Non-Tuberculous Mycobacteria in Bronchiectasis: Prevalence and Patient Characteristics. Papworth Hospital, Cambridge, UK

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1 ERJ Express. Published on June 28, 2006 as doi: / Non-Tuberculous Mycobacteria in Bronchiectasis: Prevalence and Patient Characteristics SJ Fowler 1, J French 1, NJ Screaton 2, J Foweraker 3, A Condliffe 1, CS Haworth 1, AR Exley 1, D Bilton 1 1 Lung Defence Clinic, 2 Dept of Radiology, and 3 Dept of Microbiology, Papworth Hospital, Cambridge, UK Corresponding Author Dr Stephen J Fowler, Chest Medical Unit Papworth Hospital, Cambridge, UK, CB3 8RE Phone: Fax: sjfowler@doctors.org.uk Short title: Non-Tuberculous Mycobacteria in Bronchiectasis Copyright 2006 by the European Respiratory Society.

2 ABSTRACT We aimed to investigate the prevalence and clinical associations of non-tuberculous mycobacteria (NTM) in a well characterised cohort of patients with adult-onset bronchiectasis. All patients attending our tertiary referral bronchiectasis clinic between April 2002 and August 2003 had sputum examined for mycobacteria as part of an extensive diagnostic workup. NTM positive patients subsequently had further sputa sent. A modified bronchiectasis scoring system was applied to all high resolution computed tomography (HRCT) scans from patients with NTM, and a matched cohort without. Of 98 patients attending the clinic, 10 had NTM in their sputum on first culture (NTM+), and eight had multiple positive cultures. Three were treated for NTM infection. A higher proportion of NTM+ patients were subsequently diagnosed with cystic fibrosis (CF) versus the NTM- group (2/9 vs. 2/75). On HRCT scoring, the NTM+ group had more patients with peripheral mucous plugging than the NTM- group. In our prospective study of a large cohort of patients with bronchiectasis, 10% cultured NTM in a random clinic sputum sample. Few clinical parameters were helpful in discriminating between groups, except for the higher prevalence of previously undiagnosed cystic fibrosis, and of peripheral mucous plugging on HRCT in the NTM+ group. KEYWORDS: bronchiectasis; non-tuberculous mycobacteria Page 2

3 INTRODUCTION Non-tuberculous mycobacteria (NTM), also known as opportunistic, environmental or atypical mycobacteria, are ubiquitous in the environment and have been isolated from soil, tap water, dust, and clinical laboratories (1). Guidance has been produced for the diagnosis and treatment of NTM infection (1, 2), which requires satisfaction of clinical, microbiological and radiological criteria. In pre-existing lung disease, especially where there may be frequent exacerbations such as in bronchiectasis, achieving a definition of clinical and radiological criteria specific for NTM infection is difficult. In such patients radiographic criteria favouring a diagnosis of NTM infection include pleural disease, pneumonic changes, cavitating nodules, tree-in-bud (TIB) changes away from the main sites of bronchiectasis, and mosaic perfusion (3). While there is little published information on the prevalence and significance of NTM in bronchiectasis, there are data in cystic fibrosis (CF), where NTM have been cultured from the sputum of 4-24% of screened patients (4, 5). In a large multi-centre study from the United States, culture-positive patients were older, had better lung function and were more likely to be colonised with Staphylococcus aureus, but not Pseudomonas aeruginosa (5). These findings had not been reflected in an earlier UK study, in which NTM positive sputum cultures were associated only with increased requirement for intravenous antibiotics (4). Further analysis of the cohort from the US found that lung function decline was no different in patients with multiple NTM positive sputum cultures versus those with one or no positive cultures, but they did have more characteristic features on high resolution computed tomography (HRCT) (6). Page 3

4 We therefore prospectively investigated our cohort of patients with adult-onset bronchiectasis to determine the prevalence of NTM in this group. It is currently our practice to screen all patients for NTM on referral to our tertiary referral bronchiectasis clinic. We also compared clinical indices with bronchiectasis patients who did not grow NTM over that time, to see if there was any association with disease severity, antibiotic usage, radiographic changes, or microbiology. Page 4

5 METHODS Subjects In this prospective cohort study we enrolled consecutive patients attending the Papworth Hospital Lung Defence Clinic between April 2002 and August All subjects had a clinical diagnosis of bronchiectasis, confirmed by high-resolution computed tomography. Patients had a full history taken and clinical examination performed, with selected tests to further elucidate the cause of their bronchiectasis. Investigations included α-1-antitrypsin levels, cystic fibrosis genetics and subsequent sweat testing if positive, immunoglobulin levels (including specific antibody responses), microscopic examination of the nasal cilia and fibre-optic bronchoscopy. These investigations were performed according the methodologies previously described by our group (7). Measurements As part of our clinical practice we routinely collected sputum for microscopy and bacterial culture at every clinic visit. Colonisation was defined when the same organism was cultured from sputum on at least two occasions separated by three months within one year. At the initial visit sputum was also sent for mycobacterial culture, and repeated at subsequent visits in those patients who had grown an NTM on first culture. This was our routine clinical practice at the time and as such neither approval from the Research Ethics Committee nor written patient consent were sought. All patients were further characterised using spirometry, in accordance with ATS guidelines (8). Page 5

6 Sputum processing Sputum was stained directly for Acid Fast Bacilli (AFB) and decontaminated in sodium hydroxide (2% final concentration). It was cultured by an automated liquid method using the Mycobacteria Growth Indicator Tubes (MGIT) system (Becton Dickinson) and on solid media (Lowenstein Jensen slopes) (9). MGIT tubes were incubated for 42 days and LJ slopes for 8 weeks. Mycobacteria were identified in a central reference laboratory using standard methods (10). After one Mycobacterium sp. was identified from an individual patient, at least two repeat specimens were sent for both culture and identification. HRCT scan protocol and scoring Thin (1mm) collimation CT images were obtained at 10mm intervals from lung apices to bases using during suspended full inspiration in the supine position. Images were reconstructed using a high spatial frequency reconstruction algorithm and displayed on lung parenchymal window settings (Level -700 HU, Width 1200 HU). The HRCT images were scored for the severity and extent of bronchiectasis and airways disease using a modified Bhalla system (11). Given the good interobserver agreement for the CT features of bronchiectasis previously published (12-14), CTs were evaluated by a single experienced cardiothoracic radiologist. The extent and severity of bronchiectasis and CT features of airways disease were evaluated at a lobar level with the lingula considered a separate lobe. The presence of bronchiectasis was defined using established criteria (15, 16). The severity of bronchial dilatation (0=normal, 1=1-2 x diameter of adjacent artery, 2=2-3 x diameter of adjacent artery, 3=>3 x Page 6

7 diameter of adjacent artery) (14), and of bronchial wall thickening (0=normal, 1=<0.5 x diameter of adjacent artery, 2=0.5-1 x diameter of adjacent artery, 3=>1 x diameter of adjacent artery) (14, 17), were scored for each lobe on a scale 0-3 using previously described criteria. The extent of bronchiectasis (11), and mucus plugging (18), sacculations / abscesses (11), mosaic perfusion (18), and collapse or consolidation were assessed on a lobar basis and the number of segments involved recorded. The extent of tree-in-bud opacity was scored 0-3 according to the extent of lung involved (absent = 0, <25% = 1, 25-50% = 2, >50% = 3). The distribution of bronchiectasis and mucus plugging was described for each case as central (central 50%), peripheral (outer 50%) or mixed. The total lung scores for each abnormality were defined as the mean score from all lobes in the cases of severity of bronchial dilatation, bronchial wall thickening and tree-in-bud opacity and by the total number of involved segments (0=none, 1=1-5 segments, 2=6-9 segments, 3=>9 segments) for the extent of bronchiectasis (11), mucus plugging (18), sacculations / abscesses (11) and mosaic perfusion (18). The severity of collapse or consolidation was also scored according to the total number of involved segments (0=normal, 1=1-3 segments, 2=4-6 segments, 3=>6 segments). A combined total lung score (0-24) for all CT features was derived by summing the scores for the individual CT features. High-resolution CT scans were scored as described above for all the NTM+ patients. For each, CT scans from two matched NTM- patients were also scored. Cases were matched by FEV 1, and for the presence of Pseudomonas aeruginosa in sputum where appropriate. A mean score was then calculated for each NTM- pair. Statistical Analysis Page 7

8 The patients were divided into two groups, described as NTM+ (those culturing an NTM at first visit), or NTM-. These groups were compared using the chi-squared test for clinical characteristics including number colonised with Pseudomonas aeruginosa, number on longterm (daily) antibiotics, number with mutations of the cystic fibrosis transmembrane regulator (CFTR), and CT features. Group mean spirometric values and age were normally distributed and compared using the students t-test. CT scores (total and by section) were not normally distributed, and group means were compared by the Mann-Whitney U test, and pairs (i.e. the NTM+ case and mean of its two matched NTM- controls) by the Wilcoxon signed ranks test. Page 8

9 RESULTS 98 patients (66 females) were investigated; mean (SEM) age 59.3 (1.3) years, FEV (2.3) % predicted, and FVC 85.8 (1.9) % predicted. Of these, 10 grew NTM on first sputum culture; none were smear-positive. These patients we followed up over median (range) 58 (16 to 88) weeks, and are summarised in table 1. Patient characteristics for the two groups (NTM+ versus NTM-) are shown in table 2. There was no difference between the groups in terms of age, gender, spirometry, or numbers with CFTR mutations, culturing Pseudomonas aeruginosa, or on long-term antibiotics. Four of the six patients heterozygous for CFTR mutations had high sweat sodium concentrations (range mmol/l), consistent with a diagnosis of CF. Of the remaining two, one was unable to complete the test for technical reasons, and the second was unwilling to have the test performed. Cause of bronchiectasis by group is shown in figure 1. Of the patients tested for both CFTR gene mutations and, if positive, by subsequent sweat test, more NTM+ patients had a diagnosis of CF than NTM- (2/9 versus 2/75; p < 0.01). Trends in FEV 1 for the period before and after first positive NTM culture for the NTM+ group are shown in figure 2. There was no change in FEV 1 before and after NTM was first cultured, neither in the NTM+ group as a whole, nor in the three patients who were subsequently treated. CT scores for the NTM+ group versus the matched cohort are shown in table 3. There were no differences in individual or total scores either when analysed as group means (i.e. 10 NTM+ versus 20 NTM-) or pairwise (i.e. 10 pairs of NTM+ versus the average of the two Page 9

10 matched NTM- scores). The NTM+ group did have more individuals with peripheral mucous plugging than the NTM- group (9/10 versus 5/20; p < 0.001). An example of a CT image demonstrating peripheral mucous plugging is shown in figure 3. When the data were re-analysed, excluding the two patients in the NTM+ group (subjects 1 and 3) who only cultured NTM on the first occasion, the NTM+ group had more segments affected by mucous plugging than the NTM- group (p = as group means and pairwise). Otherwise the same results were obtained (data not shown). Page 10

11 DISCUSSION We cultured NTM from the sputum of 10 of our patients with bronchiectasis out of a screened sample of 98. NTM were subsequently re-cultured in sputum from eight of these 10. The prevalence in our patients in whom CF had been definitely excluded was seven out of 80 (9%). In a recent prospective cohort, Wickremasinghe and colleagues reported multiple isolates in one of 50 screened patients with non-cf bronchiectasis and a further single isolate in one patient (19). Several groups have reported the prevalence of NTM infection in patients with CF, which has ranged from 2 to 4% in the UK (4, 20), and 6 to 24% in the US (5, 21, 22). It is also worth noting that all of our NTM culture-positive specimens were smear negative. This compares to approximately 25% of culture positives that were smear-positive in the CF-bronchiectasis study of Olivier and colleagues. The clinical relevance of this is not clear, but it demonstrates that the mycobacterial load in our patients was low. These were patients seen in a tertiary referral bronchiectasis clinic, and as such may have features of more severe bronchiectasis than is typical for the United Kingdom, in terms of recurrent infections, immune-deficiencies or symptoms, although this is not apparent from our lung function data; our patients had a mean FEV 1 of 64% predicted and FVC of 86% predicted. There were no differences in our patients with or without NTM in terms of age, sex, spirometry, co-existent sputum bacteriology or use of long-term antibiotics. In CF, Torrens et al noted that NTM+ cases had received more courses of intravenous antibiotics than controls, but no differences in respiratory function, or use of nebulised antibiotics(4). In the US, Olivier et al found that their CF patients with NTM were older, with better lung function, and more likely to culture Staphylococcus aureus, but less likely to culture Pseudomonas aeruginosa than controls(5). In a sub-group followed over 15 months, no difference in annual rate of FEV 1 decline versus NTM- controls was seen(6). The Page 11

12 investigators reported increased use of intravenous antibiotics in the NTM- group, in contrast to Torrens et al. However, increased frequency of sputum positive for Staphylococcus aureus in NTM+ was again reported. It is recognised that NTM are particularly difficult to culture in sputum colonised with Pseudomonas aeruginosa(23), and this may explain this finding. In our study six of the 10 NTM+ subjects were colonised with Pseudomonas aeruginosa, so this is less likely to be a factor. A diagnosis of CF, based on the presence of a CFTR mutation together with a positive sweat test, was more frequent in NTM+ than NTM- subjects. No other specific cause of bronchiectasis was associated with NTM isolation in our patients. NTM infection per se is a cause of bronchiectasis and it is possible, in the two subjects who were clinically deteriorating (subjects 8 and 9), that underlying NTM infection was a previously undiagnosed factor. We feel that this is less likely in the other eight patients, as non-treatment of NTMinfection causing bronchiectasis would be expected to result in further clinical deterioration, which was not evident in these cases. We treated three of our patients for NTM infection. In two cases (8 and 9) there was a significant clinical deterioration that could not be explained by factors other than NTM infection. The third (7) was under consideration for lung transplantation for bronchiectasis and the transplant team had recommended an attempt be made to eradicate the NTM. ATS microbiological criteria for NTM-infection require that three separate sputa are positive for NTM, although the time between sample collections is not specified. Two of the three (cases 7 and 8) therefore clearly achieved ATS microbiological criteria for NTM infection. It is not clear from the ATS Statement, although it seems sensible, that the three NTM should be of the same species. This is relevant to the third case (9), where Mycobacterium avium intracellulare was cultured on two occasions only, but three further sputa grew NTM that Page 12

13 could not be further identified. As the ATS radiographic criteria for diagnosis include multifocal bronchiectasis as one defining characteristic, and all our patients had known bronchiectasis, this criteria becomes largely redundant in this population. On HRCT, more NTM+ patients had peripheral mucous plugging than their matched controls. Furthermore, when the two subjects who only cultured an NTM on one occasion were excluded, the NTM+ group scored higher than the NTM- group for the component of the CT scoring system that assessed the extent of mucous plugging. Otherwise we did not find any difference in the validated CT-score in terms of either individual features or overall score. Even in the two patients with a clinical deterioration and multiple NTM positive cultures (8 and 9), there were no unique features when compared to the other 28 CT scores. In CF, Torrens et al also found no differences in Northern chest radiograph scores(4). This reinforces the view that, in pre-existing structural lung disease, it is difficult to define features with sufficient specificity to aid diagnosis of NTM infection. It is notable that even in patients with multiple NTM positive sputum cultures 58 of 78 (74%) samples were negative. We would therefore support the recommendation that a minimum of three sputum samples should be collected from a patient suspected of being infected with NTM, to minimise the risk of a false-negative result. Indeed it is likely that a proportion of our NTM- group would in fact have cultured NTM subsequently had we continued to screen their sputum for NTM during the study period. There are geographical variations in both the prevalence and causative organism in NTM infection in CF, and it is very likely that these regional and national differences apply in non- CF bronchiectasis also. The recent study by Wickremasinghe and colleagues based at the Royal Brompton Hospital in London reported one patient of 50 screened met ATS Page 13

14 microbiological criteria for NTM infection, and one more had a single isolate (19). Whilst the former prevalence (2%) is similar to our own (3%; cases 2, 7 and 8 in table 1), we had many more patients who cultured NTM on more than one occasion, but for various reasons did not fulfil the criteria for infection (cases 4, 5, 6, 9 and 10). This latter difference is difficult to explain, although it may be due partly to geographical variability and it would be very interesting to know of the prevalence of NTM in patients with bronchiectasis in other parts of the UK and internationally. Furthermore NTM can be difficult to culture, especially in the presence of other colonising organisms, so regional differences in technique could account for a degree of variability. In conclusion, we have reported a 10% prevalence of NTM (3% meeting ATS microbiological criteria for infection) in the sputum of screened patients with bronchiectasis in a specialist clinic. We did not identify any clinical features specifically associated with NTM isolation, except for the diagnosis of CF, which warrants further investigation. Previously reported radiographic hallmarks of NTM infection in bronchiectasis (3) did not appear to identify infected individuals in our population, although the presence of peripheral mucous plugging had 90% sensitivity and 75% specificity for NTM isolation. Large prospective studies are required to provide further information to assist diagnosis and management in this difficult area. We would also be interested to know if the marked geographic difference in NTM prevalence seen in CF is also the case in non-cf bronchiectasis. In both cases, standardisation of microbiological assays is essential. Page 14

15 AcknowledgmentsWe would like to thank Fay Cafferty for statistical advice Page 15

16 Reference List 1. Diagnosis and Treatment of Disease Caused by Nontuberculous Mycobacteria. Am. J. Respir. Crit. Care Med. 1997;156(2):1S Management of opportunist mycobacterial infections: Joint Tuberculosis Committee Guidelines Subcommittee of the Joint Tuberculosis Committee of the British Thoracic Society. Thorax 2000;55(3): Erasmus JJ, McAdams HP, Farrell MA, Patz EF, Jr. Pulmonary nontuberculous mycobacterial infection: radiologic manifestations. Radiographics 1999;19(6): Torrens JK, Dawkins P, Conway SP, Moya E. Non-tuberculous mycobacteria in cystic fibrosis. Thorax 1998;53(3): Olivier KN, Weber DJ, Wallace RJ, Jr., Faiz AR, Lee JH, Zhang Y, et al. Nontuberculous mycobacteria. I: multicenter prevalence study in cystic fibrosis. Am J Respir Crit Care Med 2003;167(6): Olivier KN, Weber DJ, Lee JH, Handler A, Tudor G, Molina PL, et al. Nontuberculous mycobacteria. II: nested-cohort study of impact on cystic fibrosis lung disease. Am J Respir Crit Care Med 2003;167(6): Pasteur MC, Helliwell SM, Houghton SJ, Webb SC, Foweraker JE, Coulden RA, et al. An investigation into causative factors in patients with bronchiectasis. Am J Respir Crit Care Med 2000;162(4 Pt 1): Standardization of Spirometry, 1994 Update. American Thoracic Society. Am J Respir Crit Care Med 1995;152(3): Page 16

17 9. Leitritz L, Schubert S, Bucherl B, Masch A, Heesemann J, Roggenkamp A. Evaluation of BACTEC MGIT 960 and BACTEC 460TB systems for recovery of mycobacteria from clinical specimens of a university hospital with low incidence of tuberculosis. J Clin Microbiol 2001;39(10): Gangadharam PR. Microbiology of nontuberculosis mycobacteria. Semin Respir Infect 1996;11(4): Bhalla M, Turcios N, Aponte V, Jenkins M, Leitman BS, McCauley DI, et al. Cystic fibrosis: scoring system with thin-section CT. Radiology 1991;179(3): Diederich S, Jurriaans E, Flower CD. Interobserver variation in the diagnosis of bronchiectasis on high-resolution computed tomography. Eur Radiol 1996;6(6): Grenier P, Mourey-Gerosa I, Benali K, Brauner MW, Leung AN, Lenoir S, et al. Abnormalities of the airways and lung parenchyma in asthmatics: CT observations in 50 patients and inter- and intraobserver variability. Eur Radiol 1996;6(2): Reiff DB, Wells AU, Carr DH, Cole PJ, Hansell DM. CT findings in bronchiectasis: limited value in distinguishing between idiopathic and specific types. AJR Am J Roentgenol 1995;165(2): McGuinness G, Naidich DP. Bronchiectasis: CT/clinical correlations. Semin Ultrasound CT MR 1995;16(5): Grenier P, Cordeau MP, Beigelman C. High-resolution computed tomography of the airways. J Thorac Imaging 1993;8(3): Page 17

18 17. Shah RM, Sexauer W, Ostrum BJ, Fiel SB, Friedman AC. High-resolution CT in the acute exacerbation of cystic fibrosis: evaluation of acute findings, reversibility of those findings, and clinical correlation. AJR Am J Roentgenol 1997;169(2): Helbich TH, Heinz-Peer G, Eichler I, Wunderbaldinger P, Gotz M, Wojnarowski C, et al. Cystic fibrosis: CT assessment of lung involvement in children and adults. Radiology 1999;213(2): Wickremasinghe M, Ozerovitch LJ, Davies G, Wodehouse T, Chadwick MV, Abdallah S, et al. Non-tuberculous mycobacteria in patients with bronchiectasis. Thorax 2005;60(12): Smith MJ, Efthimiou J, Hodson ME, Batten JC. Mycobacterial isolations in young adults with cystic fibrosis. Thorax 1984;39(5): Kilby JM, Gilligan PH, Yankaskas JR, Highsmith WE, Jr., Edwards LJ, Knowles MR. Nontuberculous mycobacteria in adult patients with cystic fibrosis. Chest 1992;102(1): Aitken ML, Burke W, McDonald G, Wallis C, Ramsey B, Nolan C. Nontuberculous mycobacterial disease in adult cystic fibrosis patients. Chest 1993;103(4): Bange FC, Kirschner P, Bottger EC. Recovery of mycobacteria from patients with cystic fibrosis. J Clin Microbiol 1999;37(11): Page 18

19 Figure 1: Cause of bronchiectasis. ABPA = allergic bronchopulmonary aspergillosis; CF = cystic fibrosis; RA = Rheumatoid-associated; TB = tuberculosis; GORD = gastro oesophageal reflux disease; IBD = inflammatory bowel disease. asterisk = p < 0.01 NTM+ vs. NTM-. Absolute patient numbers with each diagnosis are shown outside the bars. Figure 2: FEV1 trends before and after first NTM positive culture (at month 0). Squares indicate subjects subsequently treated for NTM infection; closed are pretreatment and open after treatment. Page 19

20 Figure 3: CT image demonstrating bilateral lower lobe bronchiectasis, with peripheral mucous plugging and tree-in-bud opacities seen particularly in the left lower lobe. Page 20

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22 Subject Age yrs NTM cultured FEV1 (%pred) Aetiology Long-term antibiotics Other microorganisms cultured 1 70 Environmental scotochromagen x 1 Negative x M. fortuitum x 3 Negative x M. fortuitum x 1 Negative x M. fortuitum x 2 M. xenopi x 1 Negative x M. malmoense x 2 Environmental scotochromagen x 1 Negative x M. xenopi x 1 M. chelonae x 1 Negative x 10 7* 48 M. xenopi x 3 Negative x 8 8* 49 M. avium intracellulare x 3 M. simiae x 1 M. species x 2 Negative x 13 9* 61 M. avium intracellulare x 2 M. species x 3 M. terrae x 1 Negative x M. avium intracellulare x 2 Negative x 2 80 Inhalation injury Nebulised colomycin P.aeruginosa 70 Rheumatoidassociated Nebulised colomycin Oral doxycycline P.aeruginosa H.influenzae S.pneumoniae S.aureus 57 CF Nebulised colomycin P.aeruginosa 76 Idiopathic None P.aeruginosa 68 Post-infective Oral doxycycline H.influenzae. S.aureus M.catarrhalis 35 Post-infective Oral amoxicillin P.aeruginosa M.catarrhalis S.pneumoniae 11 Idiopathic None P.aeruginosa 56 ABPA Nebulised colomycin P.aeruginosa 38 CF Nebulised colomycin P.aeruginosa. 108 Idiopathic Oral amoxicillin P.aeruginosa Coliform Table 1: Clinical characteristics of the ten subjects sputum culture-positive for non-tuberculous mycobacteria (NTM). Asterisk indicates the three subjects treated for NTM infection. ABPA = allergic bronchopulmonary aspergillosis; CF = cystic fibrosis Page 22

23 NTM+ (N = 10) NTM- (N = 88) Age 59.4 (3.3) 59.3 (1.4) Male 4 28 FEV1 %predicted 59.9 (8.6) 63.9 (2.3) FVC %predicted 86.0 (6.9) 85.8 (2.0) CFTR mutations 2 F508 het 2 F508 het 2 R117h het 7 none 73 none Colonisation: Long-term antibiotics: P.aeruginosa H.influenzae S.aureus 1 not known not known Colomycin (nebulised) 5 20 Gentamicin (nebulised) 0 3 Amoxicillin (oral) nebulised Azithromycin (oral) * 0 1 Ciprofloxacin (oral) * 0 1 Clarithromycin (oral) * 0 4 Doxycycline (oral) * 2 11 Metronidazole (oral) 0 1 Flucloxacillin (oral) 0 1 Trimethoprim (oral) 0 1 None 2 43 Table 2: Patient characteristics for those with (NTM+) and without (NTM-) nontuberculous mycobacteria cultured from their sputum over the study period. Mean (SEM) are shown for age and spirometric values. Two patients in the NTM- group were taking both colomycin and azithromycin long term, and one both colomycin and amoxicillin. One NTM+ subject was taking both colomycin and doxycycline. Antibiotics marked with an asterisk can have anti-mycobacterial activity. Page 23

24 NTM+ NTM- P (N = 10) (N = 20) Age FEV 1 % predicted No. colonised with P. aeruginosa CT scores: Consolidation / collapse Bronchial dilatation Bronchial wall thickening Sacculations / abscesses Mosaic perfusion Segments affected Tree-in-bud Mucous plugging Total Score CT features: Axial distribution 2c, 3m, 5p 12m, 8p 0.10 Mucus plugging 1a, 9p 15a, 5p Table 3: Mean HRCT scores each of the eight components are scores out of three as described in the methods; the total score is out of 24. Number of subjects in each group with noted features on HRCT. Key: a = absent, c = central, m = mixed, p = peripheral. P value shown from students t-test for age and FEV 1 ; Mann Whitney-U test for CT scores; chi-squared test for number colonised with Pseudomonas and CT features. Page 24