JCF-01167; No of Pages 6
Journal of Cystic Fibrosis xx (2015) xxx – xxx www.elsevier.com/locate/jcf
Original Article
Reduced risk of nontuberculous mycobacteria in cystic fibrosis adults receiving long-term azithromycin Nathalie Coolen a,b , Philippe Morand a,c , Clémence Martin a,b , Dominique Hubert a,b , Reem Kanaan a,b , Jeanne Chapron a,b , Isabelle Honoré a,b , Daniel Dusser a,b , Etienne Audureau a,d , Nicolas Veziris e,f,g , Pierre-Régis Burgel a,b,⁎ a
Université Paris Descartes, Sorbonne Paris Cité, Paris, France Service de Pneumologie, Hôpital Cochin, AP-HP, Paris, France c Service de Bactériologie, Hôpital Cochin, AP-HP, Paris, France d Service d'Informatique Médicale et de Biostatistiques, Hôpital Cochin, AP-HP, Paris, France Sorbonne Universités, UPMC Univ Paris 06, CR7, Centre d'Immunologie et des Maladies Infectieuses, CIMI, team E13 (Bacteriology), F-75013 Paris, France f INSERM, U1135, Centre d'Immunologie et des Maladies Infectieuses, CIMI, team E13 (Bacteriology), F-75013 Paris, France g AP-HP, Hôpital Pitié-Salpêtrière, Centre National de Référence des Mycobactéries et de la Résistance des Mycobactéries aux Antituberculeux, Bactériologie-Hygiène, F-75013 Paris, France b
e
Received 14 November 2014; revised 25 January 2015; accepted 16 February 2015
Abstract Background: Azithromycin reduces exacerbations in cystic fibrosis (CF) patients. Our aim was to investigate its association with nontuberculous mycobacteria isolation and macrolide susceptibility. Methods: From 2006 to 2010, all adult CF subjects at Cochin Hospital (Paris, France) harboring at least one positive NTM isolate were identified (Cases). In a nested case–control study, each Case was individually matched for age and gender with up to 4 CF adults with no NTM isolate (Controls). Clinical data at the time of first NTM isolate (index date) in Cases were compared with those of Controls using multivariate conditional regression analysis. Results: CF subjects with positive NTM isolates (Cases, n = 41) were matched to 155 Controls. Among Cases, 48.7% had isolates from Mycobacterium avium complex and 58.5% from Mycobacterium abscessus complex, and 31 Cases fulfilled the 2007 American Thoracic Society criteria for NTM infection (ATS+ Cases). Cases and ATS+ Cases were more likely to have low body mass index and colonization with Aspergillus fumigatus. Azithromycin was associated with a two-fold reduction in NTM isolates. Only one M. avium complex isolate had acquired macrolide resistance. Conclusion: These data suggest that azithromycin is a primary prophylaxis for NTM infection in CF adults. © 2015 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Keywords: Cystic fibrosis; Nontuberculous mycobacteria; Risk factors; Macrolides; Aspergillus fumigatus
1. Introduction Nontuberculous mycobacteria (NTM) have emerged as recognized pathogens in patients with cystic fibrosis (CF) [1]. NTM have been isolated from the respiratory tract of CF patients with ⁎ Corresponding author at: Service de Pneumologie, Hôpital Cochin, 27 rue du Faubourg St Jacques, 75014 Paris, France. Tel.: +33 1 58 41 23 67; fax: + 33 1 46 33 82 53. E-mail address: [email protected] (P.-R. Burgel).
prevalence values ranging from 5% to 20% [1–4]. Although an initial large scale epidemiological study did not demonstrate an association between NTM infections and worsening of lung function [5], it was subsequently reported that NTM could cause significant morbidity and mortality in CF patients [6–8]. A recent single center analysis has further suggested that NTM infection, especially with Mycobacterium abscessus, was associated with increased rate of decline in FEV1 [9]. These findings justify the growing interest in developing preventive and therapeutic interventions for NTM in CF patients.
http://dx.doi.org/10.1016/j.jcf.2015.02.006 1569-1993/© 2015 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Please cite this article as: Coolen N, et al, Reduced risk of nontuberculous mycobacteria in cystic fibrosis adults receiving long-term azithromycin, J Cyst Fibros (2015), http://dx.doi.org/10.1016/j.jcf.2015.02.006
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N. Coolen et al. / Journal of Cystic Fibrosis xx (2015) xxx–xxx
Several risk factors associated with isolation of NTM have been identified in CF subjects. NTM-positive sputum cultures were found more frequently in adolescents and in adult subjects than in young children [1,2,10], and Aspergillus fumigatus colonization was associated with NTM isolates in several studies [9–11]. An important unanswered question lies in the poorly understood interference of treatments used for CF lung disease with NTM. For example, Mussaffi et al. reported that systemic steroids, which were often used for the treatment of allergic bronchopulmonary aspergillosis (ABPA), were associated with severe NTM disease in CF subjects [11]. However, very little data exist on the impact of other potentially important therapies (e.g., inhaled steroids, long-term macrolides, inhaled and systemic antibiotics) on the incidence of NTM. Further, azithromycin may also have an impact on the incidence of macrolide-resistant NTM whether resistance is natural such as the one conferred by erm(41) gene in M. abscessus complex [12] or whether resistance is acquired such as the one conferred by rrl gene mutations in either M. abscessus complex and Mycobacterium avium complex [13]. In the present study, our goals were to examine the impact of the various drugs used in CF adults on NTM incidence and their macrolide susceptibility. We performed a case–control study, comparing adult CF patients who had NTM-positive isolates to individually matched CF patients who had no positive NTM sputum culture. Our results suggested that long-term azithromycin therapy was associated with a reduction in the incidence of NTM isolates.
2. Methods 2.1. Selection of cases and controls The Pulmonary Department at Cochin University Hospital (Paris, France) hosts an accredited CF adult center. The CF population followed in our center comprised 311 patients in 2006 and progressively increased to 347 patients at the end of the study period (May 2010). Patients followed at our center were routinely (at least once a year and often more frequently) screened for NTM. From January 2006 to May 2010, all consecutive CF adults followed in this center and who had at least one respiratory sample positive for NTM (Cases) were identified. Four patients with positive samples for Mycobacterium gordonae, which is generally considered a contaminant and not usually a clinically relevant cause of lung disease [14], were excluded from the analyses. Because NTM can be isolated due to environmental contamination, more than one culture-positive specimen for NTM is considered necessary to establish diagnosis of NTM infection [14]. The 2007 American Thoracic Society (ATS) guidelines for the diagnosis of NTM lung disease provide with compatible clinical, radiographic and bacteriologic criteria for NTM infection (i) 2 positive sputum cultures or 1 positive culture obtained through bronchial wash, lavage or lung biopsy; or (ii) at least 1 sputum or bronchial washing culture positive for NTM and evident mycobacterial histopathological features [14]. Thus, our study identified both subjects who fulfilled the 2007 ATS criteria (ATS + Cases) and those who did not (ATS − Cases).
The Control group was composed of CF subjects followed in the same institution. For each Case, we selected up to 4 Controls who had no positive respiratory sample for NTM. Controls were individually matched with Cases for gender and closest age (maximum age difference: ± 3 years) at the time of first NTM identification in the index case. All patients who were identified as Cases had previously documented negative cultures and were considered incident Cases at index date. The study conformed to the Declaration of Helsinki and was approved by the Institutional Review Board of the French learned society for respiratory medicine—Société de Pneumologie de Langue Française (approval #, 2012/013). Information was provided to patients, but written consent was not required due to the observational design of this study in accordance with French laws. 2.2. Data collection CF was diagnosed on the basis of clinical manifestations with a sweat chloride concentration exceeding 60 mM/L and/or two disease-causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene [15]. Using our local database, we collected clinical and microbiological data that were obtained at the time of first positive respiratory sample for NTM (index date) in Cases and in their Controls. Clinical data included age, gender, age at CF diagnosis, CFTR genotype, body mass index (BMI), exocrine pancreatic insufficiency, diabetes mellitus, liver cirrhosis and pulmonary function tests (forced expiratory volume in one second (FEV1, % predicted), forced vital capacity (FVC, % predicted)). Data on treatment (long-term oxygen therapy, azithromycin use, inhaled antibiotic therapy, inhaled or oral corticosteroid therapy, intravenous antibiotic therapy, and quinolones) in the 12 months before the index date were also obtained. Chronic colonization with major Gram negative (Pseudomonas aeruginosa, Burkholderia cepacia, or Haemophilus influenza) or Gram positive (methicillin-susceptible Staphylococcus aureus or methicillin-resistant S. aureus) bacteria found in CF patients were recorded. Chronic A. fumigatus colonization (defined as three or more positive respiratory samples in the past two years) and a history of ABPA were also recorded. 2.3. Processing of sputum samples for identification of NTM Sputum samples were successively processed with N-acetylcysteine/NaOH (BBL MycoPrep, BD, Franklin Lakes, NJ) and 5% oxalic acid, as previously described [16]. Before inoculation onto growing media, vancomycin, tobramycin, colistin and amphotericin B were added to each specimen at final concentrations of 2.5 mg/L, 4.0 mg/L, 4.105 UI/L and 2.5 mg/L, respectively. Specimens were inoculated onto two Coletsos (Bio-Rad, Marne-la-Coquette, France) and one Lowenstein– Jensen (LJ) slants, and one mycobacteria growth indicator tube (BBL MGIT, BD). Coletsos and LJ media were supplemented with vancomycin and amphotericin B (0.8 mg/L and 3 mg/L final concentration, respectively), and the MGIT medium was supplemented with Middlebrook OADC enrichment, as well as with polymyxin B, amphotericin B, nalidixic acid, trimethoprim,
Please cite this article as: Coolen N, et al, Reduced risk of nontuberculous mycobacteria in cystic fibrosis adults receiving long-term azithromycin, J Cyst Fibros (2015), http://dx.doi.org/10.1016/j.jcf.2015.02.006
N. Coolen et al. / Journal of Cystic Fibrosis xx (2015) xxx–xxx
azlocillin and vancomycin (4.104 UI/L, 6 mg/L, 18 mg/L, 4.5 mg/L, 4.5 mg/L and 0.5 mg/L, respectively). MGIT medium and one Coletsos slant were incubated at 37 °C, whereas LJ and one Coletsos were incubated at 30 °C. Coletsos and LJ were examined twice a week for 12 weeks and the MGIT vials were monitored in a BACTEC MGIT 960 system (BD). Identification of acid-fast organisms at the species level was performed with reverse-hybridization INNO-LiPA (Innogenetics, Gent, Belgium) or PCR-hybridization Genotype-CM (BioCentric, Bandol, France), and confirmed by sequencing of the hsp gene. 2.4. Macrolide susceptibility testing Macrolide susceptibility testing relied on measurement of clarithromycin minimum inhibitory concentrations (MICs) as recommended [14]. Clarithromycin MIC was determined by the broth microdilution method, using Sensititer® RGMYCO plates (Trek Diagnosis systems, Biocentric, France) [17]. Plates were submitted to an extended incubation as described by Nash et al. for strains belonging to the M. abscessus complex [12], with successive readings after 5, 7, 9, and 14 days of incubation at 30 °C. Mutations in the 23S rRNA gene (rrl) were sought for strains displaying clarithromycin MIC considered resistant according to CLSI at 5 day reading [17]. Mutations in the 23S rRNA gene (rrl) were sought by PCR sequencing as described by Wallace et al. [13]. Sequences were compared to the M. abscessus genome reference sequence NC_010397 [18]. 2.5. Statistical analysis Continuous variables were reported as mean (standard deviation [SD]) or median (interquartile range [IQR]) and were compared using parametric t-test or non-parametric Mann–Whitney– Wilcoxon's test to account for non-normality of the variable distributions, when demonstrated by the use of the Shapiro–Walk test for normality. Categorical variables were reported as count and percentage and were compared using Chi-squared or Fisher's exact tests, as appropriate. Multivariate conditional logistic regression was used to examine which independent factors were associated with all Cases or with ATS+ Cases, accounting for the matching between cases and controls. All variables with a P value b 0.20 in univariate analyses were entered into the models, following a stepwise backwards approach by removing non-significant variables at each step until the final model was reached. Statistical analyses were 2-sided and P b 0.05 was considered to have statistical significance. Analyses were performed using Stata V11.0 (StataCorp, College Station, TX, USA). 3. Results 3.1. Prevalence of NTM in CF adults From January 2006 to May 2010, we identified 41 adult CF subjects with at least one positive respiratory sample positive for NTM (Cases), including 31 subjects who fulfilled the 2007 ATS criteria for NTM disease (ATS + Cases) and 10 subjects who did not fulfill these criteria (ATS − Cases). The prevalence
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of Cases was 12.4% and the prevalence of ATS + Cases was 9.5%. As shown in Table 1, M. abscessus complex was the most prevalent species and was identified in 24/41 (58.5%) subjects. M. avium/intracellulare complex was found in 20/41 (48.7%) subjects. Other species (including Mycobacterium fortuitum, Mycobacterium simiae, and Mycobacterium kansasii) were found in only 5/41 (12.2%) subjects. Seven (6 ATS +) Cases had positive cultures with more than one species. M. abscessus isolates were more prevalent in ATS + Cases than in ATS − Cases (P b 0.05). The mean (SD) number of respiratory samples processed for NTM culture was 10.8 (8.0) in Cases, and cultures of respiratory samples were positive in 63% vs. 23% samples in ATS + Cases and ATS − Cases (P = 0.02). 3.2. Risk factors for incident NTM in CF adults A total of 155 Controls (mean 3.8 Controls per Case) were identified. The mean ± SD numbers of documented negative cultures per patient prior to index date were 5.2 ± 8.8 vs. 4.1 ± 7.0 in Cases and in Controls, respectively (P = 0.38). In univariate analysis (see Table 2), low BMI (b 18 kg/m2) was found in 36.6% Cases vs. 17.5% in Controls (P = 0.02), and in 41.9% ATS + Cases vs. 20.7% in their Controls (P = 0.01). Chronic A. fumigatus colonization was found in 34.1% Cases vs. 16.9% Controls (P = 0.02) and in 38.7% ATS + Cases vs. 14.7% in their Controls (P = 0.005). Cases and ATS + Cases had significantly more Haemophilus influenzae colonization than their Controls (14.6% vs. 4.5% and 16.1% vs. 4.3%, respectively; P = 0.02 and P = 0.03). Methicillinsusceptible S. aureus was found more frequently in Cases vs. Controls (P = 0.03) but not in ATS+ Cases vs. their Controls (P = 0.16). Other pathogens and other clinical manifestations (e.g., diabetes, liver cirrhosis and pancreatic insufficiency) were not associated with NTM. Treatment with azithromycin (250 mg/d) was present at index date in 87 (44.8%) of all 196 (41 Cases, 155 Controls) CF patients, with azithromycin being prescribed in 31.7% Cases vs. 48.4% Controls (P = 0.05), and in 25.8% ATS+ Cases vs. in 47.8% of their Controls (P = 0.04). The mean (SD) exposure time to azithromycin was 3.2 (2.9) years and was not different between Cases and Controls (not shown). Other treatments, including nebulized or IV antibiotics, as well as inhaled or oral steroids were not associated with NTM. Table 1 Microbiological results in adult CF subjects with NTM positive isolates (Cases). All Cases (n = 41)
ATS+ Cases (n = 31)
ATS− Cases (n = 10)
Respiratory samples per case, median [IQR] Total numbers of samples 8 [5–13] Numbers of NTM positive samples 5 [2–8]
13 [7–17]** 7 [4–9]**
6 [4–7] 1[1–26]
Mycobacteria, n (%) M. abscessus complex M. avium complex Other species ≥2 species, n (%)
22 (71.0)⁎ 14 (45.2) 2 (6.4)⁎ 6 (19.4)
2 (20.0) 6 (60.0) 3 (30.0) 1 (10.0)
24 (58.5) 20 (48.7) 5 (12.2) 7 (17.1)
⁎P b 0.05 and ⁎⁎P b 0.01 when comparing ATS+ with ATS− Cases.
Please cite this article as: Coolen N, et al, Reduced risk of nontuberculous mycobacteria in cystic fibrosis adults receiving long-term azithromycin, J Cyst Fibros (2015), http://dx.doi.org/10.1016/j.jcf.2015.02.006
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N. Coolen et al. / Journal of Cystic Fibrosis xx (2015) xxx–xxx
Table 2 Comparison of clinical characteristics between Cases and Controls. All Cases (n = 41)
Controls (n = 155)
P values
ATS + Cases (n = 31)
Controls (n = 117)
P values
27 (65.9) 26.2 [19.6–30.3] 2.0 [0.2–7.0]
102 (65.8) 26.2 [20.5–31.4] 1.5 [0.2–10.0]
1 0.95
20 (64.5) 26.1 [19.2–30.3] 2.0 [0.3–7.0]
76 (65.0) 26.0 [20.5–30.8] 2.0 [0.3–12.0]
1.00 0.88
71 (46.1) 59 (38.3) 24 (15.6) 131 (85.1) 38 (24.5) 16 (10.4) 19.7 [18.4–21.4] 27 (17.5) 56 [41–83] 77 [58–94]
0.97
0.02 0.77 0.78
14 (45.2) 11 (35.5) 6 (19.3) 25 (80.6) 7 (22.6) 40 (12.9) 18.9 [17.0–21.6] 13 (41.9) 59 [40–77] 78 [63–84]
52 (44.8) 47 (40.5) 17 (14.7) 97 (83.6) 26 (22.4) 12 (10.3) 19.7 [18.3–21.0] 24 (20.7) 55 [41–83] 77 [58–94]
0.78
BMI b 18 kg/m2, n (%) FEV1, % predicted FVC, % predicted
18 (43.9) 16 (39.0) 7 (17.1) 32 (78.0) 8 (19.5) 4 (9.8) 19.5 [17.1–20.7] 15 (36.6) 60 [46–76] 78 [68–84]
0.01 0.95 0.50
Colonization, n (%) - P. aeruginosa - MSSA - MRSA - H. influenzae - B. cepacia - A. fumigatus Oxygen therapy, n (%)
21 (51.2) 27 (65.9) 4 (9.8) 6 (14.6) 0 (0) 14 (34.1) 3 (7.3)
96 (62.3) 72 (46.8) 18 (11.7) 7 (4.5) 5 (3.2) 26 (16.9) 20 (13.0)
0.20 0.03 0.73 0.02 0.24 0.02 0.32
15 (48.4) 20 (64.5) 3 (9.7) 50 (16.1) 0 (0) 12 (38.7) 3 (9.7)
71 (61.2) 57 (49.1) 14 (12.1) 50 (4.3) 4 (3.4) 17 (14.7) 13 (11.2)
0.22 0.16 1.00 0.03 0.58 0.005 1
Antibiotics - Oral azithromycin, n (%) - Oral ciprofloxacin, n (%) - Inhaled a, n (%) - IV antibiotics, days/yr. Oral corticosteroids N 3 months, n (%)
13 (31.7) 15 (36.6) 16 (39.0) 14 [0–28] 2 (4.9)
74 (48.4) 45 (29.2) 75 (48.7) 14 [0–43] 5 (3.2)
0.05 0.37 0.27 0.33 0.62
8 (25.8) 12 (38.7) 12 (38.7) 14 [0–29] 2 (6.5)
55 (47.8) 34 (29.3) 54 (46.6) 14 [0–43] 4 (3.4)
0.04 0.39 0.54 0.20 0.61
20 (48.8)
58 (37.7)
0.20
13 (41.9)
44 (37.9)
0.69
0 [0–2000] 963
0 [0–1000] 662
0.13
0 [0–1000] 838
0 [0–1000] 694
0.53
Male, n (%) Age, yrs. Age at CF diagnosis, months CFTR mutations, n (%) - F508del/F508del - F508del/other - other/other Exocrine pancreatic insufficiency, n (%) Diabetes mellitus, n (%) Cirrhosis, n (%) BMI, kg/m2
Inhaled corticosteroids - n (%) - Beclomethasone eq., μg/d Median [IQR] Mean
0.87
0.34 0.68 1 0.33
0.99
0.79 1.00 0.75 0.65
CF: cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; BMI, body mass index; FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity; MSSA, methicillin-susceptible S. aureus; MRSA, methicillin-resistant S. aureus; IV, intravenous. Values are n (%) or median [IQR] unless otherwise specified. Bold data were for P values below 0.05 (statistical significance). a Tobramycin and/or colomycin.
In multivariate analysis (see Table 3), Cases and ATS + Cases had more often low BMI compared to their Controls (P = 0.007 and P = 0.02, respectively). Cases and ATS + Cases had increased prevalence of A. fumigatus colonization (P = 0.02 and P = 0.03, respectively). Cases were twice less likely to have received azithromycin than Controls (P = 0.05) and a similar trend was observed when ATS + Cases were compared to their Controls (P = 0.06). The distribution of mycobacterial species was not different between Cases or ATS + Cases who had received azithromycin and those who had not (see Table 4). 3.3. Macrolide susceptibility of NTM isolates Among all NTM isolates only one had mutation in rrl gene (which was previously associated with acquired macrolide
resistance): a M. avium isolate in an ATS − case who had received azithromycin prophylaxis. To further examine the association of azithromycin with macrolide resistance, we Table 3 Multivariate analysis of risk factors for NTM in adult CF subjects. OR
SE
95% CI
P values
All Cases (n = 41) vs. Controls (n = 155) 3.56 1.69 BMI b 18 kg/m2 A. fumigatus colonization 2.51 1.05 Azithromycin 0.45 0.18
1.41 – 9.02 1.11 – 5.71 0.19 – 1.00
0.007 0.03 0.05
ATS+ Cases (n = 31) vs. Controls (n = 117) 3.21 1.65 BMI b 18 kg/m2 A. fumigatus colonization 3.45 1.75 Azithromycin 0.40 0.20
1.17 – 8.78 1.27 – 9.35 0.15 – 1.05
0.02 0.02 0.06
OR, odds ratio; SE, standard error; 95% CI, 95% confidence interval.
Please cite this article as: Coolen N, et al, Reduced risk of nontuberculous mycobacteria in cystic fibrosis adults receiving long-term azithromycin, J Cyst Fibros (2015), http://dx.doi.org/10.1016/j.jcf.2015.02.006
N. Coolen et al. / Journal of Cystic Fibrosis xx (2015) xxx–xxx
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Table 4 Mycobacterial species isolated among all cases and among ATS+ Cases depending on previous azithromycin prophylaxis. All Cases (n = 41) Azithromycin
M. abscessus complex (M. abscessus + M. bolletii + M. massiliense) M. abscessus, M. bolletii M. massiliense M. avium complex Other Total a
Yes
No
4 (31%) 3 (23%) 1 (8%) 7 (54%) a 2 (15%) 13
17 (61%) 12 (43%) 5 (18%) 8 (29%) 3 (11%) 28
ATS + Cases (n = 31) P values
Azithromycin Yes
No
0.10 0.30 0.65 0.16 0.65
4 3 1 3 1 8
16 (69.5%) 11 (48%) 5 (22%) 6 (26%) 1 (4%) 23
(50.0%) (37.5%) (12.5%) (37.5%) (12.5%)
P values
0.40 0.70 1 0.66 0.26
Including 1 with acquired macrolide resistance (due to rrl gene mutation).
compared macrolide susceptibility among isolates of Cases that had received azithromycin prophylaxis and Cases that had not received azithromycin prophylaxis. Among all Cases, 37 isolates underwent susceptibility testing (4 not tested corresponded to 3 M. gordonae isolates and 1 M. fortuitum). Seventeen NTM isolates were resistant to clarithromycin: 1 M. avium (acquired resistance due to rrl mutation, see above), 1 M. simiae, 14 M. abscessus and 1 Mycobacterium bolletii (natural inducible resistance due to erm(41) gene). The proportion of (acquired + natural) resistant isolates was not different among cases that had received azithromycin compared to those who had not received azithromycin (31% vs. 46%, P = 0.50). A similar lack of difference was observed among ATS + Cases (38 vs. 52%, P = 1). 4. Discussion The present case–control study was designed to identify risk factors associated with incident NTM isolates in adult CF patients, with a special interest on the association of NTM with treatments for CF lung disease. We found that incident NTM isolates were associated with chronic A. fumigatus colonization and occurred more often in subjects with low BMI. Long-term treatment with low-dose azithromycin was associated with a two-fold reduction in the incidence of NTM, suggesting that azithromycin could be a primary prophylaxis for NTM in CF subjects. Azithromycin therapy was not associated with increased rates of NTM resistance to macrolides. We found a lower rate of NTM isolates among adult CF subjects receiving long-term azithromycin therapy. Previous studies that evaluated the association between long-term azithromycin and NTM incidence in CF patients have shown conflicting results. Renna et al. compared 14 adult CF patients with NTM to 184 CF patients without NTM and found long-term azithromycin therapy in 12/14 (86%) NTM infected patients but only in 74/184 (40%) non-infected patients [19]. In contrast with our present findings, the authors suggested that azithromycin may predispose to NTM infection [19]. Other studies found no significant association between NTM incidence and azithromycin but these studies were limited by sample size [20], by difficulties in establishing correct exposure to azithromycin in registry data [21] or by the absence of matching between Cases and Controls [10]. In a recent case–control study based on data obtained in the CF Foundation Patient Registry [22], Binder et al. found that incident NTM cases were less likely to have received
prior long-term macrolide [22]. Our results concur with those of this latter study to suggest that long-term azithromycin use could act as a primary prophylaxis for NTM in CF patients. A major concern when using long-term azithromycin monotherapy is the possible emergence of macrolide-resistant NTM isolates. To the best of our knowledge our study was the first to compare macrolide resistance in NTM cases in CF patients according to azithromycin exposure. When combining strains displaying natural inducible clarithromycin resistance (due to erm (41) gene) and acquired resistance (due to rrl gene mutations), it appeared that NTM resistance to macrolides was not increased in CF subjects treated with azithromycin. In fact the trend was rather in favor of less macrolide resistant isolates among cases that had received azithromycin prophylaxis. This trend was mainly due to the reduced occurrence of M. abscessus and M. bolletii: 20% of isolates among cases that received azithromycin vs. 40% among cases that did not receive azithromycin. However it must be underlined that the only strain with acquired macrolide resistance (due to rrl gene mutation) was isolated in an azithromycin-treated patient. This finding had no clinical consequence in this ATS − Case, in whom no NTM specific treatment was required and in whom no NTM isolate was further cultured after azithromycin discontinuation. However, acquired macrolide resistance in NTM could dramatically diminish chance for successful therapy against NTM if necessary. The small number of patients does not allow us to derive firm conclusion on the benefit or risk of longterm azithromycin therapy on prevalence of macrolide-resistant mycobacteria in CF patients. The present study was performed in a single CF center with homogeneous practices regarding drug prescription, respiratory sample collection and processing, which together strengthens data consistency. Individual matching of each Case with multiple Controls reduced potential bias related to control selection [23]. No significant difference existed in the number of respiratory samples cultured for NTM in Cases vs. Controls prior to index date, indicating that the likelihood of NTM isolation was similar in both groups of patients. In summary, macrolides have proven major beneficial effects in CF subjects (colonized or not with P. aeruginosa) [24–26], especially in the reduction in pulmonary exacerbations. Our study suggests that azithromycin also reduces the incidence of mycobacterial infection. Although the use of azithromycin was not associated with increased incidence of natural or acquired macrolide-resistant in this (relatively) small study, larger
Please cite this article as: Coolen N, et al, Reduced risk of nontuberculous mycobacteria in cystic fibrosis adults receiving long-term azithromycin, J Cyst Fibros (2015), http://dx.doi.org/10.1016/j.jcf.2015.02.006
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N. Coolen et al. / Journal of Cystic Fibrosis xx (2015) xxx–xxx
prospective studies will be required to fully understand the impact of long-term macrolide therapy on the NTM susceptibility to macrolides. Funding source None. References [1] Olivier KN, Weber DJ, Wallace RJ, Faiz AR, Lee JH, Zhang Y, et al. Nontuberculous mycobacteria. I: multicenter prevalence study in cystic fibrosis. Am J Respir Crit Care Med 2003;167:828–34. [2] Kilby JM, Gilligan PH, Yankaskas JR, Highsmith Jr WE, Edwards LJ, Knowles MR. Nontuberculous mycobacteria in adult patients with cystic fibrosis. Chest 1992;102:70–5. [3] Roux AL, Catherinot E, Ripoll F, Soismier N, Macheras E, Ravilly S, et al. Multicenter study of prevalence of nontuberculous mycobacteria in patients with cystic fibrosis in France. J Clin Microbiol 2009;47:4124–8. [4] Fauroux B, Delaisi B, Clement A, Saizou C, Moissenet D, Truffot-Pernot C, et al. Mycobacterial lung disease in cystic fibrosis: a prospective study. Pediatr Infect Dis J 1997;16:354–8. [5] 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;167:835–40. [6] Aitken ML, Limaye A, Potting P, Whimbey E, Goss CH, Tonelli MR, et al. Respiratory outbreak of Mycobacterium abscessus subspecies massiliense in a lung transplant and cystic fibrosis center. Am J Respir Crit Care Med 2012; 185:231–2. [7] Sanguinetti M, Ardito F, Fiscarelli E, LaSorda M, D'Argenio P, Ricciotti G, et al. Fatal pulmonary infection due to multidrug-resistant Mycobacterium abscessus in a patient with cystic fibrosis. J Clin Microbiol 2001;39:816–9. [8] Chalermskulrat W, Sood N, Neuringer IP, Hecker TM, Chang L, Rivera MP, et al. Non-tuberculous mycobacteria in end stage cystic fibrosis: implications for lung transplantation. Thorax 2006;61:507–13. [9] Esther Jr CR, Esserman DA, Gilligan P, Kerr A, Noone PG. Chronic Mycobacterium abscessus infection and lung function decline in cystic fibrosis. J Cyst Fibros 2010;9:117–23. [10] Levy I, Grisaru-Soen G, Lerner-Geva L, Kerem E, Blau H, Bentur L, et al. Multicenter cross-sectional study of nontuberculous mycobacterial infections among cystic fibrosis patients, Israel. Emerg Infect Dis 2008;14:378–84. [11] Mussaffi H, Rivlin J, Shalit I, Ephros M, Blau H. Nontuberculous mycobacteria in cystic fibrosis associated with allergic bronchopulmonary aspergillosis and steroid therapy. Eur Respir J 2005;25:324–8. [12] Nash KA, Brown-Elliott BA, Wallace Jr RJ. A novel gene, erm(41), confers inducible macrolide resistance to clinical isolates of Mycobacterium abscessus but is absent from Mycobacterium chelonae. Antimicrob Agents Chemother 2009;53:1367–76.
[13] Wallace Jr RJ, Meier A, Brown BA, Zhang Y, Sander P, Onyi GO, et al. Genetic basis for clarithromycin resistance among isolates of Mycobacterium chelonae and Mycobacterium abscessus. Antimicrob Agents Chemother 1996;40:1676–81. [14] Griffith DE, Aksamit T, Brown-Elliott BA, Catanzaro A, Daley C, Gordin F, et al. An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med 2007; 175:367–416. [15] Farrell PM, Rosenstein BJ, White TB, Accurso FJ, Castellani C, Cutting GR, et al. Guidelines for diagnosis of cystic fibrosis in newborns through older adults: Cystic Fibrosis Foundation consensus report. J Pediatr 2008; 153:S4–S14. [16] Whittier S, Hopfer RL, Knowles MR, Gilligan PH. Improved recovery of mycobacteria from respiratory secretions of patients with cystic fibrosis. J Clin Microbiol 1993;31:861–4. [17] Clinical and Laboratory Standards Institute. Susceptibility testing of mycobacteria, nocardiae, and other aerobic actinomycetes, approved standard. 2nd ed. Wayne, PA, USA: CLSI; 2011. [18] Ripoll F, Pasek S, Schenowitz C, Dossat C, Barbe V, Rottman M, et al. Non mycobacterial virulence genes in the genome of the emerging pathogen mycobacterium abscessus. PLoS One 2009;4. [19] Renna M, Schaffner C, Brown K, Shang S, Tamayo MH, Hegyi K, et al. Azithromycin blocks autophagy and may predispose cystic fibrosis patients to mycobacterial infection. J Clin Invest 2011;121:3554–63. [20] Verregghen M, Heijerman HG, Reijers M, van Ingen J, van der Ent CK. Risk factors for Mycobacterium abscessus infection in cystic fibrosis patients; a case–control study. J Cyst Fibros 2012;11:340–3. [21] Catherinot E, Roux AL, Vibet MA, Bellis G, Lemonnier L, Le Roux E, et al. Inhaled therapies, azithromycin and Mycobacterium abscessus in cystic fibrosis patients. Eur Respir J 2013;41:1101–6. [22] Binder AM, Adjemian J, Olivier KN, Prevots RD. Epidemiology of nontuberculous mycobacterial infections and associated macrolide use among persons with cystic fibrosis. Am J Respir Crit Care Med 2013;188:807–12. [23] Bland JM, Altman DG. Matching. BMJ 1994;309:1128. [24] Foweraker JE, Laughton CR, Brown DF, Bilton D. Phenotypic variability of Pseudomonas aeruginosa in sputa from patients with acute infective exacerbation of cystic fibrosis and its impact on the validity of antimicrobial susceptibility testing. J Antimicrob Chemother 2005;55:921–7. [25] Saiman L, Anstead M, Mayer-Hamblett N, Lands LC, Kloster M, Hocevar-Trnka J, et al. Effect of azithromycin on pulmonary function in patients with cystic fibrosis uninfected with pseudomonas aeruginosa a randomized controlled trial. JAMA 2010;303:1707–15. [26] Saiman L, Marshall BC, Mayer-Hamblett N, Burns JL, Quittner AL, Cibene DA, et al. Azithromycin in patients with cystic fibrosis chronically infected with Pseudomonas aeruginosa: a randomized controlled trial. JAMA 2003;290:1749–56.
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Journal of Cystic Fibrosis xx (2015) xxx – xxx www.elsevier.com/locate/jcf
Original Article
Reduced risk of nontuberculous mycobacteria in cystic fibrosis adults receiving long-term azithromycin Nathalie Coolen a,b , Philippe Morand a,c , Clémence Martin a,b , Dominique Hubert a,b , Reem Kanaan a,b , Jeanne Chapron a,b , Isabelle Honoré a,b , Daniel Dusser a,b , Etienne Audureau a,d , Nicolas Veziris e,f,g , Pierre-Régis Burgel a,b,⁎ a
Université Paris Descartes, Sorbonne Paris Cité, Paris, France Service de Pneumologie, Hôpital Cochin, AP-HP, Paris, France c Service de Bactériologie, Hôpital Cochin, AP-HP, Paris, France d Service d'Informatique Médicale et de Biostatistiques, Hôpital Cochin, AP-HP, Paris, France Sorbonne Universités, UPMC Univ Paris 06, CR7, Centre d'Immunologie et des Maladies Infectieuses, CIMI, team E13 (Bacteriology), F-75013 Paris, France f INSERM, U1135, Centre d'Immunologie et des Maladies Infectieuses, CIMI, team E13 (Bacteriology), F-75013 Paris, France g AP-HP, Hôpital Pitié-Salpêtrière, Centre National de Référence des Mycobactéries et de la Résistance des Mycobactéries aux Antituberculeux, Bactériologie-Hygiène, F-75013 Paris, France b
e
Received 14 November 2014; revised 25 January 2015; accepted 16 February 2015
Abstract Background: Azithromycin reduces exacerbations in cystic fibrosis (CF) patients. Our aim was to investigate its association with nontuberculous mycobacteria isolation and macrolide susceptibility. Methods: From 2006 to 2010, all adult CF subjects at Cochin Hospital (Paris, France) harboring at least one positive NTM isolate were identified (Cases). In a nested case–control study, each Case was individually matched for age and gender with up to 4 CF adults with no NTM isolate (Controls). Clinical data at the time of first NTM isolate (index date) in Cases were compared with those of Controls using multivariate conditional regression analysis. Results: CF subjects with positive NTM isolates (Cases, n = 41) were matched to 155 Controls. Among Cases, 48.7% had isolates from Mycobacterium avium complex and 58.5% from Mycobacterium abscessus complex, and 31 Cases fulfilled the 2007 American Thoracic Society criteria for NTM infection (ATS+ Cases). Cases and ATS+ Cases were more likely to have low body mass index and colonization with Aspergillus fumigatus. Azithromycin was associated with a two-fold reduction in NTM isolates. Only one M. avium complex isolate had acquired macrolide resistance. Conclusion: These data suggest that azithromycin is a primary prophylaxis for NTM infection in CF adults. © 2015 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Keywords: Cystic fibrosis; Nontuberculous mycobacteria; Risk factors; Macrolides; Aspergillus fumigatus
1. Introduction Nontuberculous mycobacteria (NTM) have emerged as recognized pathogens in patients with cystic fibrosis (CF) [1]. NTM have been isolated from the respiratory tract of CF patients with ⁎ Corresponding author at: Service de Pneumologie, Hôpital Cochin, 27 rue du Faubourg St Jacques, 75014 Paris, France. Tel.: +33 1 58 41 23 67; fax: + 33 1 46 33 82 53. E-mail address: [email protected] (P.-R. Burgel).
prevalence values ranging from 5% to 20% [1–4]. Although an initial large scale epidemiological study did not demonstrate an association between NTM infections and worsening of lung function [5], it was subsequently reported that NTM could cause significant morbidity and mortality in CF patients [6–8]. A recent single center analysis has further suggested that NTM infection, especially with Mycobacterium abscessus, was associated with increased rate of decline in FEV1 [9]. These findings justify the growing interest in developing preventive and therapeutic interventions for NTM in CF patients.
http://dx.doi.org/10.1016/j.jcf.2015.02.006 1569-1993/© 2015 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Please cite this article as: Coolen N, et al, Reduced risk of nontuberculous mycobacteria in cystic fibrosis adults receiving long-term azithromycin, J Cyst Fibros (2015), http://dx.doi.org/10.1016/j.jcf.2015.02.006
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Several risk factors associated with isolation of NTM have been identified in CF subjects. NTM-positive sputum cultures were found more frequently in adolescents and in adult subjects than in young children [1,2,10], and Aspergillus fumigatus colonization was associated with NTM isolates in several studies [9–11]. An important unanswered question lies in the poorly understood interference of treatments used for CF lung disease with NTM. For example, Mussaffi et al. reported that systemic steroids, which were often used for the treatment of allergic bronchopulmonary aspergillosis (ABPA), were associated with severe NTM disease in CF subjects [11]. However, very little data exist on the impact of other potentially important therapies (e.g., inhaled steroids, long-term macrolides, inhaled and systemic antibiotics) on the incidence of NTM. Further, azithromycin may also have an impact on the incidence of macrolide-resistant NTM whether resistance is natural such as the one conferred by erm(41) gene in M. abscessus complex [12] or whether resistance is acquired such as the one conferred by rrl gene mutations in either M. abscessus complex and Mycobacterium avium complex [13]. In the present study, our goals were to examine the impact of the various drugs used in CF adults on NTM incidence and their macrolide susceptibility. We performed a case–control study, comparing adult CF patients who had NTM-positive isolates to individually matched CF patients who had no positive NTM sputum culture. Our results suggested that long-term azithromycin therapy was associated with a reduction in the incidence of NTM isolates.
2. Methods 2.1. Selection of cases and controls The Pulmonary Department at Cochin University Hospital (Paris, France) hosts an accredited CF adult center. The CF population followed in our center comprised 311 patients in 2006 and progressively increased to 347 patients at the end of the study period (May 2010). Patients followed at our center were routinely (at least once a year and often more frequently) screened for NTM. From January 2006 to May 2010, all consecutive CF adults followed in this center and who had at least one respiratory sample positive for NTM (Cases) were identified. Four patients with positive samples for Mycobacterium gordonae, which is generally considered a contaminant and not usually a clinically relevant cause of lung disease [14], were excluded from the analyses. Because NTM can be isolated due to environmental contamination, more than one culture-positive specimen for NTM is considered necessary to establish diagnosis of NTM infection [14]. The 2007 American Thoracic Society (ATS) guidelines for the diagnosis of NTM lung disease provide with compatible clinical, radiographic and bacteriologic criteria for NTM infection (i) 2 positive sputum cultures or 1 positive culture obtained through bronchial wash, lavage or lung biopsy; or (ii) at least 1 sputum or bronchial washing culture positive for NTM and evident mycobacterial histopathological features [14]. Thus, our study identified both subjects who fulfilled the 2007 ATS criteria (ATS + Cases) and those who did not (ATS − Cases).
The Control group was composed of CF subjects followed in the same institution. For each Case, we selected up to 4 Controls who had no positive respiratory sample for NTM. Controls were individually matched with Cases for gender and closest age (maximum age difference: ± 3 years) at the time of first NTM identification in the index case. All patients who were identified as Cases had previously documented negative cultures and were considered incident Cases at index date. The study conformed to the Declaration of Helsinki and was approved by the Institutional Review Board of the French learned society for respiratory medicine—Société de Pneumologie de Langue Française (approval #, 2012/013). Information was provided to patients, but written consent was not required due to the observational design of this study in accordance with French laws. 2.2. Data collection CF was diagnosed on the basis of clinical manifestations with a sweat chloride concentration exceeding 60 mM/L and/or two disease-causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene [15]. Using our local database, we collected clinical and microbiological data that were obtained at the time of first positive respiratory sample for NTM (index date) in Cases and in their Controls. Clinical data included age, gender, age at CF diagnosis, CFTR genotype, body mass index (BMI), exocrine pancreatic insufficiency, diabetes mellitus, liver cirrhosis and pulmonary function tests (forced expiratory volume in one second (FEV1, % predicted), forced vital capacity (FVC, % predicted)). Data on treatment (long-term oxygen therapy, azithromycin use, inhaled antibiotic therapy, inhaled or oral corticosteroid therapy, intravenous antibiotic therapy, and quinolones) in the 12 months before the index date were also obtained. Chronic colonization with major Gram negative (Pseudomonas aeruginosa, Burkholderia cepacia, or Haemophilus influenza) or Gram positive (methicillin-susceptible Staphylococcus aureus or methicillin-resistant S. aureus) bacteria found in CF patients were recorded. Chronic A. fumigatus colonization (defined as three or more positive respiratory samples in the past two years) and a history of ABPA were also recorded. 2.3. Processing of sputum samples for identification of NTM Sputum samples were successively processed with N-acetylcysteine/NaOH (BBL MycoPrep, BD, Franklin Lakes, NJ) and 5% oxalic acid, as previously described [16]. Before inoculation onto growing media, vancomycin, tobramycin, colistin and amphotericin B were added to each specimen at final concentrations of 2.5 mg/L, 4.0 mg/L, 4.105 UI/L and 2.5 mg/L, respectively. Specimens were inoculated onto two Coletsos (Bio-Rad, Marne-la-Coquette, France) and one Lowenstein– Jensen (LJ) slants, and one mycobacteria growth indicator tube (BBL MGIT, BD). Coletsos and LJ media were supplemented with vancomycin and amphotericin B (0.8 mg/L and 3 mg/L final concentration, respectively), and the MGIT medium was supplemented with Middlebrook OADC enrichment, as well as with polymyxin B, amphotericin B, nalidixic acid, trimethoprim,
Please cite this article as: Coolen N, et al, Reduced risk of nontuberculous mycobacteria in cystic fibrosis adults receiving long-term azithromycin, J Cyst Fibros (2015), http://dx.doi.org/10.1016/j.jcf.2015.02.006
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azlocillin and vancomycin (4.104 UI/L, 6 mg/L, 18 mg/L, 4.5 mg/L, 4.5 mg/L and 0.5 mg/L, respectively). MGIT medium and one Coletsos slant were incubated at 37 °C, whereas LJ and one Coletsos were incubated at 30 °C. Coletsos and LJ were examined twice a week for 12 weeks and the MGIT vials were monitored in a BACTEC MGIT 960 system (BD). Identification of acid-fast organisms at the species level was performed with reverse-hybridization INNO-LiPA (Innogenetics, Gent, Belgium) or PCR-hybridization Genotype-CM (BioCentric, Bandol, France), and confirmed by sequencing of the hsp gene. 2.4. Macrolide susceptibility testing Macrolide susceptibility testing relied on measurement of clarithromycin minimum inhibitory concentrations (MICs) as recommended [14]. Clarithromycin MIC was determined by the broth microdilution method, using Sensititer® RGMYCO plates (Trek Diagnosis systems, Biocentric, France) [17]. Plates were submitted to an extended incubation as described by Nash et al. for strains belonging to the M. abscessus complex [12], with successive readings after 5, 7, 9, and 14 days of incubation at 30 °C. Mutations in the 23S rRNA gene (rrl) were sought for strains displaying clarithromycin MIC considered resistant according to CLSI at 5 day reading [17]. Mutations in the 23S rRNA gene (rrl) were sought by PCR sequencing as described by Wallace et al. [13]. Sequences were compared to the M. abscessus genome reference sequence NC_010397 [18]. 2.5. Statistical analysis Continuous variables were reported as mean (standard deviation [SD]) or median (interquartile range [IQR]) and were compared using parametric t-test or non-parametric Mann–Whitney– Wilcoxon's test to account for non-normality of the variable distributions, when demonstrated by the use of the Shapiro–Walk test for normality. Categorical variables were reported as count and percentage and were compared using Chi-squared or Fisher's exact tests, as appropriate. Multivariate conditional logistic regression was used to examine which independent factors were associated with all Cases or with ATS+ Cases, accounting for the matching between cases and controls. All variables with a P value b 0.20 in univariate analyses were entered into the models, following a stepwise backwards approach by removing non-significant variables at each step until the final model was reached. Statistical analyses were 2-sided and P b 0.05 was considered to have statistical significance. Analyses were performed using Stata V11.0 (StataCorp, College Station, TX, USA). 3. Results 3.1. Prevalence of NTM in CF adults From January 2006 to May 2010, we identified 41 adult CF subjects with at least one positive respiratory sample positive for NTM (Cases), including 31 subjects who fulfilled the 2007 ATS criteria for NTM disease (ATS + Cases) and 10 subjects who did not fulfill these criteria (ATS − Cases). The prevalence
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of Cases was 12.4% and the prevalence of ATS + Cases was 9.5%. As shown in Table 1, M. abscessus complex was the most prevalent species and was identified in 24/41 (58.5%) subjects. M. avium/intracellulare complex was found in 20/41 (48.7%) subjects. Other species (including Mycobacterium fortuitum, Mycobacterium simiae, and Mycobacterium kansasii) were found in only 5/41 (12.2%) subjects. Seven (6 ATS +) Cases had positive cultures with more than one species. M. abscessus isolates were more prevalent in ATS + Cases than in ATS − Cases (P b 0.05). The mean (SD) number of respiratory samples processed for NTM culture was 10.8 (8.0) in Cases, and cultures of respiratory samples were positive in 63% vs. 23% samples in ATS + Cases and ATS − Cases (P = 0.02). 3.2. Risk factors for incident NTM in CF adults A total of 155 Controls (mean 3.8 Controls per Case) were identified. The mean ± SD numbers of documented negative cultures per patient prior to index date were 5.2 ± 8.8 vs. 4.1 ± 7.0 in Cases and in Controls, respectively (P = 0.38). In univariate analysis (see Table 2), low BMI (b 18 kg/m2) was found in 36.6% Cases vs. 17.5% in Controls (P = 0.02), and in 41.9% ATS + Cases vs. 20.7% in their Controls (P = 0.01). Chronic A. fumigatus colonization was found in 34.1% Cases vs. 16.9% Controls (P = 0.02) and in 38.7% ATS + Cases vs. 14.7% in their Controls (P = 0.005). Cases and ATS + Cases had significantly more Haemophilus influenzae colonization than their Controls (14.6% vs. 4.5% and 16.1% vs. 4.3%, respectively; P = 0.02 and P = 0.03). Methicillinsusceptible S. aureus was found more frequently in Cases vs. Controls (P = 0.03) but not in ATS+ Cases vs. their Controls (P = 0.16). Other pathogens and other clinical manifestations (e.g., diabetes, liver cirrhosis and pancreatic insufficiency) were not associated with NTM. Treatment with azithromycin (250 mg/d) was present at index date in 87 (44.8%) of all 196 (41 Cases, 155 Controls) CF patients, with azithromycin being prescribed in 31.7% Cases vs. 48.4% Controls (P = 0.05), and in 25.8% ATS+ Cases vs. in 47.8% of their Controls (P = 0.04). The mean (SD) exposure time to azithromycin was 3.2 (2.9) years and was not different between Cases and Controls (not shown). Other treatments, including nebulized or IV antibiotics, as well as inhaled or oral steroids were not associated with NTM. Table 1 Microbiological results in adult CF subjects with NTM positive isolates (Cases). All Cases (n = 41)
ATS+ Cases (n = 31)
ATS− Cases (n = 10)
Respiratory samples per case, median [IQR] Total numbers of samples 8 [5–13] Numbers of NTM positive samples 5 [2–8]
13 [7–17]** 7 [4–9]**
6 [4–7] 1[1–26]
Mycobacteria, n (%) M. abscessus complex M. avium complex Other species ≥2 species, n (%)
22 (71.0)⁎ 14 (45.2) 2 (6.4)⁎ 6 (19.4)
2 (20.0) 6 (60.0) 3 (30.0) 1 (10.0)
24 (58.5) 20 (48.7) 5 (12.2) 7 (17.1)
⁎P b 0.05 and ⁎⁎P b 0.01 when comparing ATS+ with ATS− Cases.
Please cite this article as: Coolen N, et al, Reduced risk of nontuberculous mycobacteria in cystic fibrosis adults receiving long-term azithromycin, J Cyst Fibros (2015), http://dx.doi.org/10.1016/j.jcf.2015.02.006
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N. Coolen et al. / Journal of Cystic Fibrosis xx (2015) xxx–xxx
Table 2 Comparison of clinical characteristics between Cases and Controls. All Cases (n = 41)
Controls (n = 155)
P values
ATS + Cases (n = 31)
Controls (n = 117)
P values
27 (65.9) 26.2 [19.6–30.3] 2.0 [0.2–7.0]
102 (65.8) 26.2 [20.5–31.4] 1.5 [0.2–10.0]
1 0.95
20 (64.5) 26.1 [19.2–30.3] 2.0 [0.3–7.0]
76 (65.0) 26.0 [20.5–30.8] 2.0 [0.3–12.0]
1.00 0.88
71 (46.1) 59 (38.3) 24 (15.6) 131 (85.1) 38 (24.5) 16 (10.4) 19.7 [18.4–21.4] 27 (17.5) 56 [41–83] 77 [58–94]
0.97
0.02 0.77 0.78
14 (45.2) 11 (35.5) 6 (19.3) 25 (80.6) 7 (22.6) 40 (12.9) 18.9 [17.0–21.6] 13 (41.9) 59 [40–77] 78 [63–84]
52 (44.8) 47 (40.5) 17 (14.7) 97 (83.6) 26 (22.4) 12 (10.3) 19.7 [18.3–21.0] 24 (20.7) 55 [41–83] 77 [58–94]
0.78
BMI b 18 kg/m2, n (%) FEV1, % predicted FVC, % predicted
18 (43.9) 16 (39.0) 7 (17.1) 32 (78.0) 8 (19.5) 4 (9.8) 19.5 [17.1–20.7] 15 (36.6) 60 [46–76] 78 [68–84]
0.01 0.95 0.50
Colonization, n (%) - P. aeruginosa - MSSA - MRSA - H. influenzae - B. cepacia - A. fumigatus Oxygen therapy, n (%)
21 (51.2) 27 (65.9) 4 (9.8) 6 (14.6) 0 (0) 14 (34.1) 3 (7.3)
96 (62.3) 72 (46.8) 18 (11.7) 7 (4.5) 5 (3.2) 26 (16.9) 20 (13.0)
0.20 0.03 0.73 0.02 0.24 0.02 0.32
15 (48.4) 20 (64.5) 3 (9.7) 50 (16.1) 0 (0) 12 (38.7) 3 (9.7)
71 (61.2) 57 (49.1) 14 (12.1) 50 (4.3) 4 (3.4) 17 (14.7) 13 (11.2)
0.22 0.16 1.00 0.03 0.58 0.005 1
Antibiotics - Oral azithromycin, n (%) - Oral ciprofloxacin, n (%) - Inhaled a, n (%) - IV antibiotics, days/yr. Oral corticosteroids N 3 months, n (%)
13 (31.7) 15 (36.6) 16 (39.0) 14 [0–28] 2 (4.9)
74 (48.4) 45 (29.2) 75 (48.7) 14 [0–43] 5 (3.2)
0.05 0.37 0.27 0.33 0.62
8 (25.8) 12 (38.7) 12 (38.7) 14 [0–29] 2 (6.5)
55 (47.8) 34 (29.3) 54 (46.6) 14 [0–43] 4 (3.4)
0.04 0.39 0.54 0.20 0.61
20 (48.8)
58 (37.7)
0.20
13 (41.9)
44 (37.9)
0.69
0 [0–2000] 963
0 [0–1000] 662
0.13
0 [0–1000] 838
0 [0–1000] 694
0.53
Male, n (%) Age, yrs. Age at CF diagnosis, months CFTR mutations, n (%) - F508del/F508del - F508del/other - other/other Exocrine pancreatic insufficiency, n (%) Diabetes mellitus, n (%) Cirrhosis, n (%) BMI, kg/m2
Inhaled corticosteroids - n (%) - Beclomethasone eq., μg/d Median [IQR] Mean
0.87
0.34 0.68 1 0.33
0.99
0.79 1.00 0.75 0.65
CF: cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; BMI, body mass index; FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity; MSSA, methicillin-susceptible S. aureus; MRSA, methicillin-resistant S. aureus; IV, intravenous. Values are n (%) or median [IQR] unless otherwise specified. Bold data were for P values below 0.05 (statistical significance). a Tobramycin and/or colomycin.
In multivariate analysis (see Table 3), Cases and ATS + Cases had more often low BMI compared to their Controls (P = 0.007 and P = 0.02, respectively). Cases and ATS + Cases had increased prevalence of A. fumigatus colonization (P = 0.02 and P = 0.03, respectively). Cases were twice less likely to have received azithromycin than Controls (P = 0.05) and a similar trend was observed when ATS + Cases were compared to their Controls (P = 0.06). The distribution of mycobacterial species was not different between Cases or ATS + Cases who had received azithromycin and those who had not (see Table 4). 3.3. Macrolide susceptibility of NTM isolates Among all NTM isolates only one had mutation in rrl gene (which was previously associated with acquired macrolide
resistance): a M. avium isolate in an ATS − case who had received azithromycin prophylaxis. To further examine the association of azithromycin with macrolide resistance, we Table 3 Multivariate analysis of risk factors for NTM in adult CF subjects. OR
SE
95% CI
P values
All Cases (n = 41) vs. Controls (n = 155) 3.56 1.69 BMI b 18 kg/m2 A. fumigatus colonization 2.51 1.05 Azithromycin 0.45 0.18
1.41 – 9.02 1.11 – 5.71 0.19 – 1.00
0.007 0.03 0.05
ATS+ Cases (n = 31) vs. Controls (n = 117) 3.21 1.65 BMI b 18 kg/m2 A. fumigatus colonization 3.45 1.75 Azithromycin 0.40 0.20
1.17 – 8.78 1.27 – 9.35 0.15 – 1.05
0.02 0.02 0.06
OR, odds ratio; SE, standard error; 95% CI, 95% confidence interval.
Please cite this article as: Coolen N, et al, Reduced risk of nontuberculous mycobacteria in cystic fibrosis adults receiving long-term azithromycin, J Cyst Fibros (2015), http://dx.doi.org/10.1016/j.jcf.2015.02.006
N. Coolen et al. / Journal of Cystic Fibrosis xx (2015) xxx–xxx
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Table 4 Mycobacterial species isolated among all cases and among ATS+ Cases depending on previous azithromycin prophylaxis. All Cases (n = 41) Azithromycin
M. abscessus complex (M. abscessus + M. bolletii + M. massiliense) M. abscessus, M. bolletii M. massiliense M. avium complex Other Total a
Yes
No
4 (31%) 3 (23%) 1 (8%) 7 (54%) a 2 (15%) 13
17 (61%) 12 (43%) 5 (18%) 8 (29%) 3 (11%) 28
ATS + Cases (n = 31) P values
Azithromycin Yes
No
0.10 0.30 0.65 0.16 0.65
4 3 1 3 1 8
16 (69.5%) 11 (48%) 5 (22%) 6 (26%) 1 (4%) 23
(50.0%) (37.5%) (12.5%) (37.5%) (12.5%)
P values
0.40 0.70 1 0.66 0.26
Including 1 with acquired macrolide resistance (due to rrl gene mutation).
compared macrolide susceptibility among isolates of Cases that had received azithromycin prophylaxis and Cases that had not received azithromycin prophylaxis. Among all Cases, 37 isolates underwent susceptibility testing (4 not tested corresponded to 3 M. gordonae isolates and 1 M. fortuitum). Seventeen NTM isolates were resistant to clarithromycin: 1 M. avium (acquired resistance due to rrl mutation, see above), 1 M. simiae, 14 M. abscessus and 1 Mycobacterium bolletii (natural inducible resistance due to erm(41) gene). The proportion of (acquired + natural) resistant isolates was not different among cases that had received azithromycin compared to those who had not received azithromycin (31% vs. 46%, P = 0.50). A similar lack of difference was observed among ATS + Cases (38 vs. 52%, P = 1). 4. Discussion The present case–control study was designed to identify risk factors associated with incident NTM isolates in adult CF patients, with a special interest on the association of NTM with treatments for CF lung disease. We found that incident NTM isolates were associated with chronic A. fumigatus colonization and occurred more often in subjects with low BMI. Long-term treatment with low-dose azithromycin was associated with a two-fold reduction in the incidence of NTM, suggesting that azithromycin could be a primary prophylaxis for NTM in CF subjects. Azithromycin therapy was not associated with increased rates of NTM resistance to macrolides. We found a lower rate of NTM isolates among adult CF subjects receiving long-term azithromycin therapy. Previous studies that evaluated the association between long-term azithromycin and NTM incidence in CF patients have shown conflicting results. Renna et al. compared 14 adult CF patients with NTM to 184 CF patients without NTM and found long-term azithromycin therapy in 12/14 (86%) NTM infected patients but only in 74/184 (40%) non-infected patients [19]. In contrast with our present findings, the authors suggested that azithromycin may predispose to NTM infection [19]. Other studies found no significant association between NTM incidence and azithromycin but these studies were limited by sample size [20], by difficulties in establishing correct exposure to azithromycin in registry data [21] or by the absence of matching between Cases and Controls [10]. In a recent case–control study based on data obtained in the CF Foundation Patient Registry [22], Binder et al. found that incident NTM cases were less likely to have received
prior long-term macrolide [22]. Our results concur with those of this latter study to suggest that long-term azithromycin use could act as a primary prophylaxis for NTM in CF patients. A major concern when using long-term azithromycin monotherapy is the possible emergence of macrolide-resistant NTM isolates. To the best of our knowledge our study was the first to compare macrolide resistance in NTM cases in CF patients according to azithromycin exposure. When combining strains displaying natural inducible clarithromycin resistance (due to erm (41) gene) and acquired resistance (due to rrl gene mutations), it appeared that NTM resistance to macrolides was not increased in CF subjects treated with azithromycin. In fact the trend was rather in favor of less macrolide resistant isolates among cases that had received azithromycin prophylaxis. This trend was mainly due to the reduced occurrence of M. abscessus and M. bolletii: 20% of isolates among cases that received azithromycin vs. 40% among cases that did not receive azithromycin. However it must be underlined that the only strain with acquired macrolide resistance (due to rrl gene mutation) was isolated in an azithromycin-treated patient. This finding had no clinical consequence in this ATS − Case, in whom no NTM specific treatment was required and in whom no NTM isolate was further cultured after azithromycin discontinuation. However, acquired macrolide resistance in NTM could dramatically diminish chance for successful therapy against NTM if necessary. The small number of patients does not allow us to derive firm conclusion on the benefit or risk of longterm azithromycin therapy on prevalence of macrolide-resistant mycobacteria in CF patients. The present study was performed in a single CF center with homogeneous practices regarding drug prescription, respiratory sample collection and processing, which together strengthens data consistency. Individual matching of each Case with multiple Controls reduced potential bias related to control selection [23]. No significant difference existed in the number of respiratory samples cultured for NTM in Cases vs. Controls prior to index date, indicating that the likelihood of NTM isolation was similar in both groups of patients. In summary, macrolides have proven major beneficial effects in CF subjects (colonized or not with P. aeruginosa) [24–26], especially in the reduction in pulmonary exacerbations. Our study suggests that azithromycin also reduces the incidence of mycobacterial infection. Although the use of azithromycin was not associated with increased incidence of natural or acquired macrolide-resistant in this (relatively) small study, larger
Please cite this article as: Coolen N, et al, Reduced risk of nontuberculous mycobacteria in cystic fibrosis adults receiving long-term azithromycin, J Cyst Fibros (2015), http://dx.doi.org/10.1016/j.jcf.2015.02.006
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Please cite this article as: Coolen N, et al, Reduced risk of nontuberculous mycobacteria in cystic fibrosis adults receiving long-term azithromycin, J Cyst Fibros (2015), http://dx.doi.org/10.1016/j.jcf.2015.02.006
