms; complications of infection and thrombosis are signficant and may dominate over clinically meaningful bronchiectasis Traction bronchiectasis common, but usually asymptomatic; morbidity usually through non-respiratory complications NSIP, pulmonary hypertension and traction bronchiectasis common; primary bronchiectasis less so and perhaps more in ’diffuse’ disease Several old studies pre-1986 and pre-HRCT so likely gross overestimate Where present, traction bronchiectasis more likely than primary bronchiectasis, again usually asymptomatic NSIP and organising pneumonia common, traction bronchiectasis rarely reported and primary bronchiectasis rare Granulomatosis with polyangiitis (Wegener’s) and MPA most common of primary vasculitides; BPI-ANCA linked to Pseudomonas

Comments

[41–49]

[38–40]

[30–34] [35–37]

[25–29]

[17, 20–24]

[17–19]

[13–16]

[1–12]

[Ref.]

RA: rheumatoid arthritis; SLE: systemic lupus erythematosus; AS: ankylosing spondylitis; RP: relapsing polychondritis; MCTD: mixed connective tissue disease; PM/DM: polymoyositis/dermatomyositis; HRCT: high-resolution computed tomography; NSIP: nonspecific interstitial pneumonia; MPA: microscopic polyangiitis; BPI-ANCA: bactericidal/ permeability-increasing protein-antineutrophil cytoplasmic antibodies.

Vasculitis

PM/DM

RP MCTD

Scleroderma

AS

Various according to type

Mixed selected and unselected

2–50%

RA

Patient selection and study type

Approximate prevalence in main studies

Disease

Table 1. Summary of the features of bronchiectasis in different autoimmune diseases

D.J. DHASMANA AND R. WILSON

There are several recurring themes that are worth noting: 1) high-resolution computed tomography (HRCT) scanning, which has significantly increased the sensitivity of imaging of bronchiectasis, was not available when older studies were performed and so the diagnosis of bronchiectasis may be less certain; 2) presence of radiological bronchiectasis versus symptomatic disease; and 3) the use of retrospective data in analyses of complex often heterogeneous populations. Another theme is traction bronchiectasis that may be present in patients with lung fibrosis due to involvement of the lung parenchyma by autoimmune disease-causing fibrosis. The scarring pulls the airways apart as it contracts. The airway mucosa is normal with intact mucociliary clearance and possibly for this reason patients are not usually prone to bacterial infections. However, in some cases with traction bronchiectasis there will also be bronchiectasis in parts of the lung without fibrosis, suggesting that an inflammatory process has involved the airways and damaged the structure of the bronchial wall. These patients may be more prone to the clinical syndrome of bronchiectasis. A summary of the features of the main studies carried out are shown in table 1.

Autoantibodies should not be routinely tested for during the investigation of a patient with bronchiectasis; they should only be tested for if there are particular clinical features raising autoimmunity as a possible association [50]. In addition, rheumatoid factor is

nonspecific but high levels do characterise a group of patients with prominent small airways disease in whom immunosuppression should be considered. Anti-cyclic citrullinated peptide which is more specific for rheumatoid arthritis has not as yet been investigated in relation to bronchiectasis.

Rheumatoid arthritis The association of rheumatoid arthritis and bronchiectasis is well described [1, 51] and it is the major autoimmune condition associated with bronchiectasis. One important question which remains unanswered is how the two conditions are related and how one develops in the context of the other. One hypothesis is that the initial event is recurrent antigen stimulation from recurrent lower respiratory tract infections, and the immunopathological sequence of events that follows leads to the development of a multi-system inflammatory disorder with a predilection for arthropathy. An alternative hypothesis is that bronchiectasis arises from the immunosuppression associated with rheumatoid arthritis itself and/or its treatments.

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Prevalence The reported prevalence of rheumatoid arthritis with bronchiectasis varies considerably largely due to patient selection and study type. Reports describe between approximately 2% and 50% prevalence of bronchiectasis in the largest studies of rheumatoid arthritis published between 1967 and 2006 [1–12]. A major issue is whether radiological evidence of bronchiectasis, either by chest radiography or by HRCT scanning, represents disease that is clinically significant. Studies that have tried to explore this demonstrate poor correlation with radiology [3, 8, 9, 52]. In most studies, the prevalence is calculated on the HRCT findings rather than on clinical evidence of bronchiectasis and patients may be entirely asymptomatic with incidental HRCT findings. Most reports of prevalence have used heterogeneous populations and so carry several potential confounding characteristics including duration of illness, age (mean age of 45–64 yrs across studies), cigarette smoking history and drug-treatment schedules, which might include corticosteroids and immunosuppressants, such as methotrexate, which could influence susceptibility to infection. Moreover, the data is typically retrospective bringing with it recall and reporter bias. DESPAUX et al. [8] report prospective data on 46 unselected patients with rheumatoid arthritis (34 females, 12 males; mean age 60.1 yrs) collected over an 18-month period. In this study in which all patients had a HRCT, they found 23 (50%) patients with radiological evidence of bronchiectasis, 18 of whom were previously undiagnosed. 13 (57%) of these 18 patients were asymptomatic, thus giving a total of 22% (10 out of 46 patients) with clinically significant bronchiectasis. In two other prospective studies of 75 consecutive patients [10] and 63 consecutive patients [12] with rheumatoid arthritis, 19% and 29% of patients, respectively, were found to have bronchiectasis on HRCT, although it is not clear what proportion of these were symptomatic. A retrospective uncontrolled study of 20 life-long nonsmokers showed a high proportion of bronchiectasis with five (25%) out of 20 demonstrating basal bronchiectatic changes, but whilst three of these five gave a past history of pleurisy or pneumonia none had ongoing symptoms [3]. In other more heterogeneous studies, sub-group analysis has not been able to demonstrate a relationship between smoking and bronchiectasis in rheumatoid arthritis [8, 9, 52]. We are not aware of any study that has attempted to correlate the severity of bronchiectasis using one of the accepted scoring systems with severity of arthritis, either in terms of joint damage or immunological measures.

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The immunological diagnosis of rheumatoid arthritis may also complicate prevalence data. In particular, there may be other autoimmune diseases present within the population studied, such as Sjo¨gren’s syndrome [53, 54]. With modern day biochemical and immunological markers, there is a more robust system to better differentiate autoimmune diseases from one another, which will allow better definition of disease in the future.

Finally, the patient’s ethnic status may have an additional impact on the development of bronchiectasis with rheumatoid arthritis. This is rarely mentioned within studies. The largest cohorts are described in France close to the Alps [8] and close to the North Atlantic [4], North Africa [9], New England in the USA [2] and in central and northern England, UK [3]. The antigenic stimulation by community pathogens is likely to vary markedly in these different settings.

Pathogenesis Whilst the association of bronchiectasis and rheumatoid arthritis has long been recognised [1, 8, 51], the mechanisms of how one condition develops in the context of the other remains unclear. While the co-existence of the two separate conditions is possible, the frequency of bronchiectasis in rheumatoid arthritis is well above that found in the non-rheumatoid arthritis population and suggests that these are not chance findings [4, 7, 8]. Three mechanisms have been considered: 1) bronchiectasis gives rise to the development of rheumatoid arthritis; 2) bronchiectasis and rheumatoid arthritis are caused by similar immunological processes, or because of immunosuppression due to rheumatoid arthritis or its treatments; and 3) other diagnoses and/or comorbid conditions drive the development of rheumatoid arthritis or bronchiectasis. These will be discussed in turn, although in reality there may well be several mechanisms interacting in a particular case.

The nature of the complex immunological mechanisms present in the bronchiectatic airways has been studied. The neutrophil plays a central role in what has been called ‘‘the vicious circle hypothesis’’, but in addition abnormal mucus clearance and cellular immune responses are important [55–58]. In this context, one proposed mechanism is that persistent immunological pressure stimulated by chronic bacterial infection drives a sequence of events that leads to the formation of autoantibodies to ‘‘self’’ components and ultimately the development of a systemic inflammatory disorder. For this mechanism to operate lung disease would need to precede rheumatoid arthritis. Most reports suggest that this is the case. DESPAUX et al. [7] described from an extensive literature review that 90% of 289 reports published since 1928 document respiratory symptoms prior to articular symptoms. While this study combines old reports with variable diagnostic criteria for both rheumatoid arthritis as well as bronchiectasis, in an era before computed tomography (CT) imaging, the temporal sequence is in fact corroborated in several individual and more recent studies [4, 5, 54]. Even in newly diagnosed rheumatoid arthritis present for ,1 year, with normal chest radiographs and normal respiratory function tests, 58% of patients were found to have HRCT evidence of bronchiectasis. This study demonstrates established bronchiectasis, albeit subclinical, by the time of a formal diagnosis of rheumatoid arthritis [59]. However, since the bronchiectasis was subclinical, sufficient antigenic stimulation by bacterial infection seems unlikely.

D.J. DHASMANA AND R. WILSON

Bronchiectasis gives rise to the development of rheumatoid arthritis

Bronchiectasis is caused by similar immunological processes or by immunosuppression due to rheumatoid arthritis or its treatments HRCT has made it clear that airway disease is common in rheumatoid arthritis (fig. 1). Follicular bronchiolitis is due to lymphoid aggregates, with or without germinal centres, which lie in the wall of bronchioles and sometimes compress their lumens. This appears as centrilolobular nodules, peribronchial nodules and patches of ground-glass shadowing [60]. Airway wall thickening (indicating bronchitis without dilatation) and bronchiectasis (fig. 1a and b) are both more common in patients than matched controls [61].

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There is a recognised association of rheumatoid arthritis and obliterative bronchiolitis, also known as ‘‘constrictive bronchiolitis’’, in which bronchioles are destroyed and replaced by scar tissue (fig. 1c).

a)

b)

c)

BRONCHIECTASIS AUTOIMMUNITY

Figure 1. High-resolution computed tomography. a) Mild tubular bronchiectasis in both lower lobes, together with mosaic perfusion, in a patient with rheumatoid arthritis. b) Tree-in-bud exudative bronchiolitis is widespread in both lower lobes of a patient with rheumatoid arthritis. Small airways having thickened walls and plugged with mucus are seen as multiple white dots. c) Severe bilateral lower lobe bronchiectasis in a patient with poor lung perfusion due to a constrictive obliterative bronchiolitis.

Several associations have been observed with obliterative bronchiolitis outside of the well-known association with tissue rejection in heart and lung transplantation. Drug treatment, especially with gold and penicillamine, has been implicated in the development of obliterative bronchiolitis [62, 63] but it also occurs in patients who have had neither drug. Obliterative bronchiolitis is welldocumented post-infection and although more recognised in children, has been documented with adenovirus, measles, influenza and Mycoplasma [64–69]. Not only could such an outcome easily go unnoticed until later in life, but this could represent a plausible mechanism for the later development of formal bronchiectasis or rheumatoid arthritis. Early toxin exposure might also account for obliterative bronchiolitis and later bronchiectasis or rheumatoid arthritis in a similar step-wise mechanism [70]. Certain human leukocyte antigens (HLA) have been associated with obliterative bronchiolitis, including the presence of HLA-DR1 in obliterative bronchiolitis with rheumatoid arthritis, while a large population fail to have an identifiable cause [71–73]. Bacterial infection may complicate the picture by itself provoking inflammation in the lung and causing damage to the airway wall, as well as exciting rheumatoid arthritis-driven inflammatory processes. Mosaic perfusion and gas trapping are present on HRCT. In the context of the above, patients complain of progressive breathlessness, develop irreversible airflow obstruction and subsequently carry a poor prognosis with death due to respiratory failure [74, 75].

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It is interesting to speculate whether these different manifestations of airway disease in rheumatoid arthritis are a single inflammatory process affecting different parts of the bronchial tree, or whether they are discrete inflammatory conditions. In favour of the former suggestion, all manifestations described previously can be seen in the HRCT scan of some patients. However, it is usually necessary to postulate that constrictive obliterative bronchiolitis has been preceded by exudative bronchiolitis, rather than being able to demonstrate this by sequential radiology. However, bronchiectasis could develop in the context of additional local structural damage caused by bacterial infection as a consequence of functional immunosuppression. DEVOUASSOUX et al. [75] report a study of 25

patients with rheumatoid arthritis and obliterative bronchiolitis and demonstrate HRCT evidence of bronchiectasis in 44% of the cohort. All patients were breathless and bronchorrhoea was present in 44%. They go on to report that in a follow-up of approximately 4 years, treatment was poorly effective, chronic respiratory failure occurred in 40% and death in four patients.

Methotrexate forms part of many rheumatoid arthritis treatment regimens and despite early clinical impressions, probably does not significantly increase the infection risk in patients with poorly controlled rheumatoid arthritis [79–81]. This may be because the immunosuppressive nature of unchecked inflammation in rheumatoid arthritis in the absence of methotrexate is greater than that conferred by methotrexate itself. However, long-term corticosteroids, cyclophosphamide and azathioprine certainly do lower the threshold for opportunistic infection and with the emergence of biological agents such as anti-TNF, the complication of serious infection and ensuing bronchiectasis becomes more likely [82]. In patients with recurrent infections on rheumatoid arthritis-treatments it is difficult to define the nature of any immune paresis, and where specific functional defects are demonstrated it is difficult to ascribe them to the disease or the therapy that has been prescribed. Gold has been associated with functional antibody defects, but in a study of rheumatoid arthritis patients with and without bronchiectasis, evidence of antibody deficiency was apparent in those with bronchiectasis as well as those without, and independent of any co-incident gold therapy [83]. Other reports of late bronchiectasis may have case-specific explanations, where resistant pathogens, abnormal airways and/or impaired clearance lead to unchecked infection and inflammation and usually localised bronchiectasis [84].

Other diagnoses or comorbid conditions that drive the development of rheumatoid arthritis and bronchiectasis

D.J. DHASMANA AND R. WILSON

Rheumatoid arthritis itself is associated with increased morbidity and specifically an increased risk of infection when compared with the general population [76–78]. In a predisposed individual, regular infection with poor immunological clearance of microbes could subsequently lead to formation of bronchiectasis. In contrast to the reports described previously, SHADICK et al. [2] describes 23 patients with rheumatoid arthritis and bronchiectasis, in whom 18 (78%) patients had rheumatoid arthritis symptoms prior to the diagnosis of bronchiectasis. These patients had a mean duration of arthritic symptoms of 25 years prior to bronchiectasis, 17 out of 18 patients had used corticosteroids and respiratory symptoms were present for an average of 4.3 years prior to the formal diagnosis of bronchiectasis. When compared with the five patients who described bronchiectasis before rheumatoid arthritis, those with late bronchiectasis used more diseasemodifying agents, had more severe joint disease, were more likely to have rheumatoid nodules and carried a greater morbidity. This would support the idea that advanced rheumatoid arthritis disease and increasingly immunosuppressive medications might contribute to the development of ‘‘secondary’’ bronchiectasis as a late complication of rheumatoid arthritis.

In most cases today, a clear diagnosis of rheumatoid arthritis and bronchiectasis can be made that is based upon the history, clinical features and immunology profile. However, in older studies it is worth noting that either the diagnosis of rheumatoid arthritis may be incorrect, or there may be significant comorbid conditions that drive the disease phenotype. For example, the finding of greater numbers of abnormal Schirmer’s tests (test of tear production) by MCMAHON et al. [54] in a case-controlled study of 32 patients with rheumatoid arthritis and bronchiectasis when compared with rheumatoid arthritis without bronchiectasis did increase the possibility that Sjo¨gren’s syndrome was involved in the pathogenesis of one or both conditions, possibly by affecting mucociliary clearance in the lung. However, this finding was not reproduced by MCDONAGH et al. [52], and KELLY and GARDINER et al. [53] who found no significant difference in abnormal tear production in their rheumatoid arthritis patients with bronchiectasis (six out of 10 patients) compared with those without bronchiectasis (18 out of 30 patients).

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The cystic fibrosis transmembrane conducatance regulator (CFTR) mutation DF508 present in cystic fibrosis (CF) has been implicated through a study of a French cohort that has demonstrated

its increased presence in rheumatoid arthritis with bronchiectasis [85]. In this study, four (15.4%) out of 26 Caucasians with a median age of 59 years with rheumatoid arthritis and bronchiectasis carried the heterozygote genotype compared with none from 29 consecutive rheumatoid arthritis patients without bronchiectasis, and none from 29 patients with diffuse bronchiectasis. This is a striking difference when noted in the context of a 2.8% allelic frequency in the general Caucasian European population. In addition, those with the mutation demonstrated more frequent sinusitis, lower nasal potential differences and a trend towards more severe lower respiratory tract disease, while there was no relationship to the severity of articular features.

BRONCHIECTASIS AUTOIMMUNITY

HLA associations are well characterised for rheumatoid arthritis and the HLA-DRB1 gene locus from the DR4 ‘‘family’’ is perhaps the most closely associated susceptibility locus implicated in rheumatoid arthritis [86]. In a large case-controlled study of patients’ HLA associations in a UK cohort, HILLARBY et al. [87] demonstrated the predicted DR4 association in 79% of rheumatoid arthritis alone patients but no pattern of DR4 subtypes in those with rheumatoid arthritis and additional respiratory features, including pulmonary fibrosis and bronchiectasis. However, there was a significant association of rheumatoid arthritis and bronchiectasis with DQB1*0601, DQB1*0301, DQB1*0201 and DQA1*0501 when compared with rheumatoid arthritis alone. The group of patients with bronchiectasis in a separate prospective HRCT study of 68 consecutive rheumatoid arthritis patients showed a low prevalence of DQA1*0501 when compared with the rheumatoid arthritis group without bronchiectasis [6]. Immune dysregulation is seen in both bronchiectasis and rheumatoid arthritis, and a shared defect in both rheumatoid arthritis and bronchiectasis may impact upon the shape of the final disease phenotype. Common variable immunodeficiency (CVID) is the most common primary immunodeficiency and is frequently associated with both respiratory tract infections and autoimmune conditions including rheumatoid arthritis [88]. Defective antibody production has been recognised in rheumatoid arthritis and with rheumatoid arthritis treatments. A UK study of 80 patients was carried out and comprised of 20 patients with rheumatoid arthritis and bronchiectasis, 20 patients with each disease separately and 20 healthy matched controls. Three out of 20 from the rheumatoid arthritis-bronchiectasis group demonstrated an impaired antibody response post-immunisation, two out of 20 rheumatoid arthritis alone patients showed a poor response (both groups of patients contained individuals on gold therapy) and the control group demonstrated neither. Immunological defects, when investigated, are likely to be more common than is currently believed and may play important roles as co-factors in the developing bronchiectasis [89]. Yellow nail syndrome (YNS) is a heterogeneous disorder that includes bronchiectasis and has been associated with rheumatoid arthritis-drug therapy, particularly penicillamine. YNS does occur in rheumatoid arthritis and other autoimmune diseases independent of drug therapy and its aetiology remains unclear [90, 91]. Abnormal T-cell responses that are thought to drive disease in YNS may similarly drive a specific phenotype in the presence of rheumatoid arthritis and act as a co-factor in development of bronchiectasis.

Management of bronchiectasis in the presence of rheumatoid arthritis

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There are no specific features in the management of bronchiectasis associated with rheumatoid arthritis. We have not identified any patients requiring antibody replacement in our own group of rheumatoid arthritis-bronchiectasis patients, but it would be reasonable to measure total antibody levels and specific antibody responses to polysaccharide (pneumococcal and Haemophilus influenzae type b and protein (tetanus)). Some patients have progressive obliterative small airways disease. Our own experience is that there is a poor response in these patients to increasing immunosuppression, and this approach to treatment creates more problems by making infections worse. Once the patient is established by the rheumatologist on a regimen that may include methotrexate, we have adopted the strategy of trying to reduce the level of bronchial infection by using antibiotic prophylaxis, including the ketolide antibiotic azithromycin as a putative

immunomodulator [92], and treating exacerbations aggressively. We hypothesise that avoiding the antigenic stimulation of bacterial infections may reduce the inflammatory processes causing obliterative bronchiolitis.

The presence of bronchiectasis with rheumatoid arthritis appears to carry a significantly worse prognosis, although only one report examines mortality and morbidity in this specific context. SWINSON et al. [93] studied a UK cohort of 32 rheumatoid arthritis patients with bronchiectasis alongside matched controls with either rheumatoid arthritis alone or bronchiectasis alone. They found the mortality in the group with both diseases to be considerably higher, with a standardised mortality ratio five times and 2.4 times greater than that of the rheumatoid arthritis alone and bronchiectasis alone groups, respectively. The groups shared similar scores of physical activity and of radiological destruction (Larsen score). While several parameters carried high relative risks of mortality including grip strength and presence of rheumatoid nodules, the finding of a raised white cell count and the presence of circulating immune complexes carried the highest relative risks, the latter being the only one which demonstrated confidence intervals outside parity (relative risk 4.5, 95% CI 1.4–13.9). The 5-year survival rate in the combined rheumatoid arthritisbronchiectasis group can be calculated at 69%. Finally, it is interesting to note that those in the combined disease group did have a lower baseline forced expiratory volume in 1 s (FEV1), as well as lower forced vital capacity (FVC) and fewer patients with signs of reversibility. Airflow obstruction in the presence of lung restriction has been identified in one large bronchiectasis study as a risk factor for mortality. In this study, carried out over 13 years, 29.7% of patients with bronchiectasis of many different aetiologies died [94]. In contrast, MCMAHON et al. [54] reported no significant effect of bronchiectasis on the activity and outcome measures of arthritis when compared with those with rheumatoid arthritis alone.

Sjo¨gren’s syndrome The study of the association of Sjo¨gren’s syndrome and bronchiectasis has been made more difficult by: the presence of primary, secondary and mixed syndromes; serological overlap with systemic lupus erythematosus (SLE; in particular, Sjo¨gren syndrome-related antigen A) and also systemic sclerosis; and the inconsistencies in the literature about how the diagnosis of bronchiectasis was made. The diagnosis of Sjo¨gren’s syndrome includes the presence of dry eyes and dry mouth for 3 months, a positive Schirmer’s test, anti-Ro and anti-La autoantibodies and a minor salivary gland biopsy demonstrating a focus score .1. While the use of this definition was not clear across all studies, an international consensus was obtained to rectify the differences [95]. Clinically significant bronchiectasis is uncommon and so most information on the prevalence of bronchiectasis in Sjo¨gren’s syndrome necessarily comes from imaging studies of patients with respiratory symptoms or from studies in those who are asymptomatic. Bronchiectasis is variably reported in such studies ranging from ,10% to 46% [13–16]. In a study of 24 German patients with primary Sjo¨gren’s syndrome (excluding smokers and those with other autoimmune disease or other unrelated bronchopulmonary disorders), 19 were found to have HRCT abnormalities and 11 of these bronchiectatic changes (46% of all patients) [14]. These changes were more central, predominantly lower lobe, bilateral in eight cases and unilateral in three cases. The precise symptoms of these patients are not given but the cohort comprised of patients referred for investigation over a 10-year period to a tertiary referral centre.

D.J. DHASMANA AND R. WILSON

Prognosis

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The aetiology of Sjo¨gren’s syndrome is unknown but viral infection is implicated, including Epstein–Barr virus (EBV), cytomegalovirus and retroviruses such as HIV and human T-lymphocyte virus, with good evidence from animal studies [96]. Both B- and T-cells are recognised to infiltrate exocrine glands but the pathogenesis is likely to involve a complex interplay of glandular epithelial and endothelial cells, dendritic cells and B- and T-cells in the context of an environmental insult in a predisposed individual [97]. Hydration of the airways may be impaired

together with inspissations of secretions as a result of atrophied respiratory tract mucus glands. Bronchiectasis is proposed to develop subsequently due to recurrent bacterial infections which are predisposed to by impaired mucociliary clearance. Neutrophilic inflammation provoked by infection leads to thickened dilated lower airways and eventually bronchial wall destruction. Amyloid has been recognised in Sjo¨gren’s syndrome and may be implicated in the development of bronchiectasis with its presence confirmed in peribronchial walls, as well as the interstitium [98].

Management of bronchiectasis associated with Sjo¨gren’s syndrome There are no clinical studies reported in the literature. In our own practice we have attempted to improve mucus clearance by nebulising normal saline regularly several times per day and emphasising to patients the importance of physiotherapy. Recently we have begun to nebulise 7% hypertonic saline which has an osmotic effect, with success in individual cases. Optimal antibiotic management of lower respiratory tract infections may shorten the length of infective exacerbations and so reduce airway wall damage.

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Systemic lupus erythematosus The first reports of bronchiectasis in SLE emerged in the early 1960s with the use of bronchograms and pulmonary function tests [99–101]. With the advent of CT imaging, there has been a greater understanding of the radiological abnormalities in SLE. However, there remains some uncertainty about the significance of the reported abnormalities and the prevalence of clinically significant bronchiectasis. FENLON et al. [17] prospectively studied 34 patients with SLE with HRCT data alongside various clinical and lung function data. Of note, they found seven (21%) patients with bronchiectasis on HRCT, second only to interstitial lung disease (ILD) (11 patients), mediastinal or axillary lymphadenopathy (six patients) and pleuropericardial abnormalities (five patients). However, while the presence of HRCT abnormalities was high they found no correlation with symptoms or disease activity, and none of the patients had recurrent respiratory infections. In a separate cross-sectional study of 60 Norwegian adults of childhood-onset SLE, any HRCT abnormality was found in only five patients and in just one (,2%) was there radiological evidence of bronchiectasis; none had clinical evidence of bronchiectasis [18]. These patients had a median duration of 11 years of disease by the time of cross-sectional imaging. In contrast, BANKIER et al. [19] reported a much higher frequency of CT abnormalities with 17 out of 48 patients with SLE showing abnormalities (45 of whom had normal chest radiographs). They went on to show correlation of extent of disease radiologically with duration of clinical history (r50.93), gas transfer (r50.8) and ratio of FEV1/FVC (r50.77). However, once again there was poor correlation of bronchiectasis on CT scans and clinical symptoms of the disease. Lung fibrosis may cause traction bronchiectasis and it is not clear in reports whether bronchiectasis is present in parts of the lung not affected by fibrosis. As with other systemic diseases, it has been suggested that confounding factors might explain the association of bronchiectasis with SLE, including the increased risk of infection associated with a multi-system disease and use of immunosuppressive treatments to control the disease. Mannosebinding lectins (MBL) have been suggested to play a role in SLE in a report of two patients with SLE who went on to develop CVID [102]. The infrequent MBL haplotype 4Q-57Glu was present in both, while the haplotype 4P-57Glu in the second case was associated with recurrent respiratory infections, bronchiectasis and low circulating levels of MBL. This report raises the possibility of MBL polymorphisms in the development of autoimmune disease and significant infections which cause bronchiectasis.

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The clinical features of bronchiectasis in SLE are not described in the literature. However, it is apparent that the most common pulmonary complications are infection and vascular events [103]. While the reported frequency of clinical bronchiectasis is low, as described previously, there may be under-diagnosis of post-infective bronchiectasis in patients who have not had HRCT examination.

Respiratory function tests frequently demonstrate reduced spirometry (typically subclinical), reduced gas diffusion and, depending on severity of disease, decreased lung capacity. These changes appear to be independent of cigarette smoking [103–105]. HRCT features reported in SLE include pleuritis with or without pleural effusion, acute interstitial pneumonia and acute pulmonary haemorrhage and thrombosis [17, 106]. Morbidity and mortality in SLE are associated with infection and vascular complications [107, 108]. There is greater mortality in the first 5 years, partly linked to the use of immunosuppressive therapy in aggressive SLE disease and the subsequent complications of infection surrounding this.

There are several pulmonary manifestations of ankylosing spondylitis which include apical fibrobullous disease, secondary infection, chest wall restriction, obstructive sleep apnoea, spontaneous pneumothorax and bronchiectasis [109]. A typical course is the development of chronic bi-apical fibrobullous areas with nodules that eventually coalesce to form cysts, cavities and bronchiectasis, and later superadded infection with Aspergillus and environmental Mycobacteria species may occur. Abnormalities evident on HRCT in those either asymptomatic or with early disease are well documented with frequencies of all abnormalities in the region of 40% to 80% [20–22, 110]. However, little is published regarding bronchiectasis specifically. HRCT evidence of bronchiectasis has been found in 7–23% of ankylosing spondylitis patients in the largest cohort studies performed to date [17, 20, 21–24]; in most studies, patients do not report symptoms of bronchiectasis. Traction bronchiectasis is the most likely explanation in this context caused by pleuropulmonary fibrosis. FENLON et al. [111] reported a total of six (23%) cases of bronchiectasis from their prospective cohort study of 26 patients with ankylosing spondylitis from an out-patient setting in Ireland, of which four were primary bronchiectasis and two had traction bronchiectasis. The four with primary bronchiectasis consisted of three patients with significant smoking histories, two each with disease in the upper and lower lobes and only one with symptoms of cough and breathlessness. The latter patient with bronchiectasis had ankylosing spondylitis for significantly longer duration of 28 years, and had an abnormal plain chest radiograph (demonstrating upper lobe bronchiectasis) with restrictive respiratory function tests. Three out of four patients with bronchiectasis in a separate study from Brazil were also current smokers, although this population with several radiological abnormalities may have had other infective causes [23]. Tracheobronchomegaly or Mounier–Kuhn syndrome, which is due to a congenital cartilage abnormality, has also been reported with ankylosing spondylitis and this mechanism may influence the development of bronchiectasis in some cases [112]. HLA-B27 does not appear to correlate with general HRCT abnormalities where this has been assessed, and while it is possible that ankylosing spondylitis disease severity correlates indirectly with respiratory abnormalities in general, there are too few cases with bronchiectasis to assess any relationship with this specifically [23, 113, 114]. There is insufficient data to comment on the timing of bronchiectasis compared with the development of ankylosing spondylitis, although it appears that the majority of those found to have bronchiectasis are asymptomatic with incidental findings on imaging only [20–24, 110, 115]. Ankylosing spondylitis mortality is usually caused by non-respiratory illnesses such as cardiovascular disease, renal failure and amyloid and through complications of treatment, and only occasionally through respiratory disease [116–118].

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Ankylosing spondylitis

Scleroderma/systemic sclerosis

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Lung involvement in scleroderma or systemic sclerosis is very common. HRCT has played an important role in better characterising and following up abnormalities, and disease has also been well documented by post mortem examination with the identification of pulmonary disease in systemic sclerosis in 80% of one cohort [25–27]. The findings of an ILD, typically a nonspecific

interstitial pneumonia (NSIP) pattern and pulmonary hypertension, are quite common on HRCT. Any honeycombing is usually mild and localised and the more typical pattern is the near-confluent ground-glass opacification, fine reticular markings and associated traction bronchiectasis. Primary bronchiectasis is uncommon [28, 29], as are reports of clinically significant disease. In one of the larger studies of systemic sclerosis patients alone, ANDONOPOULOS et al. [29] investigated 22 patients with a full history, respiratory function tests, blood tests and HRCT imaging. Cylindrical bronchiectasis was evident in 13 (59%) out of 22 patients and was more common in diffuse rather than limited systemic sclerosis disease, although this finding fell short of statistical significance and did not correlate with gas transfer, ground-glass opacification or with the patient’s duration of illness. In another single case report of clinically significant bronchiectasis, there were other potential causes including Sicca syndrome and immunosuppressant treatment [119].

BRONCHIECTASIS AUTOIMMUNITY

Relapsing polychondritis The tracheobronchial tree is affected and typically leads to thickened and sometimes narrowed airways, impaired clearance and the development of airway infection and inflammation. Lower respiratory tract symptoms and significant disease developed after the initial diagnosis of relapsing polychondritis in an early and one of the largest prospective studies of 23 patients with relapsing polychondritis [30]. However, this was not the case in the only other smaller prospective series 20 years later where in six out of nine patients the respiratory symptoms were the presenting symptoms of relapsing polychondritis [31]. Cohort studies since 1966 report a prevalence of respiratory symptoms in up to 50% of those with relapsing polychondritis, although given the nature and time of these studies, accurate prevalence of bronchiectasis is not possible to estimate. A small number of cohort studies have analysed the natural history, morbidity and mortality of patients with relapsing polychondritis. Respiratory infection appears to play a significant part. Bronchiectasis is not defined by today’s standards of HRCT imaging given that these studies were carried out between 1966 and 1986. However, it can be implied that together with vasculitis and valvular heart disease, respiratory infection carries a worse prognosis [30, 32, 33]. MICHET et al. [32] describe their single-centre experience of 112 patients in the US in which they identified respiratory infection as one of the leading causes of death alongside vasculitis and cancer. Of further interest is that only 10% of deaths were directly attributed to airway involvement of the disease, that anaemia was a significant poor prognostic marker and that the use of corticosteroids did not impact on survival. BEHAR et al. [34] analysed past records of a cohort of 160 patients collected over 10 years from two referral centres and scrutinised records from 15 patients who had undergone any thoracic CT imaging. They identified increased attenuation in the tracheal walls of all 15 patients (with narrowing in one third of these patients), and also in the bronchial walls of 11 patients (73% of those scanned). Of the 11 patients who had complete lung view imaging, three were found to have bronchiectasis (two upper lobe, one diffuse), two demonstrated no significant airway stenoses and one showed widespread tracheal and bronchial stenoses. 12 (83%) out of 15 patients demonstrated thickened airway walls.

Mixed connective tissue disease

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Mixed connective tissue disease (MCTD) is a distinct clinicopathological entity with unique positive antibodies against ribonucleoprotein that shares several clinical and radiological features with SLE, systemic sclerosis and polymyositis/dermatomyositis (PM/DM). The frequency of respiratory manifestations in MCTD is reported to be between 20% and 80%, more commonly the higher end of this range, although the reports are typically based upon radiological findings rather than clinical significance [35, 120, 121]. The prevalence of bronchiectasis is not available in these older studies, once again because of the absence of HRCT. MCTD is not usually associated with

primary bronchiectasis, rather with traction bronchiectasis associated with architectural distortions and the interstitial pneumonia patterns more commonly seen in this disorder [36, 37]. KOZUKA et al. [37] analysed the abnormal HRCT imaging of 41 patients with confirmed MCTD and characterised the radiological abnormalities that were observed. They identified 18 patients with traction bronchiectasis. Primary bronchiectasis was observed in five (12%) out of 41 patients, although no clinical features were reported in this study to assess the significance of this.

Polymyositis/dermatomyositis PM/DM is typically associated with ILD with a strong correlation with anti-Jo1 antibodies, most commonly an NSIP pattern and also an organising pneumonia [38, 39]. Primary bronchiectasis is not reported and traction bronchiectasis is rarely reported, especially given that honeycombing is an infrequent finding in contrast to ground-glass opacification and patchy consolidation [38–40].

Bronchiectasis and vasculitis

The vasculitic process may be localised or involve many systems with increasing severity. The extent of disease may be such as to require aggressive immunosuppressive therapy with corticosteroids and cyclophosphamide to control the vasculitis, alongside continued antimicrobial treatment for concomitant bacterial infection [126]. Evidence of immune-mediated injury and vasculitis has been demonstrated in the context of H. influenzae and Staphylococcus aureus, as well as Pseudomonas aeruginosa [2, 42, 125]. Antineutrophil cytoplasmic antibodies (ANCA) form an important component of vasculitides of which classical ANCA (c-ANCA) against the antigen proteinase-3 and perinuclear ANCA (p-ANCA) against myeloperoxidase make up the major pathogenic types [43]. Of the primary vaculitides, granulomatosis with polyangiitis (Wegener’s) with associated c-ANCA antibodies and microscopic polyangiitis (MPA) with myeloperoxidase antibodies have been most linked with bronchiectasis. A chronic pulmonary illness typically predates the development of ANCAassociated disease in various reports and although other ANCA may exist their roles may be more specific [41, 44–46]. In a retrospective cohort study of 26 patients with MPA in Japan, nine (35%) were diagnosed with bronchiectasis, four of whom had bronchiectatic symptoms prior to the diagnosis of MPA [45]. The precise role and timing of the development of autoantibodies to selfcomponents remains unclear. FORDE et al. [47] analysed sera from a large number of patients with a wide variety of inflammatory and infective disorders in order to investigate any association of autoantibodies with acute and chronic infection. They concluded that antibodies to neutrophilic cytoplasmic components were predominantly associated with chronic bacterial infection, while antibodies to monocyte cytoplasmic components were predominantly associated with chronic granulomatous disorders such as sarcoidosis. The implication was that persistent stimulation of phagocytic cell components by bacterial infection drives the formation of autoantibodies to those components and a pathological humoral response.

D.J. DHASMANA AND R. WILSON

It has long been recognised that immune complexes and autoantibodies can accompany bronchial infection [41, 122–125]. ABRAMOWSKY and SWINEHART [123] demonstrated renal failure associated with immune complexes in patients with CF and immune complex-mediated injury was proposed in CF patients who presented with purpuric lesions late in their disease course [124]. Immune complexes adhere to the endothelium through binding with the C1q component of complement causing vasculitis and/or the complexes interfere with the intended complement-mediated clearance of pathogens.

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More recently, studies have begun to confirm the temporal relationship of immune-complex activity with infection. MAHADEVA et al. [48] identified and characterised a new antigen bactericidal/permeability-increasing protein (BPI)-ANCA in the context of Pseudomonas infection. They went on to identify this in several patient groups including those with CF and non-CF

bronchiectasis, inflammatory bowel disease and renal failure [49]. Other groups explored its behaviour in the context of Pseudomonas and proposed that high levels of BPI-ANCA correlated with chronic Pseudomonas infection and poorer prognosis [46, 127, 128]. Of note, BPI binds with high affinity to lipopolysaccharide (LPS) on Gram-negative bacteria, and the presence of high levels of circulating antibodies to BPI may interfere with clearance of LPS bacteria giving rise to concomitant severe infection. There are several other rare primary immunodeficiencies that are associated with bronchiectasis and vasculitis about which little is known. For example, an X-linked lymphoproliferative disorder linked to a specific T-cell defect in EBV immunity that is associated with multi-system vasculitis, bronchiectasis, respiratory failure and death [129] and an, as yet, poorly defined syndrome consisting of childhood dermatitis, profoundly elevated immunoglobulin E, severe pneumonia (and subsequent bronchiectasis) and multiple central neurological abnormalities [130].

The use of immunosuppressive agents and bronchiectasis

BRONCHIECTASIS AUTOIMMUNITY

There is an increasing use of immunomodulatory or immunosuppressive therapy that is proving successful in modifying autoimmune disease processes [82]. However, their availability has raised fresh concerns, mainly surrounding opportunistic infection and cancer [131–135]. In the autoimmune diseases discussed herein, those drugs used frequently include steroid-sparing agents such as azathioprine and methotrexate, alternative potent immunosuppressive drugs such as leflunomide and cyclophosphamide, biological agents that include anti-TNF agents (etanercept, infliximab and adalimumab), anti-CD20 molecules (rituximab), interleukin (IL)-6 receptor antagonists (tocizilimab) and co-stimulatory inhibitor molecule (abatacept). Reactivation of tuberculosis (TB) is a recognised risk of the use of anti-TNF therapy and the British Thoracic Society and others have issued guidelines for their use in those at risk of TB reactivation [136, 137]. TB and nontuberculous Mycobacteria [138] are pathogens that can both cause bronchiectasis and infect patients with existing bronchiectasis. Care must be taken to stratify the risk of reactivation following immunosuppressive therapies, and one should be aware that traditionally non-pathogenic strains can emerge as fatal infections [139]. Evidence for latent TB infection should be sought with the use of a detailed history, chest radiograph or CT, tuberculin skin testing and interferon-c release assays (IGRA). IGRAs are now well established and should be used to ‘‘risk-stratify’’ in the context of anti-TNF therapy. While in theory latent viruses including herpes zoster and EBV, fungus, opportunistic bacteria and parasites are all more likely to reemerge with immunosuppressive therapy, this has not been a consistent finding [140–145]. There may be a gradation of risks within this group of agents. Anti-CD20 therapy in the form of rituximab may generally be considered less aggressive. CD20 is expressed by haematopoietic progenitor cells and newly differentiated plasma cells, and while reactivation of latent virus is well documented, infection with other bacteria or parasites or TB is infrequently reported [146, 147]. Safety and long-term data are still emerging with tocilizumab, an IL-6 receptor antagonist found to be effective in rheumatoid arthritis and still being investigated for SLE [147–150]. To date, no surprising opportunistic infection data has emerged and meta-analyses have placed a figure of approximately six additional infections per 100 patient-years; those infections are mostly termed ‘‘pneumonia’’ [149, 151]. Abatacept, a newer co-stimulatory modulator that interferes with T-cell activation may not share the same documented risks of TB reactivation and may prove to be better tolerated than anti-TNF therapies, although longer term safety data on this drug is still emerging [152–155].

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In general, physicians using these agents must be diligent and counsel patients about the risks of infections, particularly if patients already have susceptibility to infection due to concomitant bronchiectasis. In this case the patient should be co-managed with a respiratory physician, sputum should be screened for Mycobacteria sp. and other opportunistic pathogens, the patient should have an antibiotic management plan if infective exacerbations develop, and antibiotic prophylaxis

should be considered if infective exacerbations become frequent. These agents often provide marked improvement in the patient’s control of their autoimmune disease, which means that when the agents are used in bronchiectasis patients with associated autoimmune disease, treatment of chronic bronchial infection and infective exacerbations of bronchiectasis should be intensified to allow the agent to be continued when this is deemed to be safe. Good communication between the rheumatologist and pulmonologist is essential.

Statement of interest None declared.

3. 4. 5. 6. 7.

8. 9. 10.

11.

12. 13. 14. 15.

16. 17. 18. 19. 20.

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Chapter 14

Antibiotic treatment strategies in adults with bronchiectasis C.S. Haworth

Summary

Keywords: Antibiotics, bronchiectasis, exacerbation, intravenous, nebulised, prophylaxis

Correspondence: C.S. Haworth, Cambridge Centre for Lung Infection, Papworth Hospital, Cambridge, CB23 3RE, UK, Email [email protected]

C.S. HAWORTH

Antibiotics play a crucial role in the management of patients with bronchiectasis by disrupting the vicious circle of infection, inflammation and airway damage central to the pathophysiology of the condition. Antibiotic use in patients with bronchiectasis can be divided into exacerbation treatment, chronic suppressive treatment and eradication treatment. Antibiotics administered during exacerbations are known to reduce serum C-reactive protein concentrations, sputum volume and bacterial density, as well as ameliorate symptoms. Clinical experience suggests that better outcomes are seen with higher dose/longer duration regimens. The prescription of long-term oral antibiotics should be considered in patients requiring exacerbation treatment at least three times per year. As patients chronically infected with Pseudomonas aeruginosa tend to have a faster rate of lung function decline, more admissions to hospital and a worse quality of life compared with bronchiectasis patients with other microorganisms, nebulised antipseudomonal antibiotics are commonly prescribed. Eradication antibiotics should be considered following identification of new growths of P. aeruginosa due to the increased morbidity associated with chronic infection.

Eur Respir Mon 2011. 52, 211–222. Printed in UK – all rights reserved. Copyright ERS 2011. European Respiratory Monograph; ISSN: 1025-448x. DOI: 10.1183/1025448x.10004410

B

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ronchiectasis is a condition characterised by irreversible dilatation of the bronchi [1] resulting from changes in the elastic and muscular components of the bronchial wall. COLE [2] proposed the vicious circle hypothesis in the 1980s to explain the pathogenesis of bronchiectasis. He suggested that an initial insult to the airway leads to bronchial wall inflammation and damage, disordered mucociliary clearance, a predisposition to chronic or recurrent infection and as a result, further airway damage. Antibiotic use in patients with bronchiectasis can be divided into

exacerbation treatment, chronic suppressive treatment and eradication treatment. Treatment of pulmonary diseases related to nontuberculous mycobacteria and fungi will be covered by other chapters in this issue.

Antibiotic treatment in exacerbations of bronchiectasis Patients with bronchiectasis may expectorate significant volumes of mucopurulent sputum when well. It is therefore important to document stable state symptoms so that exacerbations can be accurately identified. Failure to do this can result in inappropriate antibiotic treatment. Acute exacerbations of bronchiectasis are characterised by an increase in cough frequency, sputum volume, sputum purulence and viscosity. Patients may also complain of chest tightness, wheeze and breathlessness. Other common features include streaky haemoptysis, chest discomfort and temperatures. Symptoms usually progress over days, but patients can experience a more insidious decline over weeks or months.

ANTIBIOTIC TREATMENT STRATEGY

There are no randomised placebo-controlled trials evaluating the effect of antibiotic treatment during exacerbations of bronchiectasis. However, antibiotics are known to reduce serum C-reactive protein, sputum inflammatory indices, sputum volume, sputum purulence and bacterial density, as well as ameliorate symptoms [3–8]. The published literature evaluating antibiotic treatment during exacerbations of bronchiectasis is heterogeneous in terms of the class of antibiotic studied, the route of administration and the sputum microbiology of the participants. However, important management principals emerge: high doses of an antibiotic are often more effective than lower doses of the same antibiotic [3]; patients with purulent sputum after antibiotic treatment have a shorter time to next exacerbation compared with patients with mucoid sputum [3]; sputum culture sensitivity results do not necessarily predict clinical response to antibiotic treatment [9]; short courses of oral antibiotics prescribed during acute exacerbations reduce airway inflammatory indices to pre-exacerbation levels, but chronic low-grade inflammation persists [4]; and clinical improvement may not be associated with significant increases in spirometry [7, 8, 10]. Initial treatment usually involves a course of oral antibiotics unless the patient is sufficiently unwell to require intravenous treatment. The optimal dose and duration of antibiotic treatment to manage bronchiectasis exacerbations is currently undefined. Clinical experience suggests that better outcomes are seen with higher dose/longer duration regimens, which presumably reflects the difficulty of achieving adequate antibiotic concentrations within the lumen of bronchiectatic airways, particularly in the context of chronic infection where bacteria are often resistant and protected by biofilms. Expert consensus is that bronchiectasis exacerbations should be treated with 14 days of antibiotics [11]. A sputum sample should be sent for culture before starting empirical antibiotic treatment and the results can influence changes in treatment if the patient is not responding.

Oral antibiotic treatment for exacerbations of bronchiectasis

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Oral antibiotic choices should be guided, where possible, by previous sputum microbiology and suggestions for treatment are outlined in table 1. Empirical treatment may be with amoxicillin 500 mg t.i.d. or co-amoxiclav 625 mg t.i.d. in patients in whom b-lactamase-producing organisms are suspected. Doxycycline 100 mg b.i.d. is an alternative choice in the context of penicillin allergy and ciprofloxacin 750 mg b.i.d. should be prescribed if Pseudomonas aeruginosa infection is thought to be likely. Patients with a history of methicillin-resistant Staphylococcus aureus (MRSA) infection may be treated with rifampicin 600 mg q.d. and fucidin 500 mg t.i.d. The potential for antibiotic related complications such as Clostridium difficile infection need to be considered when choosing oral or i.v. antibiotic regimens to treat exacerbations of bronchiectasis.

Table 1. Oral antibiotic regimens commonly used to treat acute exacerbations of bronchiectasis in adults# First line

Second line "

Streptococcus pneumoniae

Amoxicillin 500–1000 mg t.i.d.

Haemophilus influenzae

Amoxicillin 500–1000 mg t.i.d."

Moraxella catarrhalis

Co-amoxiclav 625 mg t.i.d.

Staphylococcus aureus

Flucloxacillin 500–1000 mg q.i.d."

MRSA

Rifampicin 400–600 mg q.d.+ and fucidin 500 mg t.i.d.

Pseudomonas aeruginosa Coliforms Stenotrophomonas maltophilia Achromobacter xylosoxidans

Ciprofloxacin 750 mg b.i.d. Ciprofloxacin 750 mg b.i.d. Cotrimoxazole 960 mg b.i.d. Minocycline 100 mg b.i.d.

Clarithromycin 500 mg b.i.d. Doxycycline 100 mg b.i.d. Moxifloxacin 400 mg q.d. Trimethoprim 200 mg b.i.d. Doxycycline 100 mg b.i.d. Co-amoxiclav 625 mg t.i.d. Ciprofloxacin 750 mg b.i.d. Trimethoprim 200 mg b.i.d. Doxycycline 100 mg b.i.d. Ciprofloxacin 750 mg b.i.d. Clarithromycin 500 mg b.i.d. Clarithromycin 500 mg b.i.d. Doxycycline 100 mg b.i.d. Co-amoxiclav 625 mg t.i.d. Trimethoprim 200 mg b.i.d. Moxifloxacin 400 mg q.d. Trimethoprim 200 mg b.i.d. Doxycycline 100 mg b.i.d. Linezolid 600 mg b.i.d. Co-amoxiclav 625 mg t.i.d. Minocycline 100 mg b.i.d.

q.d.: once daily; b.i.d.: twice daily; t.i.d.: three times daily; q.i.d.: four times daily; MRSA: methicillin-resistant Staphylococcus aureus. #: recommended treatment duration 10–14 days; ": dose according to severity; + : dose according to weight.

Intravenous antibiotic treatment for exacerbations of bronchiectasis Patients with severe exacerbations or exacerbations that fail to resolve with oral antibiotic treatment may require treatment with i.v. antibiotics. This usually involves admission to hospital, but some centres run community-based i.v. antibiotic programmes, which allow patients to have all, or a proportion, of their treatment at home. This is particularly helpful for younger patients who have educational commitments or for those that do not want to take time out from work. Patients must demonstrate that they are competent at i.v. drug administration before discharge and the home environment needs to be appropriate. Most centres recommend that the first dose is administered in hospital to ensure patients can infuse the antibiotic correctly and to ensure there are no adverse events. Robust systems need to be in place to monitor drug levels in patients prescribed aminoglycosides and careful monitoring of renal function is essential in patients receiving nephrotoxic medicines.

C.S. HAWORTH

Organism

Antibiotic i.v. administration during bronchiectasis exacerbations can be achieved through use of peripheral cannulas, long lines, peripherally inserted central catheters (PICC) and totally implantable venous access devices (TIVAD) (fig. 1). PICCs and TIVADs are particularly useful in patients with difficult peripheral access who require frequent courses of i.v. treatment. However, these devices require regular flushing and potential complications include thrombosis and infection, particularly in higher risk groups such as the elderly and those with a primary or secondary immunodeficiency.

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Antibiotic i.v. choices should be based on previous sputum microbiology results and suggested regimens are outlined in table 2. Empirical antibiotic treatment may include cefuroxime or ceftriaxone, unless patients are thought to be infected with P. aeruginosa. As the efficacy of b-lactam antibiotics is related to the time above the mean inhibitory concentration, once daily antibiotics may be less effective than antibiotics taken three times a day due to the potential

problem of maintaining an adequate intraluminal antibiotic concentration in the context of structural lung damage and biofilm formation.

ANTIBIOTIC TREATMENT STRATEGY

Antibiotic i.v. treatment in patients infected with P. aeruginosa Empirical antibiotic treatment in patients with P. aeruginosa may include a b-lactam, such as ceftazidime. Monotherapy may suffice in patients infected with fully sensitive P. aeruginosa, but in the context of a resistant organism or chronic infection where patients Figure 1. Chest radiograph of a male patient with primary ciliary are likely to require repeated treatdyskinesia, dextrocardia, severe bilateral bronchiectasis and chronic ment courses in the future, many Pseudomonas aeruginosa infection who self-administers i.v. anticlinicians advocate dual therapy biotics via a totally implantable venous access device at home. with an aminoglycoside to reduce the risk of antibiotic resistance, as well as harnessing the synergistic effects between aminoglycoside and b-lactam antibiotics [12–14]. In a study evaluating the effect of i.v. azlocillin + placebo versus i.v. azlocillin + tobramycin in patients with cystic fibrosis (CF), initial clinical outcomes were comparable, but P. aeruginosa density decreased more and time to next hospitalisation was significantly longer in the group receiving dual therapy [15]. These data suggest that dual antibiotic therapy is preferable in the context of chronic P. aeruginosa infection and the absence of significant comorbidity (such as renal dysfunction). Table 2. Antibiotic i.v. regimens commonly used to treat acute exacerbations of bronchiectasis in adults# Organism

Streptococcus pneumoniae Haemophilus influenzae Moraxella catarrhalis MRSA

Pseudomonas aeruginosa

Coliforms

Stenotrophomonas maltophilia Achromobacter xylosoxidans

First line

Second line

Benzylpenicillin 1.2 g q.i.d.

Cefuroxime 1.5 g t.i.d. or Ceftriaxone 2 g q.d. Piperacillin with Tazobactam 4.5 g t.i.d. Piperacillin with tazobactam 4.5 g t.i.d. Teicoplanin" Linezolid 600 mg b.i.d. Tigecycline 50 mg b.i.d. Fosfomycin 5 g t.i.d. Aztreonam 2 g t.i.d.+ Piperacillin with tazobactam 4.5 g t.i.d.+ Meropenem 1 g t.i.d.+ Piperacillin with tazobactam 4.5 g t.i.d. Tigecycline 50 mg b.i.d. Meropenem 1 g t.i.d.

Cefuroxime 1.5 g t.i.d. or ceftriaxone 2 g q.d. Cefuroxime 1.5 g t.i.d. or ceftriaxone 2 g q.d. Vancomycin"

Ceftazidime 2 g t.i.d.+

Cefuroxime 1.5 g t.i.d. or ceftriaxone 2 g q.d. Cotrimoxazole 1.44 g b.i.d. Piperacillin with tazobactam 4.5 g t.i.d.

214

q.d.: once daily; b.i.d.: twice daily; t.i.d.: three times daily; q.i.d.: four times daily; MRSA: methicillin-resistant Staphylococcus aureus. #: recommended treatment duration 10–14 days; ": dose according to weight and drug levels; +: dual therapy with gentamicin or tobramycin may be required.

The role of antibiotic sensitivity testing in patients with bronchiectasis and chronic P. aeruginosa infection is contentious due to hypermutation and the poor correlation between in vitro antibiotic sensitivity test results and clinical outcomes [19, 20]. FOWERAKER et al. [19] studied sputum samples from patients with CF and found an average of four P. aeruginosa morphotypes per sputum sample and three distinct antibiotic sensitivity profiles per morphotype. 48 colonies with varying antibiotic sensitivity profiles were cultured from one sputum sample and it was noted that susceptibility profiles of single P. aeruginosa isolates correlated poorly with pooled cultures (the pooled cultures underestimated levels of antibiotic resistance). FOWERAKER et al. [19] also showed that sensitivity results from one sputum sample tested in duplicate by eight biomedical scientists within one laboratory and by biomedical scientists in seven other laboratories did not correlate well. These data are supported by the findings of SMITH et al. [21], who showed no correlation between the susceptibility of P. aeruginosa to ceftazidime or tobramycin and clinical response to these antibiotics in 77 chronically infected patients with CF. Furthermore, a randomised controlled trial evaluating clinical outcomes, using multiple combination bactericidal testing versus clinician preference, to guide i.v. antibiotic choices to manage CF pulmonary exacerbations showed no advantage in using the more sophisticated microbiological tests [22]. Based on the above evidence, a pragmatic approach is required when choosing antibiotic combinations for patients with bronchiectasis and chronic P. aeruginosa infection. It is common practice to choose two antipseudomonal antibiotics (usually a b-lactam in combination with tobramycin) to which the majority of morphotypes are sensitive. An alternative approach involves basing antibiotic choices predominantly on what has worked well for the patient in the past.

Nebulised antibiotic treatment for exacerbations of bronchiectasis The nebulised route enables delivery of high concentrations of antibiotic to the airways and reduces the likelihood of gastrointestinal adverse events. However, airway inflammation may lead to bronchoconstriction and drug deposition may be limited by sputum plugging.

C.S. HAWORTH

The most appropriate choice of aminoglycoside remains a matter for debate, but recent reports suggest that the risk of renal impairment, ototoxicity and vestibular damage is greater with gentamicin than tobramycin [16, 17]. While, once daily versus three times daily tobramycin dosing in children with CF appears to offer equivalent clinical outcomes and reduced renal toxicity [18], the most appropriate dosing regimen has not been established in adults with bronchiectasis.

BILTON et al. [6] tested the effect of adding inhaled tobramycin solution to oral ciprofloxacin for treatment of bronchiectasis exacerbations in the context of P. aeruginosa infection. The study involved 53 adults recruited from 17 study centres in the UK and USA. There was evidence of superior microbiological efficacy in patients receiving inhaled tobramycin and ciprofloxacin compared with those receiving ciprofloxacin alone, but superior clinical efficacy was not demonstrated. Patients treated with inhaled tobramycin and ciprofloxacin were more likely to experience respiratory adverse events, in particular wheeze (50% in the inhaled tobramycin group compared with 15% in the placebo group). Although treatment emergent wheeze was not a significant cause for withdrawal from the study, it is probable that it influenced the clinical efficacy outcome data. It is also notable that patients with ciprofloxacin resistant strains of P. aeruginosa were excluded from the study and it is possible that the inclusion of such patients, as would occur in routine clinical practice, may have resulted in more favourable outcomes in the inhaled tobramycin-treated patients.

Antibiotic prophylaxis in adults with bronchiectasis

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Antibiotics are commonly prescribed on a long-term basis in patients with bronchiectasis with a view to improving symptoms, decreasing exacerbation rates and optimising quality of life. The most likely mechanism by which antibiotics achieve these aims is by reducing bacterial load and airway inflammation. The immunomodulatory benefits of long-term macrolide antibiotics are discussed in a later chapter by SMITH et al. [23].

Antibiotics used on a long-term basis are usually administered orally or through a nebuliser. However, within the CF population, some centres advocate 3-monthly elective courses of i.v. antibiotics for patients chronically infected with P. aeruginosa [24]. This approach has not been taken up widely due to concerns about toxicity (particularly renal, vestibular and auditory), psychosocial well-being (disruption to family life, work and education), healthcare costs and those concerns relevant to all forms of antibiotic prophylaxis: increasing bacterial resistance and the creation of a niche for new organisms (both bacteria and fungi) [25, 26].

ANTIBIOTIC TREATMENT STRATEGY

Oral antibiotic prophylaxis in adults with bronchiectasis The evidence base for oral antibiotic prophylaxis in bronchiectasis dates back to the early 1950s when a number of unrandomised studies were performed [27, 28]. However, these were soon superseded by the Medical Research Council study which involved 122 subjects randomised to receive penicillin, oxytetracycline or placebo [29]. The drugs were provided as indistinguishable 0.25 g capsules and patients were asked to take two capsules four times a day on two days each week for 1 year. Outcome measures included 24-h sputum volume and the severity of cough, dyspnoea, haemoptysis and disability. Unfortunately no formal statistical analysis was performed. After 1 year, oxytetracycline treatment was associated with a reduction in sputum volume to 64% of pre-treatment levels and the purulent fraction was reduced by 50%. Treatment with oxytetracycline was also associated with fewer days off work and fewer days confined to bed. Less marked changes were evident in patients allocated to the penicillin and placebo groups. Gastrointestinal symptoms were reported by a minority of patients (five on oxytetracycline, three on penicillin and two on placebo) and one patient in the oxytetracycline and penicillin groups discontinued treatment due to antibiotic intolerance. Unfortunately, sputum microbiology data were not reported and so it is not possible to make an assessment of whether sensitivity profiles affected outcomes. Subsequent studies in the 1950s and 1960s provided further support for the use of long-term tetracycline/penicillin based antibiotic regimens in patients with bronchiectasis [30, 31]. However, the latter study also reported an increase in the isolation of Pseudomonas and Proteus species, suggesting that microbial flora of sputum may be altered by long-term antibiotic treatment. CURRIE et al. [32] performed a randomised placebo-controlled trial evaluating the effect of highdose amoxicillin in patients with bronchiectasis. 38 subjects were randomised to receive amoxicillin 3 g b.i.d. or placebo for 32 weeks. Assessment of overall response based on diary card data showed that a higher proportion of patients improved in the amoxicillin group (11 out of 17) compared with the placebo group (four out of 19). Patients in the amoxicillin group also spent significantly less time confined to bed and away from work compared with the placebo group. The frequency of exacerbation was similar in the two groups, but the exacerbations were less severe in the amoxicillin group than before the study was started. There was also a greater reduction in purulent sputum volume in the amoxicillin group (20% of pre-treatment volume) compared with the placebo group (88% of pre-treatment volume). One patient in the amoxicillin group withdrew from the study due to the development of rash and one patient from each group withdrew due to diarrhoea. There was a trend towards greater antibiotic resistance in patients treated with amoxicillin. No patients developed C. difficile-related diarrhoea. In a 16-week open-label study of oral and nebulised amoxicillin involving 10 patients with bronchiectasis and variable sputum microbiology (predominantly Haemophilus influenzae), treatment was associated with reduced sputum purulence and volume, reduced sputum inflammatory indices, improvements in lung function and improved patient well-being [33]. After cessation of treatment, sputum purulence returned after a median of 2.5 months.

216

RAYNOR et al. [34] performed a retrospective case note review of 10 patients with bronchiectasis prescribed .90 days of continuous oral ciprofloxacin. Pre-treatment sputum microbiology results from nine patients showed a variety of organisms including P. aeruginosa (n55), H. influenzae (n53) and Streptococcus pneumoniae (n51). At the end of treatment six patients had sterile sputum

cultures, of which two had previously grown P. aeruginosa, three H. influenzae and one had no pathogen. In one patient P. aeruginosa was replaced by S. pneumoniae, two patients continued to culture P. aeruginosa (which had become resistant to ciprofloxacin) and S. pneumoniae persisted in one patient. While exacerbation frequency and hospital admissions reduced with treatment, the development of ciprofloxacin-resistant strains of P. aeruginosa is of concern, particularly as this finding coincided with a relapse in symptoms requiring admission to hospital for i.v. antibiotics. In practice, the prescription of long-term oral antibiotics is considered in patients requiring exacerbation treatment at least three times per year (or in patients with fewer exacerbations but greater associated morbidity) [11]. There may also be a lower threshold to prescribe long-term antibiotics in patients with a primary or secondary immunodeficiency. Common long-term oral antibiotic regimens are outlined in table 3. Where possible, antibiotic choices should be based on sputum microbiology data. While there is no evidence currently in favour of antibiotic rotation over single agent prophylaxis in terms of the development of antibiotic resistance and efficacy, it is important to record exacerbation rates before and after starting long-term oral antibiotics and to perform regular sputum surveillance to monitor antibiotic resistance patterns and to identify treatment emergent bacteria and fungi.

Nebulised antibiotic prophylaxis in patients with bronchiectasis

Antipseudomonal nebulised antibiotic regimens evaluated to date include nebulised gentamicin, nebulised tobramycin, nebulised tobramycin in combination with nebulised ceftazidime and nebulised colistin. The largest published study was performed by BARKER et al. [36] and evaluated the microbiological efficacy and safety of inhaled tobramycin in patients with bronchiectasis infected with P. aeruginosa. Patients were randomly assigned to receive either tobramycin solution for inhalation (n537) or placebo (n537) twice daily for 4 weeks. At week 4, the tobramycin solution for inhalation group had a mean decrease in P. aeruginosa density of 4.5 log10 colony forming units per gram (CFU?g-1) of sputum compared with no change in the placebo group (p,0.01). Logistic regression analysis showed that decreases in CFU?g-1 of sputum were significant predictors of improved well-being. 2 weeks after cessation of the trial, P. aeruginosa was eradicated in 35% of the tobramycin-treated group, but was detected in all placebo patients. 62% of

C.S. HAWORTH

There have been a number of studies conducted using nebulised antibiotics in patients with bronchiectasis. The majority involve antipseudomonal agents, although earlier studies evaluated the use of nebulised amoxicillin in patients predominantly infected with H. influenzae [3, 33, 35]. While the results of the nebulised amoxicillin trials are largely positive, in practice this intervention is rarely used as high-dose oral regimens are easier and cheaper to administer.

Table 3. Oral antibiotic prophylaxis for adult patients with bronchiectasis based on sputum microbiology Organism

First line

Second line

Streptococcus pneumoniae

Amoxicillin 500 mg b.i.d.

Haemophilus influenzae

Amoxicillin 500 mg b.i.d.

Moraxella catarrhalis

Amoxicillin 500 mg b.i.d.

Clarithromycin 500 mg b.i.d. Doxycycline 100 mg q.d. Trimethoprim 200 mg b.i.d. Doxycycline 100 mg q.d. Trimethoprim 200 mg b.i.d. Doxycycline 100 mg q.d. Clarithromycin 500 mg b.i.d. Clarithromycin 500 mg b.i.d. Doxycycline 100 mg q.d. Trimethoprim 200 mg b.i.d. Doxycycline 100 mg q.d. Minocycline 100 mg b.i.d.

Staphylococcus aureus MRSA Stenotrophomonas maltophilia Achromobacter xylosoxidans

Flucloxacillin 500 mg–1 g b.i.d.

Trimethoprim 200 mg b.i.d. Cotrimoxazole 960 mg b.i.d. Minocycline 100 mg b.i.d.

217

q.d.: once daily; b.i.d.: twice daily; MRSA: methicillin-resistant Staphylococcus aureus.

tobramycin-treated patients showed improvement in their medical condition compared with 38% of the placebo patients (OR 2.7, 95% CI 1.1–6.9), but there was no significant change in lung function between the treatment groups. Tobramycin-resistant P. aeruginosa strains developed in four (11%) out of 36 of tobramycin-treated patients and one (3%) out of 32 of placebo-treated patients. Three of the four patients in the tobramycin-treated group who developed resistant P. aeruginosa strains showed no microbiological response and all four failed to improve clinically. More tobramycin-treated patients than placebo patients reported increased cough, breathlessness, wheezing and non-cardiac type chest pain, but the symptoms did not appear to limit therapy.

ANTIBIOTIC TREATMENT STRATEGY

A second trial evaluating tobramycin solution for inhalation involved 41 bronchiectasis patients infected with P. aeruginosa and employed an open-label design consisting of three treatment cycles (14 days of drug therapy and 14 days off) [37]. During the 12-week treatment period significant improvements occurred in the pulmonary symptoms severity score and in quality of life measurements. However, tobramycin-resistant strains of P. aeruginosa developed in two subjects and 10 patients dropped out due to adverse events, the most common being cough, wheeze and breathlessness. Five subjects died during the study period due to the underlying disease, one during the 12-week treatment period and four during the 40-week follow-up period. None of the deaths were considered to be related to the drug treatment. DROBNIC et al. [38] evaluated an alternative formulation of tobramycin in a double-blind placebocontrolled cross-over trial involving 30 patients. Patients received aerosolised tobramycin 300 mg or placebo twice daily for 6 months, with a 1-month wash out period between interventions. 20 patients completed the protocol as three patients withdrew from the study due to bronchospasm, five patients died from respiratory failure and two others dropped out (one failed to adhere to the study protocol and one relocated). The number of admissions and in-patient days reduced during the tobramycin period. There was also a decrease in P. aeruginosa density which persisted up until 3 months after nebulised tobramycin treatment had been stopped and there was no difference in the emergence of bacterial resistance between the two study periods. However, there was no significant difference in the number of exacerbations, antibiotic use, lung function or quality of life between the tobramycin and placebo periods. ORRIOLS et al. [39] performed a 12-month study in which patients with bronchiectasis were randomised to receive nebulised ceftazidime 1 g b.i.d. + tobramycin 100 mg b.i.d. or symptomatic treatment. One out of eight patients in the nebulised antibiotic group withdrew having developed bronchospasm and one out of nine patients in the control group died. While there were significantly less admissions and in-patient days in the nebulised antibiotic group, these findings need to be interpreted with care owing to the open-label design of the study. Interestingly, there was no difference in the use of oral antibiotics or change in lung function between the two treatment groups. There was also no difference in the emergence of antibiotic resistant bacteria between the two treatment groups. LIN et al. [40] performed a randomised controlled trial assessing the effect of aerosolised gentamicin 40 mg (n516) versus 0.45% saline (n515) administered twice daily for 3 days in patients with bronchiectasis. Gentamicin-treated patients showed significant reductions in sputum volume and sputum inflammatory indices (there was a significant correlation between the change in sputum volume and sputum myeloperoxidase) in conjunction with significant improvements in peak expiratory flow rate and 6-min walk distances.

218

MURRAY et al. [41] performed a longer term study evaluating the effect of nebulised gentamicin in patients with bronchiectasis. 65 patients were randomised to receive gentamicin 80 mg or 0.9% saline twice daily through a nebuliser for 12 months. Inclusion criteria included a history of chronic sputum colonisation with potentially pathogenic organisms when clinically stable. After 12 months, use of nebulised Gentamicin was associated with significant reductions in bacterial density with a 30.8% eradication rate in patients infected with P. aeruginosa and a 92.8% eradication rate in patients infected with other pathogens. There was reduced sputum purulence (8.7% versus 38.5%, p,0.001), greater exercise capacity (median (interquartile range) 510 (350–690) m

versus 415 (267–530) m, p50.03), fewer exacerbations (median (interquartile range) 0 (0–1) versus 1.5 (1–2), p,0.001), increased time to first exacerbation (median (interquartile range) 120 (87–161) days versus 61 (20–122) days, p50.02) and greater improvements in quality of life in patients treated with gentamicin. There were no differences between groups in 24-h sputum volume, forced expiratory volume in 1 second, forced vital capacity (FVC) or forced expiratory flow at 25–75% of FVC. There was no development of gentamicin-resistant isolates of P. aeruginosa. Small retrospective studies have evaluated the effect of nebulised colistin in patients with bronchiectasis and P. aeruginosa infection [42, 43]. Owing to the retrospective nature of the studies the results need to be interpreted with care, but the data suggest that nebulised colistin has beneficial effects in this patient population in terms of exacerbation frequency, admission rates, sputum volume and lung function. An international multicentre randomised placebo controlled trial evaluating the effect of nebulised colistin (promixin) on time to next exacerbation in patients with bronchiectasis and chronic P. aeruginosa infection is underway and will report in the next 2 years.

Eradicating new growths of specific organisms in patients with bronchiectasis There are no trials to date evaluating antibiotic eradication regimens in patients with bronchiectasis. However, in clinical practice, eradication regimens are often prescribed following identification of new growths of P. aeruginosa due to the increased morbidity associated with chronic infection [44–48]. Some of the oral and nebulised antibiotic studies in patients with bronchiectasis report variable success rates in eradicating P. aeruginosa, but these studies were not designed to address this specific issue and largely involved patients with chronic P. aeruginosa infection [34, 36–39, 41]. In the absence of conclusive trial data, many clinicians follow treatment protocols used in patients with CF, where early eradication therapy and the subsequent reduction in prevalence of chronic P. aeruginosa infection is thought to have had a major impact on survival [49]. Experience suggests that eradication of P. aeruginosa is less likely once the organism has converted to the mucoid form, which reinforces the need for early intervention [14]. In patients

C.S. HAWORTH

Patients with bronchiectasis and P. aeruginosa chronic infection tend to have more severe lung disease based on physiological and computed tomography parameters, a faster rate of lung function decline, more admissions to hospital and a worse quality of life compared with patients with other microorganisms [44–48]. Thus, nebulised antibiotics are frequently prescribed for patients with bronchiectasis and chronic P. aeruginosa infection in order to improve well-being and prevent disease progression, consistent with CF management principals [14, 25]. Common nebulised antibiotic regimens are outlined in table 4. It is important to record exacerbation rates before and after starting long-term nebulised antibiotics and to perform regular sputum surveillance to monitor antibiotic resistance patterns and treatment emergent bacteria and fungi.

Table 4. Nebulised antibiotic prophylaxis for adult patients with bronchiectasis chronically infected with Pseudomonas aeruginosa Drug and formulation# Colistin (Colomycin) Colistin (Promixin) Gentamicin 40 mg?mL-1 Tobramycin (Tobi) Tobramycin (Bramitob) Aztreonam lysine (Cayston) Ceftazidime

Dose

Diluent

2 MU b.i.d. 1 MU b.i.d. 80 mg b.i.d. 300 mg b.i.d. 300 mg b.i.d. 75 mg t.i.d. 1 g b.i.d.

4 mL 0.9% sodium chloride 1 mL water for injection 1 mL 0.9% sodium chloride

1 mL 0.17% sodium chloride 3 mL water for injection

219

MU: million units; b.i.d.: twice daily; t.i.d.: three times daily. #: unlicensed indication.

with CF and a new growth of P. aeruginosa, the prescription of ciprofloxacin + nebulised colistin resulted in 16% of treated patients developing chronic P. aeruginosa infection compared with 72% of untreated historical controls (p,0.005) after 3.5 years follow-up [50]. More recent data showed that .90% of patients with CF and early P. aeruginosa infection had negative cultures 1 month after completing a 4-week course of nebulised tobramycin (tobi 300 mg q.i.d.) [51]. In practice, many clinicians prescribe a 3-month course of nebulised colistin in combination with oral ciprofloxacin for patients with bronchiectasis and a new growth of P. aeruginosa [11, 14, 52], and offer i.v. therapy if this intervention fails. Eradication regimens are also commonly instituted in patients who culture MRSA in their sputum for the first time, due to the fact that it is a resistant organism and has significant infection control implications. Oral rifampicin and fucidin with or without nebulised vancomycin is used in some centres, but treatment regimens should be based around local policies.

Future antibiotic treatment strategies

ANTIBIOTIC TREATMENT STRATEGY

It is likely that antibiotic treatment options for patients with bronchiectasis will change significantly over the next decade. New nebulised (amikacin, aztreonam, colistin and fosfomycin in combination with tobramycin) and dry powder (ciprofloxacin, colistin and tobramycin) antibiotic formulations have been developed and may be beneficial in patients with bronchiectasis. New ways of using old antibiotics may also lead to improved outcomes. For example, due to the time-dependent antibacterial activity of b-lactam antibiotics, continuous infusions may offer superior efficacy compared with intermittent infusions [53], particularly in the context of severe structural lung damage and biofilm formation.

Conclusion Antibiotics play a crucial role in the management of patients with bronchiectasis by disrupting the infection component of the vicious circle of infection, inflammation and airway damage central to the pathophysiology of bronchiectasis. Antibiotics can be used for treatment of exacerbations, for chronic bacterial suppression and for eradication. Antibiotic choices should be based on sputum microbiological results. Careful monitoring is required regarding microbial resistance patterns and treatment emergent bacteria/fungi, gastrointestinal adverse events (C. difficile infection) and antibiotic related toxicity (particularly with aminoglycosides). In the future, antibiotic options are likely to increase through the development of new nebulised and dry powder formulations.

Statement of interest C.S. Haworth has received educational grants, speaker fees or performed consultancy work for Chiesi, Gilead, Novartis and Forest.

References

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Chapter 15

Anti-inflammatory therapies in bronchiectasis D.J. Smith*,#, A.B. Chang",+,1 and S.C. Bell*,#,+

Although the use of anti-inflammatory therapies in bronchiectasis remains an attractive proposition, there is currently insufficient evidence to support the use of inhaled and oral corticosteroids, non-steroidal anti-inflammatory drugs and macrolides. Individual patient trials may be warranted for inhaled corticosteroids and macrolides. It is hoped that recently completed and ongoing randomised control trials of macrolides will better define the use and safety in bronchiectasis. There remains an urgent need to perform adequately powered multicentre trials of other potentially useful therapies. It is anticipated that specialised bronchiectasis clinics will provide greater opportunities to study disease epidemiology and pathogenesis and allow better definition of study population for inclusion within future trials. There is a need for a more defined study population and a widely accepted definition of a pulmonary exacerbation in bronchiectasis which may be applied uniformly across studies to allow direct comparison of study outcomes. Finally, care should be taken to ensure adequate follow-up to detect potential adverse effects of new therapies, particularly on microbial resistance patterns. Keywords: Anti-inflammatory therapy, bronchiectasis, inflammation, inhaled corticosteroids, macrolides

*Dept of Thoracic Medicine, # School of Medicine, University of Queensland, The Prince Charles Hospital, Chermside, " Queensland Children’s Respiratory Centre, + Queensland Children’s Medical Research Institute, Herston, Queensland, and 1 Menzies School of Health Research, Charles Darwin University, Darwin, Northern Territory, Australia. Correspondence: S.C. Bell, Dept of Thoracic Medicine, The Prince Charles Hospital, Rode Road, Chermside, Brisbane, QLD, 4032, Australia, Email [email protected]

D.J. SMITH ET AL.

Summary

Eur Respir Mon 2011. 52, 223–238. Printed in UK – all rights reserved. Copyright ERS 2011. European Respiratory Monograph; ISSN: 1025-448x. DOI: 10.1183/1025448x.100004510

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ronchiectasis is an under-recognised condition characterised by pathological dilatation of bronchi, persistent neutrophilic airway inflammation and, in many, chronic bacterial infection. Bronchiectasis develops in the susceptible host through a vicious cycle of airway infection and inflammation [1]. The causes of non-cystic fibrosis (CF) bronchiectasis are diverse, the cohort populations are heterogeneous and the evidence to support therapies limited [2]. Factors which may have contributed to the limited evidence for treatment are likely to include population and disease severity heterogeneity, limited funding sources for clinical trials and the diverse manner that patients with bronchiectasis are managed. This appears to be changing with

the advent of specialised bronchiectasis clinics which are providing an opportunity to develop focused research programmes. There are a limited number of high-quality randomised controlled trials (RCT’s) cited in recently published management guidelines for bronchiectasis [3–5].

Airway biology in bronchiectasis Cohort studies of patients with bronchiectasis reveal Haemophilus influenzae and Pseudomonas aeruginosa to be the most frequently isolated organisms from airway secretions. Streptococcus pneumoniae, Moraxella species and nontuberculous mycobacteria (NTM) are reported less commonly [6–8]. Although infection triggers inflammation, ongoing neutrophilic infiltration of the airways is apparent even in the absence of persistent infection, suggesting dysregulation of immune responses [9]. Neutrophils are the predominant inflammatory cell found in sputum and bronchoalveolar lavage fluid (BALF) in patients with bronchiectasis [9, 10]. It is hypothesised that neutrophil apoptosis and clearance may be defective in bronchiectasis [11]. Non-apoptosed cells die by necrosis leading to exudation of toxic products (e.g. exoenzymes, oxygen free radicals, myeloperoxidase, etc.) which cause both localised tissue damage and provide an ongoing stimulus for the inflammatory response. Macrophages, lymphocytes and eosinophils are similarly present in increased number in the bronchiectatic airway, however, their role is poorly defined [12].

ANTI-INFLAMMATORY THERAPY

Acute respiratory exacerbations in patients with bronchiectasis are poorly understood but are thought to be related, in part, to increased load of existing airway bacteria and/or infection with a new bacterial pathogen. These changes provide rationale for the use of targeted antibiotics in patients with bronchiectasis during respiratory exacerbations which are discussed in detail in the chapter by FOWERAKER and WAT [13].

Targeting inflammation in bronchiectasis An alternative approach to targeting infection with antimicrobial agents is to attempt to modify the immune response to infection. In this chapter we focus on the use of anti-inflammatory agents and examine the evidence for the use and potential pitfalls of these therapies. We also explore future treatment options and studies that are in progress. Anti-inflammatory therapies will be discussed in one of three broad categories: 1) general antiinflammatory therapies which have broad immunosuppressive effects on inflammatory pathways (e.g. corticosteroids or nonsteroidal anti-inflammatory drugs (NSAIDS)); 2) novel antiinflammatory therapies which have immunomodulatory properties in addition to the cellular effects for which they are conventionally utilised (e.g. macrolides and hydroxy-methyl-glutarylcoenzymeA (HMGCoA) reductase inhibitors); and 3) targeted anti-inflammatory therapies which block a specific mediator of the immune response (e.g. anti-immunoglobulin E or anti-tumour necrosis factor (TNF)-a).

General anti-inflammatory agents Corticosteroids

224

Corticosteroids have broad anti-inflammatory effects through inhibition of inflammatory mediator synthesis and release and impairment of inflammatory cell migration [14]. Corticosteroids stimulate eosinophil apoptosis but paradoxically inhibit neutrophil apoptosis which, in part, possibly explains their variable anti-inflammatory effectiveness in different clinical settings [15]. Inhaled corticosteroids improve asthma control [16, 17] and are associated with reduction in exacerbation frequency in chronic obstructive pulmonary disease (COPD) [18], yet their withdrawal in patients with CF has minimal impact on symptoms, lung function or exacerbations [19]. Short courses of oral steroids have an established role in the treatment of exacerbations of asthma and COPD [20, 21]; however, their role in CF is more controversial [22].

Inhaled corticosteroids Recently, KAPUR et al. [23] identified six RCTs of inhaled steroids in non-CF bronchiectasis (table 1). The meta-analysis of these studies failed to provide conclusive evidence that inhaled corticosteroids result in a clinically significant improvement in lung function, affect exacerbation rates or improve quality of life in patients with bronchiectasis (fig. 1).

Two larger and longer trials studying fluticasone diproprionate (500 mg b.i.d.) in adults with bronchiectasis, demonstrated a reduction in sputum quantity [28, 29]. In a post hoc analysis TSANG et al. [28] observed that this effect was most pronounced in those patients with chronic P. aeruginosa infection. However, each of these studies had significant limitations including no placebo arm in the former and variable baseline sputum production in the treatment arms in the latter, precluding their data from being included in the assessment of this outcome measure in the Cochrane Review. Although therapy was generally well tolerated for the duration of the trials, long-term safety is uncertain in dosage regimens which would currently be considered to be high. In addition, one shortterm study [25], the data on density of total bacteria, commensal bacteria and P. aeruginosa in sputum showed an increasing trend after 4 weeks of therapy with inhaled steroids. Based on the available evidence from these published studies, KAPUR et al. [23] concluded that there is currently insufficient evidence of both benefit and safety to recommend routine use of inhaled corticosteroids in patients with bronchiectasis, however, it may be appropriate to consider a trial in severely symptomatic patients on a case by case basis, with close monitoring for adverse effects.

D.J. SMITH ET AL.

The earliest study, published in 1992 by ELBORN et al. [24], enrolled 20 patients in a 12-week crossover trial of high-dose beclomethasone diproprionate/placebo (6 weeks drug, 6 weeks placebo). Despite five patients dropping out of the study, the authors reported an 18% reduction in volume of sputum and reduced bronchoprovocation during histamine challenge testing. A subsequent study demonstrated inhaled fluticasone diproprionate reduced sputum levels of proinflammatory mediators (interleukin (IL)-8, leukotriene B4 (LTB4) and IL-1b) and sputum leukocyte density in bronchiectasis [25]. Combined with the consistent finding that inhaled steroids have no effect on sputum bacterial load [25], this suggests that any beneficial effect they may exert is most likely explained by anti-inflammatory as opposed to antimicrobial activity. Studies by TSANG et al. [26] and JOSHI and SUNDARAM [27] reported no change in exhaled nitric oxide and no change in lung function, respectively.

Oral corticosteroids There is currently no evidence supporting the use of oral corticosteroids. A Cochrane Review by LASSERSON et al. [30] failed to identify any RCTs in non-CF bronchiectasis either for short-term (during an exacerbation) or long-term use. The only evidence of potential benefit is from the paediatric CF literature in which prednisolone at a dose of 1 mg?kg-1 on alternate days was associated with reduced rate of lung function decline [22]. The long-term adverse effects including effects on growth and cataract resulted in the early termination of the trial.

NSAIDS

225

NSAIDS non-selectively block the activation of the cyclo-oxygenase pathway of pro-inflammatory prostaglandins. A landmark placebo controlled RCT examined the effects of ibuprofen in people with CF [31]. The study included 85 patients (age range 5–39 years) and demonstrated that those treated with high-dose ibuprofen (dose range 16.2–31.6 mg?kg-1) experienced a slower rate of decline in forced expiratory volume in 1 second (FEV1), as well as improved maintenance of weight when compared with control subjects over the 4-year study period. Post hoc analysis revealed these effects to be most pronounced in those participants ,13 years of age at study commencement. Ibuprofen therapy was well tolerated with only one patient withdrawing due to side-effects clearly attributable to ibuprofen (conjunctivitis and epistaxis).

226

RCT (parallel)

China (Hong Kong) India

China (Hong Kong)

T SANG [26]

T SANG [28]

Drug

Fluticasone proprionate (500 mg b.i.d. or 250 mg b.i.d.)

4 weeks

52 weeks

8 weeks (4 weeks each arm, 2 week washout) 52 weeks

36 weeks

Yes

Yes

Yes

Yes

Yes

12 weeks (6 weeks each arm, no washout)

Yes

93

86

20

60

24

20

Placebo Duration Subjects n

Findings

No change in lung function

No change in eNO

None

None reported but trend towards increased sputum density of commensal flora and P. aeruginosa Not reported

Oral candidiasis (n51)

Adverse events

Sputum volume Reduced sputum Sore throat and purulence, volume, no change in (n57) exacerbation rates, exacerbation rates, lung function sputum purulence, lung function HRQoL Improved dyspnoea, Dry mouth (n58), reduced sputum local irritation volume, reduced (n54), dysphonia b-agonist use (n54), oral (high-dose group) candidiasis (n52), aphthous ulcer (n51)

Lung function

eNO

Lung function, Improved FEV1, PD20 metacholine, improved morning sputum producPEFR, improved tion, pulmonary cough, reduced symptoms sputum volume 24 h sputum Reduced sputum (volume/leukocyte leukocyte density, counts/microbial reduced IL-1b, concentrations/ IL-8 and LTB4, IL-1/IL-8/TNF-a/ no change in sputum volume, no change in LTB4), lung function lung function

Outcome measures

DBRCT: double-blind (DB) randomised controlled trial (RCT); b.i.d.: twice daily; FEV1: forced expiratory volume in 1 second; PD20: provocative dose causing a 20% fall in FEV1; PEFR: peak expiratory flow rate; IL: interleukin; TNF-a: tumour necrosis factor-a; LTB4: leukotriene B4; P. aeruginosa: Pseudomonas aeruginosa; eNO: exhaled nitric oxide; HRQoL: health-related quality of life. #: the only blinded component of this study was for the dose of inhaled corticosteroids.

Bronchiectasis

Adults Bronchiectasis, Fluticasone (mean age nonsmokers proprionate 56 yrs) (500 mg b.i.d.) DBRCT Adults/ Bronchiectasis, Beclomethasone (crossover) children 12% improvement diproprionate (15–60 yrs) post(400 mg b.i.d.) bronchodilator FEV1 DBRCT Adults Bronchiectasis, no Fluticasone (parallel) (mean age oral/inhaled proprionate 58 yrs) corticosteroids (500 mg b.i.d.)

Fluticasone proprionate (500 mg b.i.d.)

Bronchiectasis, Beclomethasone no prior diproprionate oral/inhaled (750 mg b.i.d.) corticosteroids

Inclusion criteria

Adults Bronchiectasis (mean age .10 mL sputum 55 yrs) per 24 h

RCT-non Adults MARTINEZ- Spain DB# (parallel) (mean age GARCIA [29] 69 yrs)

J OSHI [27]

DBRCT (parallel)

China (Hong Kong)

T SANG [25]

Population

DBRCT Adults (30– (crossover) 65 yrs)

UK

E LBORN [24]

Design

Country

Study

Table 1. Randomised controlled trials of inhaled corticosteroids in bronchiectasis

ANTI-INFLAMMATORY THERAPY

ICS Mean±SD

Total

Placebo Total Weight % Mean±SD

FEV1 L# 0.011±0.11 -0.045±0.14 JOSHI [27] 10 0.064±0.154 MARTINEZ [29] 29 0.038±0.107 0.2±0.87 0±0.739 TSANG [25] 12 Subtotal (95% Cl) 51 Heterogeneity: χ2 = 0.59, df = 2 (p = 0.74); I2 = 0% Test for overall effect: Z = 3.04 (p = 0.002) FVC L# JOSHI [27] 10 0.038±0.16 -0.067±0.16 MARTINEZ [29] 29 -0.062±0.181 0.025±0.104 TSANG [25] 12 0.1±1 0±1 Subtotal (95% Cl) 51 Heterogeneity: χ2 = 0.05, df = 2 (p = 0.98); I2 = 0% Test for overall effect: Z = 2.66 (p = 0.008) Peak flow L.min-1# JOSHI [27] 10 17±27.36 -7.8±47.82 12 TSANG [25] 35±111 -2±122.58 Subtotal (95% Cl) 22 Heterogeneity: χ2 = 0.06, df = 1 (p = 0.81); I2 = 0% Test for overall effect: Z = 1.60 (p = 0.11) Diffusion capacity % pred¶ 84.2±10 29 86.9±10 MARTINEZ [29] 71.8±28.63 12 70±21.86 TSANG [25] Subtotal (95% Cl) 41 Heterogeneity: χ2 = 0.01, df = 1 (p = 0.93); I2 = 0% Test for overall effect: Z = 1.03 (p = 0.30) RV % pred¶ 108±10 29 106±29.2 MARTINEZ [29] 109±48.11 12 135.8±59.46 TSANG [25] Subtotal (95% Cl) 41 Heterogeneity: χ2 = 1.44, df = 1 (p = 0.23); I2 = 31% Test for overall effect: Z = 0.28 (p = 0.78) TLC % pred¶ MARTINEZ [29] 89.6±10 86.4±10 29 TSANG [25] 83.8±19.32 87.5±20.83 12 Subtotal (95% Cl) 41 Heterogeneity: χ2 = 0.64, df = 1 (p = 0.42); I2 = 0% Test for overall effect: Z = 1.01 (p = 0.31)

Mean difference IV, fixed, 95% Cl

10 27.7 28 71.5 12 0.8 50 100

0.06 (-0.05_0.17) 0.10 (0.03_0.17) 0.20 (-0.45_0.85) 0.09 (0.03_0.15)

10 23.0 28 76.3 12 0.7 50 100

0.11 (-0.04_0.25) 0.09 (0.01_0.16) 0.10 (-0.70_0.90) 0.09 (0.02_0.16)

10 88.2 24.80 (-9.35_58.95) 12 11.8 37.00 (-56.56_130.56) 22 100 26.23 (-5.84_58.31)

28 93.9 12 6.1 40 100

2.70 (-2.49_7.89) 1.80 (-18.58_22.18) 2.65 (-2.39_7.68)

10 84.6 2.00 (-16.46_20.46) 12 15.4 -26.80 (-70.08_16.48) 22 100 -2.43 (-19.41_14.55)

28 12 40

3.20 (-1.99_8.39) 90.5 9.5 -3.70 (-19.77_12.37) 2.55 (-2.39_7.49) 100

Mean difference IV, fixed, 95% Cl

◆

◆

D.J. SMITH ET AL.

Study or subgroup

◆

◆

-50 -25 0 25 50 Favours placebo Favours ICS

Figure 1. Forest plot of lung function indices comparing adults with bronchiectasis (in stable state) on inhaled corticosteroids (ICS) versus controls. FEV1: forced expiratory volume in 1 second; FVC: forced vital capacity; % pred: % predicted; RV: residual volume; TLC: total lung capacity. #: end study minus baseline values; ": end of study values. Reproduced from [23] with permission from the publisher.

227

A Cochrane Review by LANDS and STANOJEVIC [32] of NSAIDs in CF, including four RCTs, concluded that high-dose ibuprofen is capable of slowing disease progression; whilst NSAIDs are an attractive potential therapy in patients with bronchiectasis the benefits of treatment

demonstrated in patients with CF cannot necessarily be extrapolated. This has been demonstrated with the use of human recombinant DNase, which when trialled in non-CF bronchiectasis resulted in increased pulmonary exacerbations and greater decline in lung function [33]. Two recent Cochrane Reviews of oral and inhaled NSAID therapy in non-CF bronchiectasis were able to identify only one study suitable for inclusion [34, 35]. In this study 25 adults with chronic lung disease (eight bronchiectasis, 12 chronic bronchitis and five diffuse panbronchiolitis) received inhaled indomethacin or placebo for 14 days. In the treatment group (inhaled indomethacin) compared with placebo, there was a significant reduction in sputum production over 14 days (difference -75 g?day-1; 95% CI -134.61– -15.39) and significant improvement in dyspnoea score (difference -1.90; 95% CI -3.15– -0.65). There was no significant difference between groups in lung function or blood indices [36].

Novel immunomodulatory agents Macrolides

ANTI-INFLAMMATORY THERAPY

Macrolides have been in clinical use as antimicrobial agents for .50 years. There are three classes of macrolides based on the central ring structure: 14-membered ring macrolides (e.g. erythromycin, roxithromycin and clarithromycin); 15-membered ring macrolides (also known as ‘‘azolides’’, e.g. azithromycin); and 16-membered ring macrolides (e.g. spiramycin and josamycin) (fig. 2). The variation in structure of each class influence pharmacokinetic and pharmacodynamic properties [38]. Importantly, compared with other classes, the 15-membered ring structure azolides have less drug interaction, improved gastrointestinal tolerance and enhanced ability to concentrate within the neutrophil [39].

Antimicrobial properties Macrolides exert their antimicrobial action against Gram-positive, Gram-negative and intracellular organisms by binding to ribosomal subunits required for protein replication. Of particular relevance to their use in bronchiectasis is their antimicrobial activity against H. influenzae, Moraxella catarrhalis and S. pneumoniae. Similarly their activity against ‘‘atypical’’ respiratory pathogens (including Legionella pneumophila, Chlamydia spp. and Mycoplasma pneumoniae) has led to their widespread usage in the treatment of community-acquired pneumonia [40, 41]. At least two compounds (clarithromycin and azithromycin) have demonstrated activity in NTM infection and are important components of multi-drug regimes for treatment of Mycobacterium avium complex [42]. If adherence to treatment regimens is poor or if macrolide monotherapy is administered, NTM species may develop resistance. This may result in poorer clinical outcome [43]. This is a major concern when macrolides are prescribed in disease processes where mycobacterial infections can co-exist. The recently published Australia and New Zealand bronchiectasis guidelines recommend screening for NTM prior to initiation of macrolide therapy and regular sputum surveillance during treatment [5].

Anti-pseudomonal properties

228

The reported prevalence of P. aeruginosa infection in bronchiectasis varies from 12% to 33% [8] and is associated with radiological disease severity [44], increased lung function decline [45] and mortality [46]. Mucoid transformation of P. aeruginosa allows alginate secretion and biofilm production which provides a physical barrier from the immune system and contributes to persistent airway infection and inflammation [47]. P. aeruginosa within biofilms can communicate through quorum sensing systems (las and rhl) which are important in coordination of the expression of virulence factors and biofilm maturation [48]. Azithromycin has been shown to suppress both lasI and rhlI in vitro [49].

Macrolides have also been shown to suppress various P. aeruginosa virulence factors including protease, elastase, leucocidin, pyocyanin, phospholipase C and exotoxin A [51–53]. Suppression of P. aeruginosa virulence varied depending on P. aeruginosa strains studied and the specific macrolide used. In general, azithromycin has been shown to be more effective than other macrolides [51, 52]. Azithromycin has also been shown to inhibit P. aeruginosa antibiotic efflux pumps thereby potentially contributing to synergy and increasing the efficacy of other classes of antimicrobials [54]. Although these studies suggest macrolides are capable of impairing P. aeruginosa virulence, it is important to highlight that most of these studies were performed with laboratory strains of P. aeruginosa using in vitro systems.

a) O

CH3 HO

H3C

OH CH3 O

HO

N O

CH3

HO H3C O CH3

CH3 O CH3

O CH3

OH

O

CH3 O CH3 H3C

b) CH3

H3C

OH CH3 O

HO

CH3 N

HO

N H3C

O

CH3

HO H3C

CH3

O CH3

O

CH3

O

CH3 CH3

O

OH

O CH3

CH3

c) N O

Anti-inflammatory properties Anti-inflammatory properties of macrolides were first considered in the 1970s when observational studies noted that steroid-dependent asthmatics were able to reduce their dose of oral corticosteroid dose while prescribed erythromycin and triacetyloleandomycin [55]. The steroid sparing effect was later confirmed in prospective studies in patients with severe corticosteroid dependent asthma [56]. Furthermore, a reduction in bronchial hyperreactivity in asthmatic subjects was seen in patients receiving erythromycin, clarithromycin or roxithromycin [57–59].

CH3

H3C

CH3

D.J. SMITH ET AL.

P. aeruginosa is considered to be inherently resistant to macrolides as the in vitro minimal inhibitory concentration (MIC) is significantly higher than the concentration achievable in vivo [50]. However sub-MIC concentrations of macrolides may inhibit P. aeruginosa virulence. Type IV pili on the surface membrane of P. aeruginosa increase the organism’s motility and are believed to be critical in adhesion of P. aeruginosa to epithelial cells and colony expansion, and in facilitating biofilm formation. Sub-MIC concentrations of clarithromycin inhibit adherence of P. aeruginosa to cell surface pili and retard biofilm maturation in vitro [50].

CH3

O

CH3

CH3

OH

CH3

CH3 CH3 OH N O H3C

O

O

O

OH O

OH CH3

OH

O

O

CH3

CH3

Figure 2. Structure of macrolides (representative examples). a) 14-membered ring (erythromycin); b) 15-membered ring (azithromycin); and c) 16membered ring (spiramycin I). Reproduced from [37] with permission from the publisher.

229

However, it was in the 1980s when use of macrolides revolutionised the treatment of diffuse panbronchiolitis (DPB) that their immunomodulatory properties came under closer scrutiny. DPB is an idiopathic inflammatory airway condition found almost exclusively within the South East Asian populations (especially in Japan), which histologically is characterised by intense neutrophilic inflammation of the bronchioles [60]. Its typical onset is in the second to fifth decade of life which, when untreated, progresses to severe bronchiectasis, chronic airway infection and

ultimately respiratory failure. Prior to the introduction of macrolides in the mid 1980s, 10-year survival rates were low (,33%) [61], and even lower in those patients with chronic P. aeruginosa infection [62]. Since the introduction of erythromycin and subsequently other macrolides, survival has improved dramatically achieving 10-year survival rates .90% [61].

Immunomodulatory properties Herin we briefly review the supportive evidence with more comprehensive reviews in the literature [63, 64]. While anti-inflammatory actions of macrolides are well established, the differences seen in some studies are probably attributable to variance in methodology, model system used and the macrolide agent studied.

ANTI-INFLAMMATORY THERAPY

Endotoxins produced by invading bacteria stimulate human epithelial cells both directly and through toll-like receptors, triggering an inflammatory cascade leading to the activation of nuclear factor (NF)-kb [65]. NF-kb is central in regulating transcription of genes which encode proinflammatory mediators, including IL-6, IL-8, TNF-a (cytokines) and the intercellular adhesion molecule-1 (ICAM-1). In vitro studies have demonstrated both erythromycin and clarithromycin to be capable of inhibiting NF-kb activation [66, 67] and complimentary studies have independently demonstrated release of lower levels of IL-1, IL-6, IL-8 and ICAM-1 from activated bronchial epithelial cells when exposed to macrolides [68–70]. Neutrophils recruited to the site of inflammation become activated allowing phagocytosis of microorganisms and production of proteases (including neutrophil elastase and matrixmetalloproteinases (MMP)-9), and reactive oxygen species (ROS) responsible for the ‘‘oxidative burst’’ believed to be fundamental to killing the phagacytosed microorganism [71, 72]. In the setting of infection, spillage of these proteases and ROS from necrotic neutrophils contributes towards localised tissue damage and provides ongoing stimulus to the inflammatory process. Macrolides are able to modulate neutrophil function by several mechanisms. In an animal model of bronchiectasis, macrolides inhibit ICAM-1 expression which may reduce neutrophil migration to the site of inflammation [64]. Various 14-membered macrolides have been shown to inhibit the oxidative burst [72] and similarly erythromycin and flurythromycin inhibit the release of neutrophil elastase [73]. Interestingly, macrolides are associated with increased neutrophil degranulation [63]. A shortterm study of the effect of azithromycin (3 days) in healthy volunteers demonstrated an immediate increase in neutrophil degranulation and circulating ROS, but decreased IL-8. This was followed by a delayed inhibitory effect on oxidative burst, myeloperoxidase, IL-6 and increased neutrophil apoptosis [74]. These in vitro studies provide impetus for studying the potential impact of macrolides on neutrophil dominated airway diseases such as bronchiectasis.

Macrolides and mucus hypersecretion Mucus hypersecretion is a hallmark of bronchiectasis, which in combination with impaired mucociliary clearance produces a local environment conducive to chronic infection. Mucins (macromolecular glycoproteins) are major constituents of mucus and are encoded by a number of genes. One such gene, MUC5AC is specifically expressed by bronchial epithelial goblet cells [75] and in vitro studies demonstrate erythromycin and clarithromycin attenuate lipopolysaccharide-induced increased MUC5AC gene expression [64]. Azithromycin demonstrates similar effects on the MUC5AC gene in P. aeruginosa quorum sensing mediator stimulated human epithelial cells [76]. These effects are supported by in vivo responses to macrolides in varied animal models [77, 78].

230

In summary, the potential benefits of macrolide therapy in patients with bronchiectasis may result from antimicrobial properties and effects on biofilm development in patients with P. aeruginosa infection, by down-regulating acute and chronic inflammatory responses and limiting mucus hypersecretion.

Clinical trials of macrolides To date there have been limited studies examining the effectiveness of macrolides for treatment of non-CF bronchiectasis. Those published studies have been performed in small patient populations and have varied considerably in study design including duration, dose and specific macrolide, outcome measures and whether a control group was used as a comparator (table 2). The first double-blind, placebo-controlled RCT of macrolides in non-CF bronchiectasis compared the effect of roxithromycin (4 mg?kg-1 b.i.d.)/placebo for 12 weeks in children with a clinical diagnosis of bronchiectasis and evidence of airway hyperreactivity [79]. There was a significant reduction in sputum purulence, leukocyte concentration and a reduction in airway reactivity (provocative dose causing a 20% fall in FEV1 to metacholine). However, there was no change in lung function when compared with placebo.

A double-blind RCT of erythromycin in adults with non-CF bronchiectasis compared 8 weeks of erythromycin (500 mg b.i.d., n514) with placebo (10 patients) during a period of clinical stability [81]. Three patients, each receiving erythromycin withdrew (adverse effect n51, poor adherence n52). A ‘‘per protocol’’ analysis based on those who completed the trial demonstrated an improvement in lung function (mean increase in FEV1 and forced vital capacity of 140 mL and 120 mL, respectively) and decreased sputum production in those receiving erythromycin. There were no differences in levels of inflammatory cytokines (IL-8, TNF-a or LTB4) in sputum. Several uncontrolled studies have also been reported. An open label, randomised, crossover study of 6 months of azithromycin 500 mg twice weekly and standard treatment in 12 patients (11 included in analysis) demonstrated a reduction in the number of exacerbations requiring antibiotics (five versus 16, p,0.019) and sputum volume during azithromycin therapy and no change in lung function [82]. Notably, the investigators aimed to recruit 30 subjects for the study based on pre-study power estimates.

D.J. SMITH ET AL.

In a second macrolide trial also in children with stable non-CF bronchiectasis, YALCIN et al. [80] compared the impact of clarithromycin with conventional treatment administered for 3 months on immune mediators within BALF. The study demonstrated greater reduction in sputum volume and BALF total cell counts, neutrophil ratios and IL-8 levels in the clarithromycin group. Interestingly, there was no significant change in sputum microbiology. This study had the major limitation of the lack of a placebo.

A prospective cohort study of azithromycin in adult patients with frequent pulmonary exacerbations (.4 in the year prior to enrolment), employed a treatment protocol of azithromycin 500 mg?day-1 for 6 days, then 250 mg?day-1 for 6 days, followed by maintenance treatment of 250 mg three times per week [83]. Six (15%) of the 39 patients recruited withdrew due to adverse effects. Analysis based on those who tolerated therapy demonstrated a reduction in exacerbation rate (from 0.71 to 0.13 per month, p,0.001), reduction in number of courses of antibiotics (0.08 to 0.003 per month, p,0.001) and a trend to improvement in lung function parameters. Respiratory symptoms improved in those treated with azithromycin over a mean follow-up period of 20 months (in-house symptom questionnaire). Finally a cohort study of 56 adult patients treated with azithromycin 250 mg three times per week, of which 50 patients completed a minimum of 3 months (mean duration 9.1 months), demonstrated a reduction in exacerbation rate and sputum production (compared with the 6 months prior to treatment) and an improvement in FEV1 (only 29 patients assessable) [84].

231

In summary, these small studies have demonstrated that macrolide therapy is generally well tolerated and reduces sputum volume, however, effect on pulmonary function is unclear. Several studies have reported significant participant dropout due to gastrointestinal adverse events. Routine use of macrolides cannot be supported based on current evidence and there is an urgent need for large randomised placebo controlled trials to assess tolerability, clinical impact, which

232

DBRCT Adult (mean Bronchiectasis Erythromycin (parallel) age 55 yrs) .10 mL (500 mg b.i.d.) sputum per 24 h

Open label Adult Bronchiectasis Azithromycin (crossover) (mean age (500 mg b.i.d.) 71 yrs)

Cohort

Cohort

China (Hong Kong)

USA

UK

UK

T SANG [81]

C YMBALA [82]

D AVIES [83]

A NWAR [84]

No

No

No

Yes

No

Yes

204 weeks

52 weeks (26 weeks each arm, 4 weeks washout) Mean 80 weeks

8 weeks

12 weeks

12 weeks

56

39

12

24

34

25

Placebo Duration Subjects n

Findings

Diarrhoea (n53)

Withdrew due to rash (n51)

None

None

Adverse events

Withdrew (n56); abnormal liver function (n52), diarrhoea (n52), rash (n51), tinnitus (n51) Exacerbation rates, Reduced sputum Withdrew lung function, volume, reduced (n56)"; diarrhoea (n53), abdominal sputum volume/ exacerbation rates, microbiology reduced positive sputum cramps (n52), skin rash (n52) microbial cultures

Exacerbation Reduced exacerbation rates, antibiotic rate, reduced antibiotic usage, lung function usage, improved DL,CO, no change in FEV1, FVC

Reduced sputum Sputum purulence/leukocyte purulence/WCC, counts, reduced airway FEV1, PD20 metacholine reactivity, fall in FEV1 Sputum volume, Reduced sputum lung function, volume, reduced BALF BALF (leukocyte neutrophil ratio, IL-8, counts, microbial increased FEF25–75, No change in FEV1 cultures, IL-8, IL-10, TNF-a) 24 h sputum Reduced sputum (volume/WCC/ volume, improved FEV1 and FVC, no change in microbial concentrations/ microbial concentration, immune mediators#), no change in immune lung function mediators Sputum volume, Reduced sputum exacerbation volume, reduced rates, lung function exacerbations, no change in lung function

Outcome measures

DBRCT: double-blind randomised controlled trial (RCT); b.i.d.: twice daily; WCC: white cell count; FEV1: forced expiratory volume in 1 second; PD20: provocative dose causing a 20% fall in FEV1; BALF: bronchoalveolar lavage fluid; IL: interleukin; TNF: tumour necrosis factor; FVC: forced vital capacity; FEF25–75%: forced expiratory flow at 25–75% FVC; q.d.: once daily; DL,CO: diffusing capacity of the lung for carbon monoxide; MWF: Monday, Wednesday, Friday. #: immune mediators: IL-1a, TNF-a and leukotriene B4; ": seven adverse events in six patients.

Adult Bronchiectasis, Azithromycin (18–77 yrs) .4 (500 mg q.d. 6 days, exacerbations prior 52 weeks 250 mg q.d. 6 days, 250 mg MWF) Adult Bronchiectasis, Azithromycin (mean age o3 (250 mg MWF) 63 yrs) exacerbations prior 26 weeks

Children Bronchiectasis, Clarithromycin (7–18 yrs) no antibiotics in (15 mg?kg-1 b.i.d.) prior 16 weeks

RCT (parallel)

Turkey

Drug

Children Bronchiectasis, Roxithromycin (mean age airway (4 mg?kg-1 b.i.d.) 13 yrs) hyperreactivity

Inclusion criteria

Y ALCIN [80]

DBRCT (parallel)

Population

South Korea

Country Design

K OH [79]

Study

Table 2. Clinical trials of macrolide therapy in bronchiectasis

ANTI-INFLAMMATORY THERAPY

macrolide is most beneficial and to assess the risk of macrolide resistant infections. This latter point is important given the emerging evidence of macrolide resistance in Europe [85–87] and in the CF population [88–90]. Several studies have either recently been completed, are actively recruiting or about to commence, which will hopefully address some of these important issues (table 3).

HMGcoA reductase inhibitors HMGcoA reductase inhibitors (‘‘statins’’) have established clinical utility as lipid lowering agents in patients with hyperlipidaemia. They also have widely recognised anti-inflammatory and immunomodulatory properties. In vitro studies of HMGCoA reductase inhibitors have demonstrated inhibition of neutrophil migration and epithelial cell production of chemoattractants and proteases and potentiation of macrophage efferocytosis [72].

There are currently no studies of the use of HMGCoA reductase inhibitors for bronchiectasis, however, the findings of the studies in other airway diseases suggest that future studies are worthwhile.

Targeted agents There are currently no phase III trials of targeted therapies in inflammatory airway diseases, however, a number of potential candidate agents specifically targeting neutrophilic inflammation are under investigation.

D.J. SMITH ET AL.

In animal models of COPD, simvastatin has been shown to inhibit airway remodelling, lower TNF-a and MMP-9 levels and reduce peribronchial and perivascular inflammation [91, 92]. A recent systematic review identified nine studies using HMGCoA reductase inhibitors in patients with COPD [93], however, only one of these was a prospective RCT. Collectively, these studies demonstrated beneficial effects on pulmonary function, exacerbation rates and mortality and provide the foundation for further study. Large, prospective RCTs are currently underway. Studies in asthmatic subjects have yielded more variable results. Reduction in airway hyperreactivity has been seen in one study [94], no benefit in another [95] and one retrospective review even suggested HMGCoA reductase inhibitor use was associated with poorer clinical outcomes [96]. A recent placebo-controlled, double-blind RCT of simvastatin 40 mg?day-1 in patients with steroid responsive (eosinophilic) asthma failed to demonstrate any clinically significant steroid sparing effect from the addition of simvastatin [97].

The CXC chemokines and their associated receptors (CXCR1/CXCR2) are believed to have a key role in neutrophilic inflammation in pulmonary disease and recently a number of agents which inhibit this pathway have been developed [98]. A phase II study of an anti-CXCL8 monoclonal antibody in COPD has demonstrated safety and improvement in dyspnoea scores over 3 months [99]. In a complimentary in vitro study ELR-CXC antagonists inhibited neutrophil chemotactic factors in the sputum of bronchiectatic patients [100]. These studies suggest that further investigation of these agents may be valuable. Anti-TNF-a agents have an established role in treatment of systemic inflammatory diseases, including rheumatoid arthritis [101] and Crohns disease [102]. In short-term trials of anti-TNF-a agents in inflammatory lung diseases variable efficacy has been reported. While improvement in exacerbation rates in asthma have been demonstrated [103], no effect was seen in patients with COPD [104]. The major concerns associated with the use of these agents in patients with pulmonary disease are the potential for the emergence of opportunistic infections, in particular the re-activation of mycobacterial disease [105] and their possible association with acute deterioration of fibrotic lung disease [106].

233

With the emerging array of anti-inflammatory monoclonal antibodies and targeted receptor blocker drugs, new therapeutic options will potentially become available. Carefully conducted trials will be required to support the use and examine adverse consequences. Although manipulation of the immune response is an attractive prospect for treatment of a range of

234

DBRCT (parallel)

DBRCT (factorial design), stratified by P. aeruginosa status

Australia

DBRCT (parallel), stratified by P. aeruginosa status

DBRCT (parallel)

DBRCT (parallel)

Design

New Zealand

The Netherlands

Australia

International multicentre study (Australia, New Zealand)

Country

Confirmed bronchiectasis (HRCT)

Confirmed bronchiectasis (HRCT), o2 exacerbations in prior 52 weeks, daily productive cough, clinically stable (4 weeks)

o1 pulmonary exacerbation prior 52 weeks, confirmed bronchiectasis or chronic SLD

Inclusion criteria

Azithromycin (250 mg q.d.)

140

130

26 weeks

72

26 weeks

52 weeks

118

Erthromycin (400 mg b.i.d.)

48 weeks

Subjects n 88

Duration

104 weeks Azithromycin (30 mg?kg-1?week-1)

Drug

Confirmed Azithromycin bronchiectasis (HRCT), (500 mg MWF) clinically stable, o1 exacerbations in prior 52 weeks Adults (18– Bronchiectasis (HRCT + Azithromycin 80 yrs clinical), clinically (250 mg q.d.) or including stable, o2 weeks hypertonic saline 7% or both indigenous since antibiotics for adults) exacerbation

Adults (18– 80 yrs)

Adults (.18 yrs)

Adults (18–80 yrs)

Indigenous children (1–8 yrs)

Population

Completed

Study completed, yet to report

Ongoing

Exacerbations Study completed, (time to first/rate/severity), yet to report change in lung function, HRQoL, change in sputum cell count HRQoL, exacerbation rate, Recruitment to change in lung function, commence change in symptoms score, early 2011 change in airway microbiology, sputum inflammatory markers, adverse events

Exacerbation rate, change in lung function, change in symptom scores, change in airway microbiology, sputum inflammatory markers, HRQoL, adverse events

Exacerbation rate, antibiotic usage, HRQoL, sputum volume/inflammatory markers

Exacerbations (time to first/ Recruitment until rate/severity), safety/adverse Dec 2010 events, antimicrobial resistance

Outcome measures

BIS: bronchiectasis intervention study; BLESS: bronchiectasis and low-dose erythromycin study; BAT: bronchiectasis and long-term azithromycin treatment; EMBRACE: effectiveness of macrolides in patients with bronchiectasis using azithromycin to control exacerbations; DBRCT: double-blind randomised controlled trial; SLD: suppurative lung disease; HRCT: high-resolution computed tomography; b.i.d.: twice daily; HRQoL: health-related quality of life; q.d.: once daily; P. aeruginosa: Pseudomonas aeruginosa; MWF: Monday, Wednesday, Friday.

EMBRACE

BAT

BLESS

BIS

Study acronym

Table 3. Registered trials of macrolide therapy in bronchiectasis

ANTI-INFLAMMATORY THERAPY

inflammatory medical conditions, history advocates caution. In March 2006, six healthy volunteers enrolled in a phase I trial were administered a first-in-man anti-CD28 humanised monoclonal antibody (TG1412) designed to modulate regulatory T-cells. Within hours of administration each volunteer experienced a severe cytokine storm resulting in multi-organ failure [107]. Although all six survived, the most severely affected subject required intensive care support for 3 weeks. Similarly, in a recent study in children and adults with CF the use of an LTB4 antagonist (BIIL284) resulted in increased respiratory exacerbations resulting in the study being prematurely terminated after interim data analysis [108]. These studies highlight that in conditions characterised by infection associated with inflammation, anti-inflammatory therapies may be associated with adverse consequences and require very careful and detailed analysis.

Conclusion Evidence for the use of anti-inflammatory therapies in bronchiectasis is limited and more adequately powered studies are required [109, 110]. There is currently insufficient evidence to support the use of inhaled and oral corticosteroids, NSAIDs and macrolides. Individual patient trials may be warranted for inhaled corticosteroids and macrolides and other therapies remain unproven with no evidence to support use as anti-inflammatory therapy in bronchiectasis.

Statement of interest

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

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Chapter 16

Pharmacological airway clearance strategies in bronchiectasis P.T. Bye*,#,", E.M.T. Lau*,#," and M.R. Elkins*,"

Impaired mucociliary clearance and mucus retention contribute to the chronic cycle of airway inflammation, infection and damage in bronchiectasis. There is a strong rationale for the use of pharmacological strategies to aid airway clearance, often in combination with chest physiotherapy. Despite the availability of many candidate mucoactive agents, the evidence base for recommending these agents is currently limited. Recent research and trials have focused particularly on osmotic agents (hypertonic saline and mannitol), which increase airway hydration, and early studies appear promising for both of these agents. Dornase alfa is not effective in non-cystic fibrosis (CF) bronchiectasis, which underscores the importance of conducting high quality and adequately powered trials that specifically address the therapeutic options for non-CF bronchiectasis. Keywords: Bronchiectasis, mucoactive, mucociliary clearance, mucus

*Dept of Respiratory and Sleep Medicine, Royal Prince Alfred Hospital, # Sydney Medical School, University of Sydney, Camperdown, and " Woolcock Institute of Medical Research, Glebe, Australia. Correspondence: P.T. Bye, Dept of Respiratory and Sleep Medicine, Royal Prince Alfred Hospital, Missenden Road, Camperdown, NSW 2050, Australia, Email [email protected]

P.T. BYE ET AL.

Summary

Eur Respir Mon 2011. 52, 239–247. Printed in UK – all rights reserved. Copyright ERS 2011. European Respiratory Monograph; ISSN: 1025-448x. DOI: 10.1183/1025448x.10004610

N

239

on-cystic fibrosis (CF) bronchiectasis is a heterogeneous disorder defined by irreversible dilatation of the airways [1]. Although a wide variety of underlying pathological processes can initiate the development of bronchiectasis, the final common pathophysiological pathway is one characterised by the vicious cycle of chronic infection and inflammation leading to progressive airway damage [2]. Impaired mucociliary clearance is a feature of the abnormal bronchiectactic airway [3, 4], and may represent the primary abnormality in conditions such as primary ciliary dyskinesia. Mucus retention, the result of defective mucociliary clearance, not only produces the classic symptom of chronic productive cough but also causes airflow obstruction and ventilation/ perfusion mismatch and forms a nidus for ongoing infection. Therefore, interventions aimed at promoting clearance of excess mucus may be beneficial in patients with non-CF bronchiectasis.

The normal mucociliary escalator forms an essential element of the innate host defence mechanism against inhaled pathogens. The complex physiology of mucociliary clearance in health and disease has been reviewed in detail elsewhere [5–7]. Briefly, this process is dependent upon normal ciliary function, optimal rheological properties of the airway mucus and an adequate volume of airway surface liquid (ASL). The lung has the additional mechanism of cough for airway mucus clearance, although the effectiveness of cough clearance itself is also dependent upon the viscoelastic properties of mucus [8]. Agents that are intended to facilitate airway mucus clearance are termed mucoactive drugs. A classification of mucoactive agents, based on their mechanism of action, is summarised in table 1. Despite these agents having been available for many years, limited high-quality clinical trials have been undertaken exploring the efficacy of mucoactive agents in non-CF bronchiectasis. Indeed, since the mid-2000s, multiple authors have called for a coordinated approach in order to establish multicentre clinical trials and for funding bodies to consider support for this disease, highlighting the huge unmet needs in non-CF bronchiectatic therapy [9–11]. The present chapter reviews the current pharmacological strategies available for enhancing airway clearance in non-CF bronchiectasis.

Hypertonic saline

PHARMACOLOGICAL AIRWAY CLEARANCE

Hypertonic saline is a sterile salt solution with a higher concentration of salt (typically 3–7%) than plasma (0.9%), and is delivered by inhalation via a nebuliser. Hypertonic saline accelerates mucociliary clearance in both healthy subjects and patients with cystic fibrosis (CF), as demonstrated in radioaerosal studies [12–15]. It is thought to enhance airway clearance by altering the viscoelastic properties of mucus, increasing hydration of the ASL and also directly stimulating cough [15–18]. The hydrating effect of hypertonic saline on mucociliary function has been best characterised in the CF airway. In health, ASL is present as a bilayer, with a superficial mucus layer and a layer of periciliary liquid (PCL) interposed between the mucus and the epithelium. The PCL layer approximates the height of the cilia and provides a low-viscosity fluid in which the cilia beat [5]. A critical depth of PCL is crucial for ciliary function and mucociliary transport [6]. CF transmembrane conductance regulator dysfunction leads to airway dehydration and depletion of the PCL layer of the ASL [19]. The addition of hypertonic saline to the CF epithelium rapidly restores the depth of the ASL by creating an osmotic gradient and drawing water across the Table 1. Common mucoactive drugs and their mechanisms of action Agent Expectorants Hypertonic saline Mannitol Mucolytics N-Acetylcysteine Nacystelyn Dornase alfa Mucoregulators Carbocisteine Glucocorticoids Macrolide antibiotics Anticholinergics Mucokinetics b2-Agonists Surfactant

240

Modified from [8].

Predominant mechanism Increases airway hydration; stimulates cough

Interrupts disulfide bonds linking mucin polymers; anti-inflammatory and antioxidant effects Interrupts disulfide bonds; increases chloride secretion Cleaves DNA polymers Modulates mucus content; anti-inflammatory and antioxidant effects Reduces airway inflammation and mucin secretion Reduces airway inflammation and mucin secretion Decreases volume of secretions Increases cilia beat frequency; improves cough clearance by increasing expiratory flows Decreases mucus adherence to epithelium

respiratory epithelium [15]. Restoration of the depth of ASL not only optimises ciliary function but also causes excess water entering the airway to be stored in the mucus layer, making its rheological properties more favourable for clearance [18]. The efficacy of long-term inhalation (48 weeks) of hypertonic saline has previously been demonstrated in a randomised placebo-controlled trial for patients with CF [20]. Regular hypertonic saline inhalation significantly improved lung function and reduced pulmonary exacerbations. These changes were accompanied by prescription of fewer courses of antibiotics, reduction in absenteeism from school and work, and improved quality of life. A recent Cochrane review, which included 12 trials (442 participants aged 6–46 years), indicated that hypertonic saline is a safe, low-cost and effective therapy in CF [21].

More recently, NICOLSON et al. [23] reported, in abstract form, the results of a randomised controlled trial on the effect of long-term hypertonic saline inhalation. A total of 40 patients were randomised to hypertonic saline (6%) or isotonic saline (0.9%) though an Aeroneb1 Go nebuliser (Aerogen, Galway, Ireland) twice daily for 12 months while performing the ACBT. The mean forced expiratory volume in 1 second (FEV1) of the study group was 83% of the predicted value. No differences in lung function, number of exacerbations or quality of life were observed at 3, 6 and 12 months between the hypertonic and isotonic saline groups. Both the hypertonic saline and isotonic saline groups demonstrated clinically significant improvement in health-related quality of life compared to baseline. However, this study was substantially underpowered to examine the effect of hypertonic saline relative to isotonic saline. As clinically worthwhile benefits were not excluded by the confidence intervals (CIs), further investigation of this promising agent is warranted.

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Preliminary evidence suggests that hypertonic saline may be clinically effective in non-CF bronchiectasis. In a randomised crossover trial, KELLETT et al. [22] evaluated the effect of hypertonic saline as an adjunct to physiotherapy in 24 stable bronchiectatic patients. Subjects were allocated to receive four different single-session treatments in random order: 1) active cycle of breathing technique (ACBT) alone, 2) nebulised terbutaline followed by ACBT, 3) nebulised terbutaline followed by isotonic saline (0.9%) and then ACBT, and 4) nebulised terbutaline followed by hypertonic saline (7%) and then ACBT. Each single-treatment session was followed by a 1-week washout period. When hypertonic saline was used, physiotherapy yielded greater sputum weight, increased the ease of sputum expectoration and reduced sputum viscosity. Although encouraging, this study has clear limitations. The study only included patients who were minimal sputum producers (,10 g?day-1), a phenotype which is clearly distinct from high sputum producers. Patient blinding was incomplete (taste masking not performed), and the results only represented the effect of a single treatment dose.

Hypertonic saline appears to be well tolerated by patients with bronchiectasis. In 50 administrations of hypertonic saline to patients with acute exacerbations, no major bronchoconstriction (fall in FEV1 of .20%) or oxygen desaturation occurred [24]. Routine premedication with a bronchodilator is recommended (typically 200–400 mg salbutamol delivered via a spacer device). We generally recommend that spirometry is performed and oxyhaemoglobin saturation measured before and after delivery of the first dose.

Mannitol

241

Mannitol is a six-carbon monosaccharide (sugar alcohol), and is commercially available in an encapsulated stable dry powder formulation for inhalation. Similar to hypertonic saline, creation of an osmotic gradient causing influx of water into the airway and increasing the ASL layer is considered to be its primary mechanism of action [25]. In addition, mannitol may cause the release of mediators that may stimulate ciliary beat frequency [26, 27], although direct evidence that mannitol stimulates the cilia has not been established. Mannitol may also alter the viscoelastic properties of mucus by breaking the hydrogen bonds between mucins [28]. Mannitol (160–480 mg) increases mucociliary clearance in a dose-dependent manner in radioaerosal studies [29–31].

In a phase-3 randomised double-blind placebo-controlled trial in CF, inhalation of mannitol (400 mg b.i.d.) for 6 months resulted in an early and sustained improvement in FEV1 compared to placebo (118 mL change from baseline to week 26; p,0.001) [32]. The benefit in FEV1 was seen irrespective of the concurrent use of dornase alfa. The study was not sufficiently powered to show a reduction in the secondary end-point of exacerbations. Results from the 12-month open-label phase of the study have also been reported. The improvement in lung function with mannitol appeared to be maintained for up to 18 months of treatment [33]. The full results of this study are yet to be published. There is emerging evidence that mannitol is an effective treatment in non-CF bronchiectasis. In an open-label pilot study, DAVISKAS et al. [34] treated nine patients with bronchiectasis with 400 mg mannitol daily for 12 days. Lung function was unchanged by treatment apart from an improvement in forced expiratory flow (FEF). However, health-related quality of life had improved at the end of the treatment period and was maintained for 1 week thereafter. Mannitol reduced the surface tension, increased the wettability and reduced the cohesiveness and solids content of sputum. Cough transportability, measured by an in vitro simulated cough machine, also increased. All subjects tolerated treatment well, without report of any adverse events.

PHARMACOLOGICAL AIRWAY CLEARANCE

A phase-3 multicentre randomised controlled trial has recently been completed and its data presented in abstract form [35]. Subjects with bronchiectasis and mild-to-moderate lung function impairment (FEV1 of .50% pred and o1 L) were randomised to 320 mg inhaled mannitol (n5185) or placebo (n595), given twice daily for 3 months. Subjects treated with mannitol exhibited a significant reduction in the St George’s Respiratory Questionnaire total score of 3.9 units compared to 2.0 units in the placebo group. In the mannitol group, the time to first antibiotic use was longer and total antibiotic use was less than for placebo. The full report of this study is awaited with interest.

Dornase alfa Dornase alfa is a proteolytic enzyme that cleaves DNA polymers [8]. DNA is released into the airway mucus in large amounts by degenerating neutrophils, and neutrophilic inflammation is a feature of both CF and non-CF bronchiectasis. Purulent airway secretions, particularly in CF, show an abundance of highly polymerised DNA, which contributes to mucus hyperviscosity and adhesiveness [36]. Daily inhalation of dornase alfa is a well-established therapy in CF bronchiectasis, resulting in improvement in lung function and reduction in exacerbations, in both mild and severe disease [37–40]. In contrast, clinical studies in non-CF bronchiectasis have shown that dornase alfa is of no benefit, and may even be harmful. In a short-term study of WILLS et al. [41], dornase alfa was not associated with any improvement in lung function and quality-of-life measures in patients with non-CF bronchiectasis. Indeed in vitro sputum transportability fell following the addition of dornase alfa to non-CF bronchiectatic sputum. A subsequent international multicentre study randomised 349 patients with stable non-CF bronchiectasis to either dornase alfa or placebo over a 24-week period (and remains the largest therapeutic trial in non-CF bronchiectasis to date) [42]. Pulmonary exacerbations were more frequent, and FEV1 decline was greater in patients who received dornase alfa. The reasons for this difference in response between patients with CF and non-CF bronchiectasis remain unclear. The biological rationale for the use of dornase alfa in non-CF bronchiectasis was strong, but the unexpected detrimental finding highlights the importance of performing welldesigned studies that address the therapeutic options for non-CF bronchiectasis, rather than merely extrapolating the results of trials involving patients with CF.

N-Acetylcysteine, carbocisteine and other thiol derivatives 242

N-Acetylcysteine (NAC) is the classic mucolytic agent, and disrupts the disulfide bonds in mucus when delivered via the aerosolised route [8]. In addition to reducing sputum viscosity, NAC

demonstrates antioxidant, anti-inflammatory and potentially antibacterial properties [43–45]. NAC exhibits extremely low bioavailability, and is not readily detectable in bronchoalveolar lavage fluid following oral administration [46]. Thus the mechanism of action of oral NAC is unlikely to be mediated via its mucolytic properties. Carbocisteine, although commonly regarded as a mucolytic, has a mechanism of action that differs from that of the classical mucolytics. Mucus produced under the action of carbocisteine shows an increase in sialomucin content. Sialomucins, which are structural components of mucus, influence the viscoelastic properties of mucus [47]. Similar to NAC, carbocisteine also exerts anti-inflammatory actions, and, in pre-clinical studies, it has been shown to decrease levels of the cytokines interleukin (IL)-6 and IL-8 and reduce neutrophil influx into the airway lumen [48, 49].

The evidence supporting the use of NAC and thiol derivative in bronchiectasis is even more limited. There are several studies of oral and inhaled NAC in CF, but most studies have only evaluated changes in the rheologicaal properties of CF sputum [52]. The few controlled clinical studies in CF performed to date have consistently shown no clinical benefit [53–55]. There are currently no well-designed studies of NAC and thiol derivatives in non-CF bronchiectasis. This is supported by a Cochrane review, which concluded that there is insufficient evidence to evaluate the routine use of these agents in non-CF bronchiectasis [56].

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The majority of clinical studies of NAC and thiol derivatives have been performed in chronic obstructive pulmonary disease (COPD), with conflicting results. The Bronchitis Randomized on NAC Cost–Utility Study (BRONCUS), which randomised 523 patients (Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage 2 and 3) to 600 mg oral NAC daily or placebo, showed that NAC was ineffective at reducing pulmonary exacerbations and decline in lung function over a 3-year period [50]. This is in contrast to the large Chinese Preventive Effects on Acute Exacerbations of COPD with Carbocisteine (PEACE) study, which randomised 709 patients (GOLD stage 2, 3 and 4) to receive carbocisteine or placebo for 1 year [51]. The primary end-point of exacerbation rate over the 1-year period was met, with carbocisteine demonstrating a significant reduction in exacerbations (risk ratio 0.74; 95% CI 0.61–0.89). The discrepant findings between these two large randomised controlled trials may have been explained by the different rates of inhaled corticosteroid usage (less in the PEACE study) and phenotypic differences in COPD across ethnicities.

Bronchodilators b2-Agonists are commonly prescribed to treat airflow obstruction and bronchial hyperreactivity, and as an adjunct to physiotherapy in patients with bronchiectasis. Between 20 and 46% of patients with bronchiectasis display bronchodilator reversibility [57, 58]. b2-Agonists may facilitate airway clearance by increasing ciliary beat frequency via stimulation of b2-receptors and downstream increase in cyclic adenosine monophosphate (cAMP) signalling [59]. cAMP is a regulator of ciliary beat frequency in human airway epithelia [60, 61]. The bronchodilatory effect of b2-agonists may serve to increase expiratory flow rates and thus enhance cough clearance. Two small studies have demonstrated that nebulised terbutaline, given immediately prior to physiotherapy, yields greater sputum production [22, 62], and also improved mucociliary clearance in a radioaerosal study [62]. Although it seems reasonable and logical that b2-agonists be used to treat airflow limitation (particularly if objective bronchodilator reversibility is demonstrated), and as an adjunct to chest physiotherapy in non-CF bronchiectasis, this is currently not supported by the evidence. The relevant Cochrane reviews found no randomised controlled trials of the use of shortacting or long-acting b2-agonists in non-CF bronchiectasis [63, 64].

Surfactant 243

A thin layer of airway surfactant phospholipid separates the PCL layer and the mucus gel layer, and effectively functions as a lubricant to facilitate mucus transport [8]. Furthermore, depletion of

the PCL layer leads to entanglement and adhesion of mucus to the underlying epithelial surface. Surfactant is a potential therapeutic candidate for enhancing mucociliary clearance by reducing the molecular interactions that bind mucus to the airway. Patients with CF display alterations in the composition of the pulmonary surfactant system, with a reduction in the surface-active fractions, such as phosphatidylcholine and phosphatidylglycerol [65, 66]. This suggests that surfactant dysfunction may contribute to impaired mucociliary function in CF. Preliminary clinical studies of exogenous surfactant therapy have only been performed in COPD and CF populations. A single randomised controlled trial of 66 patients with COPD and symptoms of chronic bronchitis showed that aerosolised surfactant for 2 weeks increased in vitro sputum transportability, improved FEV1 and forced vital capacity (FVC) by .10%, and reduced gas-trapping [67]. The result of a phase-2 study of pulmonary surfactant in CF was recently reported in abstract form. In this placebo-controlled crossover trial, 16 subjects (aged .14 years and with an FEV1 of .40% pred) were assigned to five doses of nebulised surfactant or five doses of nebulised saline (0.9%) over a 24-hour period, with a washout period of 2 weeks. Aerosolised surfactant was well tolerated and not associated with any serious adverse events. No difference in mucociliary clearance (quantified by radioaerosal labelling) was observed between surfactant and saline (0.9%) treatment.

PHARMACOLOGICAL AIRWAY CLEARANCE

Humidification Humidification is commonly used to relieve sputum retention. CONWAY et al. [68] performed a small crossover study evaluating the role of humidification as an adjunct to chest physiotherapy in seven subjects with moderate-to-severe bronchiectasis. Humidification with cold water via a jet nebuliser for 30 minutes prior to chest physiotherapy was compared to chest physiotherapy alone. Radioaerosal clearance and sputum weight both increased when humidification was performed prior to chest physiotherapy. In a recent study of REA et al. [69], long-term domiciliary humidification was evaluated in a randomised placebo-controlled trial. A total of 108 subjects with COPD (n563) or bronchiectasis (n545) were randomly assigned to humidification or usual care for 12 months. Fully saturated humidified air at 37uC was delivered via nasal cannulae at a flow rate of 20–25 L?min-1 via a humidifier and flow source. Patients were encouraged to use humidification for o2 hours?day-1. The primary end-point of the study, exacerbation frequency during the study period, was nonsignificant but showed a trend favouring the humidification group (3.36 versus 2.97; p50.067). However, patients on long-term humidification therapy showed significantly fewer exacerbation days and increased time to first exacerbation compared to usual care. Quality-of-life scores and lung function had also improved significantly with humidification therapy at 3 and 12 months. The authors hypothesised that improvement in mucociliary clearance with humidification was one of the main mechanisms accounting for the observed benefit. The limitations of this study include the absence of a placebo, which resulted in subjects and investigators being unblinded to the intervention assignment. The study population included both COPD and bronchiectasis, which are clearly two very distinct disorders. Compliance with therapy was poor (mean 1.6 hours?day-1), but, despite this, the secondary outcomes of the study were still significantly in favour of humidification therapy. The high flow rate of the humidification system was equivalent to the delivery of 1– 3 cmH2O of positive end-expiratory pressure (PEEP). PEEP, even at this low pressure, may by physiologically relevant in reducing the work of breathing by offsetting intrinsic PEEP, recruiting alveolar units to improve ventilation/perfusion matching and providing partial stabilisation of the upper airway if used during sleep. Thus the mechanisms via which long-term high flow humidification might be beneficial in obstructive airways disease remain uncertain.

Conclusion 244

Bronchiectasis is increasingly recognised as a major cause of respiratory morbidity. Research projects are required in order to establish therapies for this under-investigated, under-recognised and

undertreated disease. Such trials should focus on the experimental agent’s effects on quality of life, use of healthcare resources and participation. Hypertonic saline, NAC and carbocisteine are promising candidates for such trials. There is proof of concept for the use of bronchodilators in combination with physiotherapy, but trials with clinically important outcome measures are needed. Mannitol appears effective, but clinicians must await publication of the full results of the most recent trials and commercial availability of the dry powder formulation. Humidification also appears effective. Dornase alfa has detrimental effects and should not be used in non-CF bronchiectasis.

Statement of interest M.R. Elkins has received financial assistance for travel to the European Cystic Fibrosis Conference from Praxis Pharmaceuticals.

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Chapter 17

Surgery for bronchiectasis D.C. Mauchley* and J.D. Mitchell*,#

SURGICAL MANAGEMENT

Summary Surgical resection for bronchiectasis should be reserved for patients with localised disease who have failed medical management and have persistent symptoms that negatively affect their quality of life. Patients with unilateral segmental disease have the best outcomes. The key to successful surgical intervention includes: 1) complete resection of all affected areas; 2) relatively early intervention to prevent development of resistant organisms and spread to adjacent lung segments; 3) pre-operative targeted antimicrobial therapy based on in vitro sensitivities; 4) continuation of antimicrobial therapy postoperatively; 5) pre-operative nutritional supplementation when indicated; and 6) anticipation of potential complications that may alter the surgical approach. Surgical resection can be accomplished with minimal morbidity and mortality and it can usually be completed with a video-assisted thoracoscopic approach. The only surgical option for diffuse bronchiectasis is bilateral lung transplantation and is mainly employed when treating patients with cystic fibrosis. Keywords: Bronchiectasis, lobectomy, lung transplantation, pulmonary infections, segmentectomy, video-assisted thoracic surgery

*Dept of Surgery, Division of Cardiothoracic Surgery, Section of General Thoracic Surgery and Center for the Surgical Treatment of Lung Infections, University of Colorado Denver, Aurora, and # National Jewish Health, Denver, CO, USA. Correspondence: J.D. Mitchell, Section of Thoracic Surgery, Division of Cardiothoracic Surgery, C-310, University of Colorado, Denver School of Medicine, 12631 E. 17th Avenue, C310, Aurora, CO 80045, USA, Email [email protected]

Eur Respir Mon 2011. 52, 248–257. Printed in UK – all rights reserved. Copyright ERS 2011. European Respiratory Monograph; ISSN: 1025-448x. DOI: 10.1183/1025448x.10004710

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ince its first description by LAENNEC [1] in 1819, bronchiectasis continues to be recognised as a cause of considerable respiratory illness. This disease is characterised by abnormal dilation of bronchi and is usually the result of recurrent pulmonary infections. Patients suffer from chronic cough, excessive sputum production, a progressive decline in respiratory function and haemoptysis that can be life threatening. The majority of patients can be treated medically, but those that fail or become intolerant of medical treatment may be eligible for surgical management.

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Initial attempts at surgical treatment of bronchiectasis were fraught with complications. Postoperative bronchopleural fistula (BPF) and empyema occurred in f50% of cases [2, 3]. Perioperative mortality was as high as 46% [3]. By 1950, the introduction of effective antibiotics in addition to improvements in surgical technique led to a dramatic decline in perioperative morbidity and mortality. Currently, surgical intervention is mainly reserved for patients with focal disease that remain symptomatic despite optimal medical management. Diffuse bronchiectasis may be treated with bilateral lung transplantation and is mainly limited to patients with cystic fibrosis (CF).

General principles Once thought to be in decline, the incidence of non-CF related bronchiectasis is now felt to be on the rise in North America and throughout the world [4]. Patients present with recurrent pulmonary infections accompanied by copious sputum production and occasional bouts of haemoptysis. Traditional treatment paradigms have consisted of rotating schedules of targeted antibiotic therapy along with manoeuvres to promote secretion clearance. Surgical resection for bronchiectasis is reserved for patients who demonstrate disease progression despite optimal medical treatment, or become intolerant of medical therapy. Failure of such treatment represents the most common reported indication for surgical resection [5–13]. The basic concept behind surgical resection for bronchiectasis is to remove permanently damaged areas of lung parenchyma that antibiotics penetrate poorly, and thus serve as a reservoir for microbes leading to recurrent infection. Resection of diseased segments will alter the pattern of repeated bouts of infection, and provide significant symptom relief regarding cough and excess sputum production. Patients with concomitant cavitary lung disease or recurrent bouts of haemoptysis may also benefit from surgery.

Medical therapy should always be attempted prior to entertaining the idea of surgery as the vast majority of patients will improve. There have not been any prospective randomised trials comparing the short- or long-term efficacy of medical treatment and surgery [15]. However, retrospective studies comparing patients requiring hospitalisation treated either medically or surgically found that those in the surgical group were more likely to be symptom-free at the time of follow-up. They also had fewer yearly hospital days and an overall trend toward decreased mortality [16, 17].

Pre-operative assessment Patients with bronchiectasis most commonly present with recurrent pulmonary infections. Symptoms associated with these infections include productive cough, foul-smelling sputum, haemoptysis, fever and dyspnoea on exertion. The presence of a nonproductive cough is suggestive of upper lobe involvement. Adequate pulmonary reserve is determined through standard pre-operative pulmonary function testing and occasionally split function perfusion testing when appropriate.

D.C. MAUCHLEY AND J.D. MITCHELL

The ideal candidate for surgical therapy should have truly localised disease that is amenable to anatomic lung resection. Non-anatomic (wedge) resections should be avoided if possible as this strategy frequently results in incomplete removal of the affected area. Incomplete resection has overwhelmingly been found to be the greatest predictor of symptomatic failure in these patients [5, 7, 8, 10, 12–14]. The diseased areas of lung tend to contribute little to the patient’s overall lung function, thus supporting an aggressive surgical approach.

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The diagnosis and location of bronchiectasis is made using standard radiographic techniques. Chest radiographs are often abnormal demonstrating focal areas of consolidation, atelectasis and occasional evidence of thickened bronchi. High-resolution computed tomography (HRCT) scanning has replaced contrast bronchography as the gold standard for radiologic diagnosis of bronchiectasis. This imaging modality can detect the distribution of bronchiectatic alterations with only 2% false-negative and 1% false-positive rates [18]. Findings suggestive of the disease include bronchial dilation such that the internal diameter of the affected bronchus is greater than the accompanying bronchial artery, and a lack of bronchial tapering on sequential slices [4]. The extent of disease seen on HRCT scans has been correlated to quality of life and subsequent functional decline [19, 20]. The left lung is more commonly affected than the right and the dependent lower lobes tend to harbour more disease than the upper lobes (fig. 1). Middle lobe and lingular disease is often associated with nontuberculous

a)

mycobacterial disease (fig. 2). Upper lobe involvement is suggestive of CF or allergic bronchopulmonary aspergillosis. Bronchoscopy is performed preoperatively, primarily to identify the offending organisms and to rule out concomitant endobronchial pathology. When patients present with active haemoptysis, bronchoscopy can be utilised to localise the source within the bronchial tree to the segmental or even subsegmental level. Sputum and bronchoalveolar lavage specimens are collected to allow identification of the microbial pathogens involved. Culture results should include in vitro susceptibility testing appropriate for the cultured organism to assist in pre-operative antimicrobial therapy.

SURGICAL MANAGEMENT

b)

Many patients who have been suffering with chronic lung infections will have lost weight and can be significantly malnourished at presentation. If this is the case, an aggressive pre-operative regimen of nutritional supplementation is recommended. This may require the placement of a nasojejeunal feeding tube or a percutaneous gastrostomy. We have found that this is typically not necessary for those with limited, focal parenchymal disease.

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At our institution (National Jewish Health, Denver, CO, USA), we have employed a multimodality treatment approach where patients appropriate for surgical therapy are discussed at a weekly multidisciplinary conference attended by surgeons, pulmonologists and infectious disease physicians with speFigure 1. a) Axial and b) coronal high-resolution computed cialisation in respiratory infectious tomography images of a patient with severe left lower lobe disease. This approach ensures that bronchiectasis. the patients receive the appropriate antimicrobial therapy and assists in optimal timing of surgical intervention. In fact, the timing of resection should be dependent on the pre-operative antimicrobial regimen, allowing enough time to produce a bacterial nadir at the time of surgery. We feel this is critical to minimise the risk profile in the perioperative period.

Surgical technique A standard anaesthetic technique utilised for thoracic surgical procedures is employed. Single-lung ventilation is accomplished with the use of a double-lumen endotracheal tube, or rarely a single lumen endotracheal tube with the use of a bronchial blocker. Early lung isolation may also limit dispersion of purulent secretions of uninvolved areas of the lungs. A thoracic epidural may be placed for post-operative analgesia when a thoracotomy is planned. This is usually not necessary in the event Figure 2. Axial high-resolution computed tomography image of a of a thoracoscopic approach, where patient with right middle lobe and lingular bronchiectasis in the post-operative analgesia is provided setting of nontuberculous mycobacterial disease termed Lady Windermere syndrome. by intercostal administration of 0.25% bupivicaine at multiple levels placed at the end of the procedure by the operative team. An arterial line and urinary catheter are placed and intra-operative fluid administration is limited as with other forms of extensive lung resection. Extubation at the end of the procedure is planned.

Surgical approach Bronchoscopy is routinely performed prior to initiation of the surgical procedure, clearing the airway of secretions to optimise ventilation during the operation. It is important to rule out bronchial obstruction secondary to a tumour or aspirated foreign body prior to attempting resection. If severe airway inflammation is found at the time of bronchoscopy, surgical therapy may be delayed until infection control is optimised. Finally, there is always the possibility that the patient may have normal variations in bronchial anatomy which would be helpful to know prior to attempting anatomic resection. Surgical resection for bronchiectasis is classically approached via lateral thoracotomy, tailored for the targeted segment or lobe. In the setting of significant disease, a full posterolateral thoracotomy may be employed. The mobilisation of muscle flaps should be accomplished at the onset of the thoracotomy, for transposition into the hemithorax later in the case after completion of the resection. An extrapleural dissection plane, if needed, may be initiated prior to placement of the rib spreader.

D.C. MAUCHLEY AND J.D. MITCHELL

Anaesthesia

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Several important differences exist between anatomic resection for infectious lung disease and resection for malignancy. Pleural adhesions are frequently present, and in some cases can be extensive and vascular in nature. They are typically localised to the involved segment(s) of lung, but can be scattered throughout the hemithorax. In upper lobe predominant disease, the adhesions to the overlying parietal pleura can be significant. The presence of dense adhesions can be predicted on the pre-operative HRCT, but the amount of pleural symphysis is often underestimated. Pleural adhesions can usually be divided safely with a thoracoscopic approach, often with improved visibility compared with open thoracotomy. During division of dense adhesions, care must be taken to avoid adjacent vital structures such as the phrenic nerve or great vessels.

The bronchial circulation is frequently hypertrophied in cases of longstanding bronchiectasis, and particular care must be taken to assure haemostasis. Bronchial arteries should be ligated with clips if enlarged. Significant lymphadenopathy is also usually present, and in the setting of chronic granulomatous disease can make dissection at the pulmonary hilum and around vessels hazardous. When dividing pulmonary fissures with stapling devices, we advocate a line of division just on the side of the uninvolved lobe. This will assure complete resection and will avoid a staple line in infected, devitalised tissue.

SURGICAL MANAGEMENT

The pulmonary vessels and bronchus are divided and sealed using standard stapling devices. Once the resection is completed, the diseased segment or lobe should be placed in a bag for removal, unless the thoracotomy is generous enough to avoid contamination with the specimen. In the setting of routine cases of anatomic resection for bronchiectasis, we typically do not buttress the bronchial closure with autologous tissue. The intrathoracic space is irrigated and then drained with one or two 28 French thoracostomy tubes. Portions of the specimen are sent for culture, and the remainder for pathologic analysis. Despite the fact that the majority of published series [5–10, 12, 16] of surgery for bronchiectasis describe resection using an open (thoracotomy) approach, a video-assisted thoracoscopic (VATS) approach has been successfully employed in some studies, and is the preferred approach at our institution [21, 22]. A standard VATS technique uses two 10-mm ports and a 4-cm utility incision centred over the anterior hilum. No rib spreading is used with this technique. The two 10-mm ports are placed first with one in the seventh intercostal space in the anterior axillary line, and the other just posterior to the scapular tip. Once the feasibility and safety of a VATS approach are confirmed, the utility incision is then made. We employ a wound protector for the utility incision to avoid contamination and retract the soft tissues of the chest wall. Modifications can be made to this technique to better serve the specifics of the planned resection. Adhesions are well visualised, and are typically easier to lyse thoracoscopically, although the presence of dense adhesions or complete pleural symphysis may suggest conversion to thoracotomy. The planned resection is then completed in a manner analogous to an open approach.

Use of tissue flaps Although not routinely performed, tissue transposition should be considered in any patient who is at increased risk for breakdown of the bronchial stump. Indications for autologous tissue coverage of the bronchial stump would include poorly controlled infection prior to surgery, resection in the setting of significant drug resistance or in the rare case of pneumonectomy for bronchiectasis [23]. We favour use of either a latissimus dorsi or intercostal muscle flap for coverage of a bronchial stump and omentum for use after a right pneumonectomy [24]. We avoid the use of a serratus muscle flap as there tend to be problems with wound healing in these characteristically thin patients related to the winged scapula following serratus transposition. Mobilisation of the latissimus is performed at the initiation of the procedure, and the muscle is transposed through the second or third intercostal space. When using an omental flap, the omentum is mobilised via a midline laparotomy prior to thoracotomy and tacked to the undersurface of the right hemidiaphragm for retrieval later during lung resection. Occasionally, significant intrathoracic space issues may result after resection, and may be at least partially addressed with latissimus transposition.

Post-operative management

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Management of patients after surgery for bronchiectasis is similar to that of any patient who has undergone anatomic lung resection. Emphasis is placed on early mobilisation, aggressive pulmonary toilet, chest physiotherapy and nutritional supplementation. Chest tube management is routine. Appropriate antimicrobial therapy is maintained in the post-operative period and is often continued for several months, depending on the isolated organism. In patients who present

with bilateral disease and consequently are left with unilateral disease post-operatively, bronchoscopy may be necessary to help with mobilisation and clearance of secretions. Those who are treated with a thoracoscopic approach can leave the hospital as early as second or third postoperative day, while those who undergo thoracotomy often stay for up to a week.

Complications

Although it is rare, the development of BPF is a source of significant morbidity, particularly after pneumonectomy. It occurs more commonly on the right side, after completion pneumonectomy, and in the setting of patients who have persistently positive sputum cultures for organisms such as multidrug-resistant Mycobacterium tuberculosis [22, 24]. When presented with a patient at high risk of development of BPF, prevention is paramount. Appropriate antimicrobial coverage should be given before surgery; a tension-free technique used to close the bronchus and muscle or omentum used to buttress the closure. Typical findings of a BPF after pneumonectomy include fever, cough productive of serous followed by purulent sputum, contralateral lung infiltrates and a dropping air–fluid level on chest radiograph. Management begins with prompt drainage of the infected space to prevent further damage to the remaining lung. If the BPF is diagnosed very early after resection, primary repair of the bronchial stump with rebuttressing may be attempted. When diagnosis is delayed management usually requires rib resection and creation of an Eloesser flap followed by BPF closure and subsequent Clagett procedure. As mentioned previously, intrathoracic space problems are somewhat more common after surgery for bronchiectasis, mainly due to the fact that the remaining lung is often unable to fully expand. This leaves residual space that is usually not a problem, but can lead to development of empyema in cases that involve significant pleural soilage or parenchymal injury. Again, prevention is key and patients with these potential problems should be anticipated. Liberal use of muscle flaps to minimise the space can help prevent complications.

D.C. MAUCHLEY AND J.D. MITCHELL

The complications that accompany lung resection for bronchiectasis mirror those that follow lung resection for other indications with a few exceptions. Overall morbidity following resection ranges from 9% to 25% depending on the series. The most common complications after surgery for bronchiectasis include atelectasis requiring therapeutic bronchoscopy, prolonged air leak, space problems, empyema, BPF and wound infection (table 1) [5, 7–13, 22]. Absence of pre-operative bronchoscopy, forced expiratory volume in 1 second of ,60% of the predicted value and incomplete resection have all been associated with the development of post-operative complications [25].

Table 1. Summary of morbidity after surgical resection for bronchiectasis First author [ref.] D OGAN [9] AGASTHIAN [5] FUJIMOTO [10] P RIETO [13] K UTLAY [12] B ALKANLI [8] G URSOY [11] B AGHERI [7] Z HANG [22]

Prolonged air Atelectasis Empyema/ leak/space BPF issues 0 4.5 5.6 5.9 1.7 2.5 9.8 3.2 2.7

1.4 6.7 6.7 0 2.3 2.9 3.2 3.6 2

1.8 4.5 6.7 0 1.2 1.7 0 3.2 1

Wound Bleeding Arrhythmia Overall infection morbidity 7.4 0 0 0 0 0 3.3 5.7 0

0 3 1.1 3.4 1.7 1.7 0 0 1.1

0 2.2 0 3.4 0 0 0 0 4

10.6 24.6 19.6 12.6 11.4 8.8 16.3 15.8 16.2

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Data are presented as % of all patients in each reference. BPF: bronchopleural fistula.

Table 2. Summary of patient characteristics and operative mortality after surgical management of bronchiectasis First author [ref.] D OGAN [9] A GASTHIAN [5] F UJIMOTO [10] P RIETO [13] K UTLAY [12] B ALKANLI [8] G URSOY [11] B AGHERI [7] Z HANG [22]

Study period

Patients n

1976–1988 1976–1993 1990–1997 1988–1999 1990–2000 1992–2001 2002–2007 1985–2008 1989–2008

487 134 90 119 166 238 92 277 790

Age years 25.5 48 44.7 42.2 34.1 23.7 38.7 34.7 41.6

(2–56) (4–89) (9–75) (11–77) (7–70) (15–48) (10–67) (8–65) (6–79)

Males

Left-sided disease

Complete resection

Operative mortality

57 41 49 40 45 86 41 72 59

64 Not stated 59 Not stated 59 Not stated 74 70 Not stated

Not stated 80.6 83.3 90.8 88.5 64.7 90.2 82.7 89

3.5 2.2 0 0 1.7 0 1.1 0.7 1.1

Data are presented as mean (range) or %, unless otherwise stated.

SURGICAL MANAGEMENT

Results Perioperative mortality after resection for bronchiectasis is very low with contemporary rates ranging from 0% to 3.5% (table 2). Completion pneumonectomy remains a highly morbid procedure and leads to many of the deaths related to surgical treatment of this disease [5, 24]. Renal failure and advanced age (.70 years) are associated with post-operative mortality in this group of patients [22]. Mean age at the time of surgery ranges from 25.5 to 48 years and more female patients seem to be affected than male patients. Female predominance is not as consistent in reports from developing countries [7–9, 22]. The most common indication for surgical intervention is failure of medical therapy. Left-sided disease predominates and complete resection of disease is usually possible 80–90% of the time. The most commonly performed procedure is lobectomy, followed by segmentectomy, lobectomy with segmentectomy, and pneumonectomy. Very few patients undergo bilobectomy for bronchiectasis (table 3). The most common reported reason for incomplete resection is bilateral disease, although the majority of these patients should be candidates for contralateral resection at a later date. The vast majority of patients are either asymptomatic or are symptomatically improved at follow-up (table 4). Lack of symptomatic improvement is most commonly associated with incomplete resection [5, 7, 8, 10, 12, 13, 22, 25], but has also been associated with saccular bronchiectasis, history of sinusitis and tuberculous infection [10, 22]. The results of VATS lung resection for bronchiectasis have been examined in two studies in the last decade. WEBER et al. [26] described thoracoscopic lobectomy using five trocars with subsequent mini-thoracotomy in 76 patients with benign lung disease. 49 of the patients had bronchiectasis or Table 3. Summary of operative procedures performed for the surgical management of bronchiectasis First author [ref.] D OGAN [9] A GASTHIAN [5] A SHOUR [6] F UJIMOTO [10] P RIETO [13] K UTLAY [12] B ALKANLI [8] G URSOY [11] B AGHERI [7] Z HANG [22]

Lobectomy

Pnemonectomy

Segementectomy/ wedge

41.5 64.2 64.7 54.3 62 63.4 79.4 39.1 42.2 62.9

39 15.7 16.5 6.5 8 7.5 5.5 10.9 7.9 11.3

0 13.4 18.8 33.7 13 12.2 2.1 Not stated 6.5 4.7

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Data are presented as % of total resections for each reference.

Lobectomy + Bilobectomy segment 14.8 6.7 0 0 14 10.5 13 34.8 23.5 14

4.7 0 0 5.4 3 6.4 0 Not stated 19.9 7.1

Table 4. Summary of pulmonary symptoms after surgical resection for bronchiectasis First author [ref.] D OGAN [9] A GASTHIAN [5] A SHOUR [6] F UJIMOTO [10] P RIETO [13] K UTLAY [12] B ALKANLI [8] G URSOY [11] B AGHERI [7] Z HANG [22]

Mean follow-up time years

% Follow-up

Asymptomatic

Symptomatic improvement

No change in symptoms/worse

4.6 6 3.8 6.1 4.5 4.2 0.75 1.3 4.5 4.2

Not stated 76.9 100 87.8 90.8 89.2 96.2 81.5 100 89.4

71 45.5 74.1 40 61.3 66.9 79.4 68.5 68.5 60.5

Not stated 22.4 22.4 33.3 26.1 18.7 12.2 8.7 23.8 14.1

Not stated 9 3.5 14.5 3.4 3.6 4.6 4.3 7.5 14.8

chronic lung infection. The mortality rate was 0%, morbidity rate was 18.7% and 12 (15.3%) cases were converted to open procedure. Reasons for conversion to open procedure included dense adhesive disease as well as upper lobe-predominant disease. Compared with those who underwent open thoracotomy during the same time period, patients undergoing VATS resection suffered fewer post-operative complications, had less blood loss and a shorter hospital stay. More recently, ZHANG et al. [27] reported 52 patients who underwent VATS lobectomy using two 12-mm trocars and a 4–5-cm incision. Overall, they had similar findings with no mortality, a morbidity rate of 15.4% and conversion to thoracotomy in 13.5% of patients. Furthermore, those who were treated with a VATS approach had less morbidity and a shorter hospital stay compared with a cohort of patients who underwent open thoracotomy for resection during the same time period. Pain scores based on an 11 point pain scale were also lower in the VATS group. The conclusions of both reports were that benign lung disease, including bronchiectasis, could feasibly be resected using a VATS approach with negligible mortality and lower morbidity than with thoracotomy.

Lung transplantation Lung transplantation in patients with bronchiectasis is only indicated for those with diffuse disease that is not amenable to segmental surgical resection and declining lung function despite maximal medical therapy. The vast majority of transplants for bronchiectasis are performed on patients with CF. Bronchiectasis develops in nearly all cases of CF and leads to chronic cough, expectoration of abnormal mucus, progressive airflow obstruction and persistent respiratory tract infections. Those with advanced bronchiectasis have poor quality of life and are at increased risk of death secondary to declining lung function. Lung transplantation has been shown to both improve quality of life and prolong survival in appropriately selected patients with advanced bronchiectasis [28, 29].

D.C. MAUCHLEY AND J.D. MITCHELL

Data are presented as % of all patients (including those lost to follow-up) from each reference, unless otherwise stated.

CF is the third most common indication for which lung transplantation is performed [30]. The current recommendation is for bilateral lung transplant in those with suppurative lung disease secondary to CF, even in those with heterogeneous disease. Single lung transplantation would risk contamination of the new graft by the old lung in an immunocompromised patient. Some centres will perform a single lung transplant in conjunction with contralateral pneumonectomy to avoid this risk.

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The guidelines for referral of patients with CF and bronchiectasis for transplantation are listed in table 5 [30]. Additionally, patients should be considered for transplantation if there is a ,50% likelihood of survival over 2 years without transplant, if quality of life is likely to be improved as a result of transplant, there are no contraindications to transplant, and they are informed of the risks and benefits of the operation and committed to proceeding with evaluation and listing. Young females with CF are considered for early referral if they suffer rapid deterioration in pulmonary

Table 5. Guidelines for lung transplantation in diffuse bronchiectasis (both cystic fibrosis and non-cystic fibrosis) Guidelines for referral to a transplant centre

Guidelines for transplantation

FEV1 ,30% predicted or a rapid decline in FEV1, particularly in young female patients Exacerbation of pulmonary disease requiring ICU stay Increasing frequency of exacerbations requiring antibiotic therapy Refractory and/or recurrent pneumothorax Recurrent haemoptysis not controlled by embolisation Progressive decline in lung function Oxygen-dependent respiratory failure Hypercapnia Pulmonary hypertension

FEV1: forced expiratory volume in 1 second; ICU: intensive care unit.

SURGICAL MANAGEMENT

status as they have a particularly poor prognosis [30]. Finally, several studies in the 1990s described infection with Burkholderia cepacia in prospective CF transplant candidates to be associated with significant post-transplantation infectious complications and poor outcomes [31, 32]. This has led to the presence of B. cepacia infection to be a relative contraindication to lung transplantation in the CF population, although some centres continue to offer transplantation therapy in this setting. More recent evidence suggests that some, but not all subspecies within the B. cepacia complex confer an increased risk [33]. A number of complications may occur after lung transplantation for CF and bronchiectasis, including haemorrhage, pulmonary oedema, primary graft dysfunction, anastomotic dehiscence and various infectious complications. Bacterial infections are common after transplant for bronchiectasis as numerous pathogens chronically dwell in respiratory tract secretions of these patients. Antibacterial regimens guided by pre- and perioperative cultures are used post-operatively in addition to standard prophylactic medications given for viral and fungal pathogens [34]. Patients with CF and bronchiectasis can expect a dramatic improvement in pulmonary function after lung transplant as well as the ability to perform activities of daily living without limitations. Long-term survival has been demonstrated in a review of 123 patients with CF who underwent either bilateral lung transplantation or bilateral lower lobe transplant from living donors [35]. Survival rates were 81% at 1 year, 59% at 5 years and 38% at 10 years. A sustained improvement in quality of life after transplantation can be expected for at least 1–3 years [34]. Transplantation for non-CF bronchiectasis is rare and specific referral guidelines have not been developed. For this reason, the guidelines used for those with CF bronchiectasis are generally used [30].

Statement of interest None declared.

References

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1. Laennec RTH. De l’Ausculation Mediate ou Traite du Diagnostics des Maladies des Poumons et du Coeur. [On Mediate Ausculation or Treatise on the Diagnosis of the Disease of the Lungs and Heart]. Paris, Brosson and Chaude´, 1819. 2. Lindskog GE, Hubbell DS. An analysis of 215 cases of bronchiectasis. Surg Gynecol Obstet 1955; 100: 643–650. 3. Ochsner A, DeBakey M, DeCamp PT. Bronchiectasis; its curative treatment by pulmonary resection; an analysis of 96 cases. Surgery 1949; 25: 518–532. 4. O’Donnell AE. Bronchiectasis. Chest 2008; 134: 815–823. 5. Agasthian T, Deschamps C, Trastek VF, et al. Surgical management of bronchiectasis. Ann Thorac Surg 1996; 62: 976–978. 6. Ashour M, Al-Kattan K, Rafay MA, et al. Current surgical therapy for bronchiectasis. World J Surg 1999; 23: 1096–1104.

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7. Bagheri R, Haghi SZ, Fattahi Masoum SH, et al. Surgical management of bronchiectasis: analysis of 277 patients. Thorac Cardiovasc Surg 2010; 58: 291–294. 8. Balkanli K, Genc O, Dakak M, et al. Surgical management of bronchiectasis: analysis and short-term results in 238 patients. Eur J Cardiothorac Surg 2003; 24: 699–702. 9. Dogan R, Alp M, Kaya S, et al. Surgical treatment of bronchiectasis: a collective review of 487 cases. Thorac Cardiovasc Surg 1989; 37: 183–186. 10. Fujimoto T, Hillejan L, Stamatis G. Current strategy for surgical management of bronchiectasis. Ann Thorac Surg 2001; 72: 1711–1715. 11. Gursoy S, Ozturk AA, Ucvet A, et al. Surgical management of bronchiectasis: the indications and outcomes. Surg Today 2010; 40: 26–30. 12. Kutlay H, Cangir AK, Enon S, et al. Surgical treatment in bronchiectasis: analysis of 166 patients. Eur J Cardiothorac Surg 2002; 21: 634–637. 13. Prieto D, Bernardo J, Matos MJ, et al. Surgery for bronchiectasis. Eur J Cardiothorac Surg 2001; 20: 19–23. 14. Stephen T, Thankachen R, Madhu AP, et al. Surgical results in bronchiectasis: analysis of 149 patients. Asian Cardiovasc Thorac Ann 2007; 15: 290–296. 15. Corless JA, Warburton CJ. Surgery vs non-surgical treatment for bronchiectasis. Cochrane Database Syst Rev 2000; 4: CD002180. 16. Annest LS, Kratz JM, Crawford FA Jr. Current results of treatment of bronchiectasis. J Thorac Cardiovasc Surg 1982; 83: 546–550. 17. Sanderson JM, Kennedy MC, Johnson MF, et al. Bronchiectasis: results of surgical and conservative management. A review of 393 cases. Thorax 1974; 29: 407–416. 18. Young K, Aspestrand F, Kolbenstvedt A. High resolution CT and bronchography in the assessment of bronchiectasis. Acta Radiol 1991; 32: 439–441. 19. Eshed I, Minski I, Katz R, et al. Bronchiectasis: correlation of high-resolution CT findings with health-related quality of life. Clin Radiol 2007; 62: 152–159. 20. Sheehan RE, Wells AU, Copley SJ, et al. A comparison of serial computed tomography and functional change in bronchiectasis. Eur Respir J 2002; 20: 581–587. 21. Mitchell JD, Bishop A, Cafaro A, et al. Anatomic lung resection for nontuberculous mycobacterial disease. Ann Thorac Surg 2008; 85: 1887–1892. 22. Zhang P, Jiang G, Ding J, et al. Surgical treatment of bronchiectasis: a retrospective analysis of 790 patients. Ann Thorac Surg 2010; 90: 246–250. 23. Shiraishi Y, Nakajima Y, Katsuragi N, et al. Pneumonectomy for nontuberculous mycobacterial infections. Ann Thorac Surg 2004; 78: 399–403. 24. Sherwood JT, Mitchell JD, Pomerantz M. Completion pneumonectomy for chronic mycobacterial disease. J Thorac Cardiovasc Surg 2005; 129: 1258–1265. 25. Eren S, Esme H, Avci A. Risk factors affecting outcome and morbidity in the surgical management of bronchiectasis. J Thorac Cardiovasc Surg 2007; 134: 392–398. 26. Weber A, Stammberger U, Inci I, et al. Thoracoscopic lobectomy for benign disease – a single centre study on 64 cases. Eur J Cardiothorac Surg 2001; 20: 443–448. 27. Zhang P, Zhang F, Jiang S, et al. Video-assisted thoracic surgery for bronchiectasis. Ann Thorac Surg 2011; 91: 239–243. 28. Courtney JM, Kelly MG, Watt A, et al. Quality of life and inflammation in exacerbations of bronchiectasis. Chron Respir Dis 2008; 5: 161–168. 29. Loebinger MR, Wells AU, Hansell DM, et al. Mortality in bronchiectasis: a long-term study assessing the factors influencing survival. Eur Respir J 2009; 34: 843–849. 30. Orens JB, Estenne M, Arcasoy S, et al. International guidelines for the selection of lung transplant candidates: 2006 update – a consensus report from the Pulmonary Scientific Council of the International Society for Heart and Lung Transplantation. J Heart Lung Transplant 2006; 25: 745–755. 31. Egan JJ, McNeil K, Bookless B, et al. Post-transplantation survival of cystic fibrosis patients infected with Pseudomonas cepacia. Lancet 1994; 344: 552–553. 32. Snell GI, de Hoyos A, Krajden M, et al. Pseudomonas cepacia in lung transplant recipients with cystic fibrosis. Chest 1993; 103: 466–471. 33. Murray S, Charbeneau J, Marshall BC, et al. Impact of burkholderia infection on lung transplantation in cystic fibrosis. Am J Respir Crit Care Med 2008; 178: 363–371. 34. Hayes D Jr, Meyer KC. Lung transplantation for advanced bronchiectasis. Semin Respir Crit Care Med 2010; 31: 123–138. 35. Egan TM, Detterbeck FC, Mill MR, et al. Long term results of lung transplantation for cystic fibrosis. Eur J Cardiothorac Surg 2002; 22: 602–609.

Chapter 18

Conclusions and future developments R.A. Floto Correspondence: R.A. Floto, Cambridge Institute for Medical Research, University of Cambridge, Addenbrooke’s Hospital, Hills Road, Cambridge, CB2 0XY, UK, Email: [email protected]

FUTURE DEVELOPMENTS

T

his Monograph represents a ‘‘state-of-the-art’’ review of non-cystic fibrosis (CF) bronchiectasis and highlights many recent advances in our understanding of this condition and how best to manage it. However, it is clear, that many important unanswered questions remain about the patho-physiology, investigation and treatment of bronchiectasis. The management of these patients remains hampered by a paucity of clinical trial evidence which, through necessity, requires us to draw on potentially misleading data from CF or chronic obstructive pulmonary disease studies. Furthermore, inadequate education and training of physicians remain barriers to optimal delivery of care to patients with non-CF bronchiectasis, something which this Monograph may hopefully begin to address. There are a number of potential future developments, discussed below, which may significantly contribute to our understanding of why non-CF bronchiectasis develops in particular individuals, how to best investigate possible aetiological factors and how to optimally manage these patients.

Why does bronchiectasis develop? As described in the chapter by BILTON and JONES [1], a large number of conditions leading to impaired host immunity and/or defective muco-ciliary clearance have been implicated in the development of non-CF bronchiectasis. However, using established investigative approaches, outlined by DRAIN and ELBORN [2], we are currently unable to define an obvious triggering cause in a large percentage of adults with bronchiectasis.

Immunity and inflammation Developments in our understanding of the mechanisms controlling lung inflammatory and immune responses [3] and their resultant effects on lung tissue [4] may allow researchers to focus on specific critical host responses that may qualitatively or quantitatively vary within a population predisposing particular individuals to bronchiectatic lung damage. Future developments in immunological testing [2, 5] may focus on identifying aberrant responses in patients’ immune cells to inflammatory or infective stimuli. Furthermore, more detailed analysis of patients with bronchiectasis associated with inflammatory bowel disease [6] and systemic autoimmunity [7] may provide important insights into how aberrant immunological responses might lead to bronchiectasis.

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Microbiology A better understanding of the role of bacteria and how they interact with epithelial cells and viruses [8] is likely to permit more targeted treatments and more effective prophylaxis. Nonculture-based microbial detection methods [9] are likely to provide mechanistic insights into the possible protective role of commensal microbial flora, the role of anaerobic and intracellular organisms and the dynamic interplay between bacterial species in specific lung niches. For fungal diseases [10] and nontuberculous mycobacterial infection [11], future research into the basic mechanisms of disease may permit development of novel diagnostic tools and more effective treatment strategies.

Mucociliary clearance Recent work has suggested that nonclassical or secondary ciliary dysfunction [12] and epithelial channel mutations [13] may be important determinants in compromising mucociliary clearance (MCC) and, thus, predisposing to bronchiectatic damage. Novel developments in lung imaging [14], including quantification of global and regional MCC, may permit more detailed investigation of bronchiectasis patients and assessment of the impact of specific physiotherapy techniques and mucolytic therapies.

Novel genetic approaches

1) Genome-wide association scans (GWAS) may, as in other conditions [15], identify novel disease-associated, single-nucleotide polymorphisms and potentially uncover critical pathways involved in bronchiectasis in an unbiased, ‘‘hypothesis-free’’ way. Challenges in undertaking GWAS studies include the large number of patient DNA samples required (usually several thousand), as well as the problem of multiple initiators for the development of bronchiectasis leading to reduced signal discrimination.

R.A. FLOTO

There are potentially three ways in which new developments in genetics might be exploited to better understand the patho-physiology of non-CF bronchiectasis.

2) Candidate gene approaches could also be used to identify diseased-associated polymorphisms in more well defined subsets of patients. Obvious candidates would include known genetic modifiers of CF [16], genes involved in lung inflammation and those encoding proteins that are critical for epithelial cell function. 3) Whole exome analysis using massive parallel sequencing can now permit rapid sequencing of the entire expressed genome of individuals [17], potentially permitting detection of gene mutations in small cohorts of patients with familial disease.

Can we improve the treatment of patients with non-CF bronchiectasis? It is reasonable to anticipate a number of future developments which may impact on the management of patients with non-CF bronchiectasis.

New antibiotic strategies

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As highlighted in the chapter by HAWORTH [18], the development of new nebulised or inhaled formulations of single or combination antibiotics may significantly impact on our ability to provide adequate prophylaxis for patients. In addition, a number of novel approaches are being developed for the treatment of Pseudomonas aeruginosa which may prove useful, including novel b-lactamase inhibitors, blockers of bacterial efflux pumps (which normally remove otherwise toxic antibiotics), antimicrobial peptides and species-specific bacteriophage-based therapy [19].

Novel anti inflammatory agents As discussed in the chapter by SMITH et al. [20], anti-inflammatory therapy may be of considerable benefit in bronchiectasis. Novel agents that may have a future role include nonantibiotic macrolides, HMG-CoA inhibitors (statins) and peroxisome proliferator-activated receptor-c agonists. The difficulty will be to balance control of inflammation with compromise of host defence.

Mucolytic strategies The potential benefits of improved airway clearance [21] are likely to be vast. Future therapies that reduce mucus viscosity (by altering mucin production or blocking subsequent cross-linking), increase airway-surface liquid (through osmosis or altered epithelial channel activity) or improve ciliary function may all have potential benefit.

Surgery and lung repair

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FUTURE DEVELOPMENTS

More research will be needed to define the precise role of surgery in the management of non-CF bronchiectasis [22]. Anticipated improvement in surgical techniques and reductions in perioperative morbidity will impact on when surgery in considered and in whom. Future developments in stem cell biology (including studies re-programming induced pluripotent stem cells and overcoming engraftment difficulties) may open the door for therapeutic lung repair and regeneration. Over the next few years we can optimistically look forward to greater advances in our understanding of the patho-physiology and genetic determinants of non-CF bronchiectasis, the development of more sophisticated methods for investigation of patients and an increasing number of clinical trials focusing on improving evidence-based treatment of this challenging condition.

Statement of interest None declared.

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