- Email: [email protected]
Mycobacterium bolletii Lung Disease in Cystic Fibrosis
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Mycobacterium bolletii Lung Disease in Cystic Fibrosis
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Virginie Rollet-Cohen, MD; Anne-Laure Roux, MD; Muriel Le Bourgeois, MD; Guillaume Sapriel, PhD;
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Manele El Bahri, MD; Jean-Philippe Jais, MD; Beate Heym, PhD; Faiza Mougari, PhD; Laurent Raskine, PhD; Q1
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Nicolas Véziris, PhD; Jean-Louis Gaillard, MD; and Isabelle Sermet-Gaudelus, MD
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The cystic fibrosis (CF) pathogen, Mycobacterium abscessus complex, covers three subspecies: M. abscessus, M. massiliense, and M. bolletii. There are no clinical outcome data concerning M. bolletii. Our aim was to characterize M. bolletii lung infections in patients with CF. BACKGROUND:
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We included patients with M. bolletii lung infection recorded between 1994 and 2012 in France. Data were collected from the CF registry, medical records, and questionnaires submitted to the CF primary physician. Strains were typed by multilocus sequence typing analysis.
METHODS:
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RESULTS: Fifteen cases were identified in nine CF centers. Nine patients (60%) presented with nontuberculous mycobacterial pulmonary disease. Follow-up of 13 patients showed a trend to more rapid decline in FEV1 in the first year of colonization (–9.4%; SD 19.3) in comparison with noninfected control subjects (–2.3%; SD 12.1) (P ¼ .16). Twelve patients were treated, and 11 received oral macrolides. Treatment-induced eradication occurred in five patients (41.7%). Four patients died (26.7%), including one patient with fatal nontuberculous mycobacterial pulmonary disease. Inducible macrolide resistance was demonstrated in all strains. Patients always harbored unique strains.
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Our study reports the largest study cohort of CF patients infected with M. bolletii. M. bolletii infection affects both children and young adults, is most often symptomatic, and may be fatal. Macrolide-based therapies have poor effectiveness. There is no evidence of patient-to-patient transmission. CHEST 2019; -(-):---
CONCLUSIONS:
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clarithromycin; cystic fibrosis; erm41; macrolide resistance; multilocus sequence typing analysis; Mycobacterium bolletii; Mycobacterium abscessus; Mycobacterium massiliense; nontuberculous mycobacteria KEY WORDS:
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Q2 ABBREVIATIONS:
ATS = American Thoracic Society; CF = cystic fibrosis; MABSC = Mycobacterium abscessus complex; MLST = multilocus sequence typing; NTM = nontuberculous mycobacteria; NTMPD = nontuberculous mycobacterial pulmonary disease AFFILIATIONS: From the Centre de Ressources et de Compétences pour la Mucoviscidose, Pneumologie et Allergologie Pédiatriques (Drs Rollet-Cohen, Le Bourgeois, and Sermet-Gaudelus), Hôpital Necker Enfants Malades, Paris, France; UMR 1173 (Drs Roux, Sapriel, Heym, and Gaillard), UFR des Sciences de la Santé Simone Veil, Université de Versailles Saint-Quentin-en-Yvelines, Montigny-le-Bretonneux, France; the Laboratoire de Microbiologie (Drs Roux, Heym, and
Gaillard), Hôpital Ambroise Paré, Boulogne, France; the Atelier de Bioinformatique (Dr Sapriel), Institut Systématique Evolution Biodiversité, UMR 7205, Paris, France; the Département de Biostatistiques (Drs El Bahri and Jais), Hôpital Necker Enfants Malades, Paris, France; the Laboratoire de Bactériologie (Drs Mougari and Raskine), Hôpitaux Lariboisière-Saint Louis, Paris, France; the Centre National de Référence des Mycobactéries et de la Résistance des Mycobactéries aux Antituberculeux (Drs Mougari and Raskine), Laboratoire de Bactériologie, Hôpitaux Universitaires Pitié-Salpetrière, Paris, France; and Sorbonne Université (Dr Véziris), Centre d’Immunologie et des Maladies Infectieuses, and Centre National de Référence des
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Mycobacterium abscessus complex (MABSC) is a rapidly growing, nontuberculous mycobacterium (NTM) that is increasingly recognized as a respiratory pathogen in people with cystic fibrosis (CF).1–4 MABSC may infect patients with CF at any age, but infection peaks between 11 and 15 years of age,5–7 with a prevalence from 3% to 6%.1,3,6–8 MABSC infection has been associated with a decline in lung function,7,9 severe pseudotuberculous lung disease, and fatal dissemination.10,11 However, MABSC can also transiently, intermittently, or permanently colonize the lungs of patients with CF without causing pulmonary disease.12 MABSC is a single species comprising three “subspecies”: M. abscessus, M. massiliense, and M. bolletii.13,14 Previous studies have focused on M. abscessus and M. massiliense, which are responsible for > 90% of MABSC cases in CF.15,16 Major clinical differences have been reported between these two subspecies. M. abscessus and M. massiliense target different CF subpopulations, with young and malnourished subjects being particularly susceptible to M. massiliense.17 Infections linked to M. abscessus are
more severe than M. massiliense and generally refractory to clarithromycin-based combination therapies, due to the expression of a functional erm41 gene that causes macrolide resistance (“T28 erm41 genotype”) in most strains. M. massiliense carries a nonfunctional, truncated erm41 gene and is susceptible to macrolides,18 inducing therefore more frequent success in eradication. Finally, patient-to-patient transmission in CF centers has exclusively been reported for M. massiliense.19,20 It is still unknown whether M. bolletii displays a specific profile for infection and therapeutic susceptibility in CF. Very few cases of M. bolletii lung disease have been reported and exclusively in non-CF adult patients.21 M. bolletii was described as resistant to clarithromycin, consistent with the expression of functional erm41 in this species.15,21,22 In this study, we present the largest cohort of CF patients infected with M. bolletii ever published and analyze the characteristics and outcome of 15 cases of M. bolletii lung infection in patients with CF, in association with comprehensive strain characterization.
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Methods Study Population We included all M. bolletii lung infection cases recorded between 1994 and 2012 in the French CF Registry. Cases were defined according to the American Thoracic Society (ATS) bacteriologic criteria for NTM lung infection (ie, positive culture results from at least two separate expectorated sputum samples).23,24 NTM pulmonary disease (NTMPD) was defined according to the ATS/Infectious Diseases Society of America (IDSA) 2007 statement.24 NTM eradication was defined by at least three negative cultures for M. bolletii over at least 1 year. Clinical and radiologic data were collected from medical records and the French CF Registry with the permission of an international review board. All the patients enrolled in the French CF Registry had given informed consent for data use.
multicenter database created in 2008 (PHRC P100201), according to age categories, sex, Pseudomonas aeruginosa colonization, and FEV1 at entry in the study. We focused on the first year of colonization to determine whether the colonization was a risk factor for immediate degradation. Bacteriologic Methods All strains were sent to the National Reference Center (NRC) for Mycobacteria and cryoconserved at –80 C. rpoB-based identification, erm41 genotyping, and drug susceptibility testing were performed by the NRC (see the online article). Genetic analysis of strains was performed by the microbiology laboratory of Ambroise Paré Hospital, using multilocus sequence typing (MLST) (see the online article).
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Statistical Methods
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Qualitative variables were analyzed by c2 test or Fisher exact test, and continuous variables by Student t test. Statistical significance was accepted for P < .05.
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DFEV1 (FEV1 change over 1 year) between the infected patients and
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the control group was compared using a linear mixed model (LME4 package of R v3.4; R Foundation) to take into account the paired design and the variable number of control subjects per stratum. Significance was defined as p < .05.
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To investigate the impact of M. bolletii on the evolution of respiratory function, colonized patients were matched with NTM-negative control subjects issued from the French CF Registry and a prospective
Mycobactéries et de la Résistance des Mycobactéries aux Antituberculeux, Département de Bactériologie, AP-HP, Hôpitaux Universitaires de l’Est Parisien, Paris, France. 157 Drs Rollet-Cohen and Roux contributed equally to this manuscript. 158 FUNDING/SUPPORT: This work was supported by the association 159 Q3 Q4 Vaincre la Mucoviscidose. 160 CORRESPONDENCE TO: Isabelle Sermet-Gaudelus, MD, Centre de 161 Ressources et de Compétences pour la Mucoviscidose, Pneumologie et Allergologie Pédiatriques, Hôpital Necker Enfants Malades, Paris 162 Q5 75014, France; e-mail: [email protected] 163 Copyright Ó 2019 Published by Elsevier Inc under license from the 164 American College of Chest Physicians. 165 DOI: https://doi.org/10.1016/j.chest.2019.03.019 156
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Results
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Cases Included
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Fifteen M. bolletii lung infection cases were identified between 1994 and 2012. All cases fulfilled the ATS bacteriologic criteria for NTM lung infection, and nine
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TABLE 1
] Demographic Characteristics of Patients at Patients (n ¼ 15)
Characteristics Sex ratio, men/women, No. (%)
6/9 (0.67)
Age, y, mean (SD)
15.9 (10.7)
227
F508del/F508dela: No. (%)
228
BMI, kg/m2, mean (SD)
229
FEV1, % predicted, mean (SD)
230
Pancreatic insufficiencya
231 232 233 234 235 236 237 238 239 240
Treated ABPA
7 (46.7) 17.5 (2.6) (n ¼ 14)
243
13 (86.7)
a
3 (20.0)
Low-dose azithromycina
6 (42.8) (n ¼ 14)
Staphylococcus aureus colonizationa,b
12 (80.0)
Pseudomonas aeruginosa colonizationa,b
11 (73.3)
ABPA ¼ allergic bronchopulmonary aspergillosis. a Number/total number evaluated (%). b At least one positive sample within the previous year.
(60.0%) had NTM-PD. The mean follow-up was 7.8 years (2-19 years).
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Patient Characteristics
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Demographic characteristics of patients at the first isolation of M. bolletii are presented in Table 1. Nine of 15 (60%) were younger than 15 years. Six patients (40%) had a negative BMI z-score value. Two patients (5 and 36 years old) received a diagnosis of CF during the search for an underlying cause of M. bolletii infection.
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Clinical and Radiologic Presentation
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Clinical and radiologic findings are presented in Table 2. Eleven patients (73%) were symptomatic at the first isolation of M. bolletii. CT scan modifications were
256 257 258 259 260
TABLE 2
] Clinical and Radiologic Presentation at First
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Isolation of Mycobacterium bolletii
Presentation
No. (%)
Clinical signs and symptoms Any
11 (73.3)
Respiratory deterioration
11 (73.3)
267
Weight loss
7 (46.7)
268
Fever
4 (26.7)
269
CT scan abnormalities
270
Any
9 (60.0)
271
Tree-in-bud
5 (33.3)
272
Consolidation
5 (33.3)
Nodules
4 (26.7)
Cavities
3 (20.0)
273 274 275
278 279 280
0
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–20 NTM-negative control subjects n = 32
M. bolletii infected patients n = 13
Figure 1 – FEV1 follow-up 1 year after the first isolation of Mycobacterium bolletii. Absolute change in FEV1 (% predicted) in 13 patients infected with M. bolletii and 32 NTM-negative control subjects with cystic fibrosis. Each rectangle spans the first quartile to the third quartile (the interquartile range). Median and minimum/maximum range are shown. NTM ¼ nontuberculous mycobacteria.
284 285 286 287 288 289
Q21
290 291
Q16
292 293
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+20
71.4 (19.6)
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First Isolation of Mycobacterium bolletii
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FEV1 change (%)
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observed in nine patients (60%). Worsening of respiratory symptoms was defined by breathlessness, increased cough and/or sputum production. CT scan abnormalities involved at least two lobes in all patients. Two or more abnormalities were associated in four patients (27%), most frequently consolidations and nodules associated with cavities.
297 298 299 300 301 303
Follow-up of respiratory function was not available for two patients. The 13 remaining patients were compared with 32 CF control subjects negative for NTM. Infected patients presented a trend to a more rapid decline in FEV1 (–9.4%; SD 19.3) during the first year of colonization in comparison with the control subjects (–2.3%; SD 12.1) (delta estimate, 6.54; SD 4.6) (Fig 1). Q9 The difference was, however, not significant (P ¼ .16).
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Response to Antimycobacterial Treatment
Twelve patients were treated with antimycobacterial combination therapies during the follow-up period. Treatment was implemented after a mean (SD) delay of 21 (28.3) months after the first isolation of M. bolletii, and the mean (SD) duration of treatment was 15 (24.8) months. Three patients (20%) had “spontaneous” eradication of M. bolletii from their sputum and were not treated. All treated patients received combination therapies based on oral macrolides (clarithromycin, nine patients; clarithromycin followed by azithromycin, two patients), except one patient, who was treated with moxifloxacin, ethambutol, and amikacin. Drugs combined with macrolides were inhaled (n ¼ 7) and/or intravenous amikacin (n ¼ 7), ethambutol (n ¼ 4), linezolid (n ¼ 3),
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Evolution of Respiratory Function
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314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330
331
TABLE 3
] Comparison of Patients With or Without Eradication of Mycobacterium bolletii
332 333 334
Sex ratio, men/women
335
Age, y, mean (SD)
336
BMI, kg/m2, mean (SD)
337
F508del/F508dela
338
FEV1, % predicted, mean (SD)
339 340 341
Pseudomonas aeruginosa colonization, No. (%) a
NTM pulmonary disease, No. (%)
342
Months before antimycobacterial treatment, mean (SD)
343
Months of antimycobacterial treatment, mean (SD)
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Eradication (n ¼ 5)
No Eradication (n ¼ 7)
1/4
4/3
.29
9.6 (6.4)
21.4 (12.8)
.09
P Value
387 388 389 390
.5
391
4 (80.0)
2 (28.6)
.24
392
65.4 (17.1)
73.4 (23.8)
.54
393 394
16.5 (2.7)
a
386
17.4 (1.5)
4 (80.0)
5 (71.4)
1
2 (40.0)
6 (85.7)
.22
25.7 (36.0)
19.7 (25.0)
.77
10 (4.3)
31.9 (35.1)
.26
395 396 397 398 399
NTM ¼ nontuberculous mycobacteria. a At least one positive sample within the previous year.
400
346
401
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402
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moxifloxacin (n ¼ 2), tigecycline (n ¼ 2), cefoxitin (n ¼ 2), imipenem (n ¼ 2), ciprofloxacin (n ¼ 1), and trimethoprim/sulfamethoxazole (n ¼ 1). Total eradication of M. bolletii following treatment was obtained in five of 12 patients (42%), vs three of 15 nontreated patients (20%) who underwent spontaneous eradication. This difference was not significant (eTable 1). Although no factor was significantly associated with eradication, patients who experienced eradication tended to be younger (mean [SD] age, 9.6 [6.4] vs 21.4 [12.8] years; not significant) (Table 3).
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Mortality
Four of the 15 patients (27%) died during the study period. Two patients died following lung transplant. One patient was determined to have end-stage CF disease after being excluded for lung transplant because of repeated positive cultures under antimycobacterial treatment. The last patient was a young boy who developed severe M. bolletii lung disease treated with clarithromycin and imipenem in association with intravenous and inhaled amikacin. The patient died at 9 years of age with respiratory insufficiency and blood cultures positive for M. bolletii. Lung Transplant
Four patients underwent lung transplant during the study period. M. bolletii was eradicated in three of those for at least 12 months before transplant. The last patient was transplanted while being treated for M. bolletii lung disease and remained NTM positive after lung transplant without receiving any antimycobacterial treatment. Two of the lung transplant recipients were still alive 2 and 4 years after transplant, including the patient who failed to culture convert; the other two
4 Original Research
patients died because of organ rejection at 8 and 9 months posttransplant.
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Drug Susceptibility Testing Results
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Inducible macrolide resistance was observed in all strains, with minimal inhibitory concentrations $ 16 mg/L after extended incubation (e-Table 2). Sequencing of erm41 showed that all strains displayed a “T28” genotype, including the two M. bolletii/ M. massiliense “mosaic” strains identified by MLST analysis. The proportions of strains susceptible to amikacin, linezolid, and tigecycline were, respectively, 10 of 12 (83.3%), 5 of 12 (41.7%), and 10 of 10 (100%); 2 of 12 (16.7%) and 7 of 12 (58.3%) of the strains were susceptible and intermediate in response to cefoxitin, respectively; no strain showed susceptibility to moxifloxacin (e-Table 2).
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Genetic Analysis of Strains
MLST analysis of the M. bolletii strains isolated from the 15 patients showed that each patient harbored a Q10 sequence type (ST) that differed from all other STs; there was no genetic proximity among the strains isolated from patients attending the same CF center (Fig 2). Although harboring M. bolletii rpoB and erm41 sequences, the strains from two patients appeared to be closer to M. massiliense (Fig 3). These “mosaic” M. bolletii/M. massiliense strains were eradicated (one without treatment, and the other after antimycobacterial course).
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Discussion
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This study, based on the analysis of 15 cases of M. bolletii infection, is the largest series published to date, even outside the context of CF. Surprisingly,
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Figure 2 – Minimum spanning tree (MST) of Mycobacterium bolletii population. MST was generated from MLST data, using PHYLOViZ (a platformindependent Java tool; http://www.phyloviz.net/). Red circles, NTM-PD strains; green circles, non-NTM-PD strains. MLST ¼ multilocus sequence typing; NTM-PD ¼ nontuberculous mycobacterial pulmonary disease.
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although M. bolletii is considered to be genetically closer to M. massiliense,25 we found that the main characteristics of infection with M. bolletii were close to those of M. abscessus infection.25 As for M. abscessus, M. bolletii was isolated not only from children and adolescents, but also from young adults. Moreover, 60% of our patients presented with NTM-PD, as assessed by ATS/IDSA criteria.24 We reanalyzed the data from our previous study to classify cases according to the criteria used in this study.23,24 This analysis yielded figures of 39% (9/23) of NTM-PD for M. abscessus and 7% (1/14) for M. massiliense. Infection severity is thus similar for M. bolletii and M. abscessus. This is also supported by the fact that patients infected with M. bolletii underwent a trend to more rapid decline in
lung function by almost 10% in contrast to control subjects whose FEV1 declined by 2.3%, a change similar to reports from European and US registries.26 Although the difference from the control group was not significant because of the low number of patients, this difference is highly relevant and supports the initiation of an eradication therapy as early as possible. In contrast, M. massiliense infections were much less severe and more likely to be treated successfully than infections with M. bolletii or M. abscessus. This supports the hypothesis that M. massiliense is a much less pathogenic member among MABSCs, at least in the context of CF.17 Q11 Indeed, our two cases of infection with a mosaic strain consisting mostly of M. massiliense were eradicated, even spontaneously in one case.
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Figure 3 – Multiple correspondence analysis (MCA) of Mycobacterium bolletii population. Each ST was projected onto the first two axes of MCA, based on the MLST allele profile. Red dots represent the 15 strains included in the present study. *The two mosaic strains. MLST ¼ multilocus sequence typing; ST ¼ sequence type.
Our study clearly demonstrates the inefficacy of macrolides for treating M. bolletii infections. This finding is consistent with previous studies showing that M. bolletii consistently displays inducible resistance to macrolides, due to the presence of a genotype T28 erm41 gene.18,22 This genotype is similar to that of almost all M. abscessus strains.22,28,29 Indeed, we found a bacteriologic success rate (total eradication from the sputum) on treatment in 40% of patients infected with M. bolletii, a figure not significantly different from the spontaneous eradication rate of 20%. This rate is close to the eradication pattern of M. abscessus.17 Indeed, in our previous cohort study, macrolide-based therapies were also ineffective against M. abscessus (eradication rate, 25% with treatment vs 33.3% without; P ¼ .65), and highly effective against M. massiliense (eradication rate, 85.7% with treatment vs 28.6% without; P ¼ .03). M. bolletii isolates tested in our study showed patterns of susceptibility to amikacin, cefoxitin, linezolid, tigecycline, and moxifloxacin similar to those reported for MABSC isolates.27 This suggests that antibiotic
6 Original Research
regimens recommended for treatment of MABSC-PD23 should also be used for M. bolletii pulmonary disease.
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The consensus of the US CF Foundation and the European CF Society clarifies the course of action in cases of lung transplant, a problem that has been the subject of several studies in CF and non-CF patients.30,31 The largest and most comprehensive study, carried out by the University of North Carolina Chapel Hill between 1990 and 2003, showed that MABSC infection before transplant was a significant risk factor for the recurrence of NTM after transplant, with a significant impact on morbidity but not mortality.11 In the current consensus, it is strongly recommended not to exclude patients with current or previous respiratory tract samples positive for NTM from the transplant program.23 Our observations are consistent with these recommendations. The two transplant patients who died were NTM negative for over 1 year. The cause of death was severe lung graft rejection, whereas the sole patient who remained
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positive for M. bolletii after lung transplantation did not develop any complications linked to NTM infection.
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MLST genetic analysis showed that the strains of M. bolletii were extremely diverse. Each patient, including those monitored at the same center, carried a unique strain. This is consistent with most of the molecular typing studies carried out to date in the context of CF32 and supports the hypothesis of patient contamination from diverse environmental sources.33 This eliminates the possibility of cross-transmission between patients or from a common environmental source within the hospital. However, this result must be interpreted with caution. Indeed, two studies using whole-genome sequencing identified outbreaks of M. massiliense cases in a UK CF center, suggesting that person-to person transmission of MABSC strains may occur.19,20
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MLST analysis also confirmed the existence of mosaic strains of MABSC, resulting from horizontal genetic transfers involving chromosomal regions of various sizes between the three subspecies, probably allowing them to adapt to their environmental niches.34 We currently have no data concerning the relationship of these processes to differences in pathogenicity in humans. However, the existence of these mosaic strains poses two practical problems. The first is related to the possible presence of the rpoB gene (or of any other target gene used for the diagnosis of subspecies) in the transferred region, leading, for example, to a mosaic strain with an
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“M. massiliense” genetic background. In contrast, carrying the rpoB gene of M. bolletii, as in our series, leads to an “erroneous” diagnosis of M. bolletii infection. The other issue is the prediction of strain sensitivity to macrolides. Indeed, genetic transfers between subspecies of MABSC may involve the erm41 gene, resulting in the replacement of the endogenous gene with a gene from a different subspecies. For any MABSC strain, it is therefore prudent (1) to sequence the erm41 gene to ensure consistency between the genotype found and the type of subspecies; and (2) to determine macrolide sensitivity in vitro, which can detect both inducible resistance involving erm41 and high-level acquired resistance due to mutations of the rrl gene (23S rDNA gene).18,22,35
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Q13
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Conclusion This study is the largest, to date, that specifically addresses lung infection due to M. bolletii in the setting of CF. It shows that M. bolletii lung infection shares a number of similarities with M. abscessus infection such as a rapid decline in respiratory function and inefficiency of macrolides, due to the T28 erm41 genotype. This is in contrast to the high success rate of eradication obtained against M. massiliense, which carries a nonfunctional erm41 gene. The erm41 genotype should be determined before any treatment, due to the possibility of horizontal genetic transfers within MABSC.
735 736 737 738 739 740 741 742 743 744 745 746 747 748 750
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709
721 722
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719 720
733
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717 718
734
694
706
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Acknowledgments Author contributions: Guarantor of the article: I. S.-G. had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis, including and especially any adverse effects. I. S.-G. and J.-L. G. designed the study, participated in acquisition of the data, analyzed the results, and wrote the manuscript. V. R.-C., A.-L. R., and M. L. B. participated in the design of the study, analyzed the results, and participated in the writing of manuscript. G. S., E. Cambau, A. Ferroni, B. H., F. M., L. R., and N. V. performed the bacteriological study, analyzed the results, and participated in the writing of the manuscript. M. E. B. and J.-P. J. performed the statistical analysis and participated in the writing of the manuscript. P. R. Burgel, T. Gendry, H. Corvol, L. Couderc, D. Grenet, A. Haloun, V. Houdouin, F. Huet, S. Marchand, I. Pin Isabelle, and S. Quetant provided medical records and reviewed the manuscript. L. Lemonnier provided French CF Registry data. A. Edelman analyzed the results and
participated in the writing of the manuscript.
analysis of the data, or the preparation of the manuscript.
Financial/nonfinancial disclosures: The authors have reported to CHEST the following: I. S.-G. has no relevant conflict with the data included in this article. She declares the following funding from the Vertex Pharmaceuticals Innovation Award funding program in 2016 (Use of Nasal Epithelial Cells to Investigate the Relationship Between CF Mutation, Clinical Phenotype and CFTR Dysfunction in Cystic Fibrosis) and 2017 (ATP12A as a Novel Therapeutic Target in Cystic Fibrosis Lung Diseases). She was PI in clinical trials for Vertex Pharmaceuticals VX-809-103, VX-809-109, VX-661-108, and VX-661-109. She took part on scientific advisory boards organized by Vertex Pharmaceuticals in June 2017, September 2017, October 2017, and June 2018. None declared (V. R.-N., A.-L. R., M. L. B., G. S., M. E. B., J.-P. J., B. H., F. M., L. R., N. V., J.-L. G.). Role of sponsors: The sponsor had no role in the design of the study, the collection and
Other contributions: The authors thank all the clinicians for their contributions: PierreRégis Burgel, MD; Thierry Gendry, MD; Harriet Corvol, MD; Laure Couderc, MD; Dominique Grenet, MD; Alain Haloun, MD; Véronique Houdouin, MD; Frédéric Huet, MD; Sophie Marchand, MD; Isabelle Pin, MD; Lydie Lemonnier, PhD; Sébastien Quetant, MD; Emmanuelle Cambau, MD; and Agnès Ferroni, MD.
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Additional information: The e-Appendix and e-Tables can be found in the Supplemental Materials section of the online article.
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References
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1. Levy I, Grisaru-Soen G, Lerner-Geva L, et al. Multicenter cross-sectional study of nontuberculous mycobacterial infections among cystic fibrosis patients, Israel. Emerg Infect Dis. 2008;14(3):378-384. 2. Radhakrishnan DK, Yau Y, Corey M, et al. Non-tuberculous mycobacteria in children with cystic fibrosis: isolation,
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prevalence, and predictors. Pediatr Pulmonol. 2009;44(11):1100-1106.
Int J Syst Evol Microbiol. 2006;56(Pt 1): 133-143.
Mycobacterium massiliense and Mycobacterium bolletii be united and reclassified as Mycobacterium abscessus subsp. bolletii comb. nov., designation of Mycobacterium abscessus subsp. abscessus subsp. nov. and emended description of Mycobacterium abscessus. Int J Syst Evol Microbiol. 2011;61(Pt 9): 2311-2313.
3. Roux A-L, Catherinot E, Ripoll F, et al. Multicenter study of prevalence of nontuberculous mycobacteria in patients with cystic fibrosis in France. J Clin Microbiol. 2009;47(12):4124-4128.
15. Koh W-J, Jeong B-H, Kim S-Y, et al. Mycobacterial characteristics and treatment outcomes in Mycobacterium abscessus lung disease. Clin Infect Dis. 2017;64(3):309-316.
4. Seddon P, Fidler K, Raman S, et al. Prevalence of nontuberculous mycobacteria in cystic fibrosis clinics, United Kingdom, 2009. Emerg Infect Dis. 2013;19(7):1128-1130.
16. Zelazny AM, Root JM, Shea YR, et al. Cohort study of molecular identification and typing of Mycobacterium abscessus, Mycobacterium massiliense, and Mycobacterium bolletii. J Clin Microbiol. 2009;47(7):1985-1995.
26. Kerem E, Viviani L, Zolin A, et al. Factors associated with FEV1 decline in cystic fibrosis: analysis of the ECFS patient registry. Eur Respir J. 2014;43(1):125-133.
17. Roux A-L, Catherinot E, Soismier N, et al. Comparing Mycobacterium massiliense and Mycobacterium abscessus lung infections in cystic fibrosis patients. J Cyst Fibros. 2015;14(1):63-69.
27. Cowman S, Burns K, Benson S, Wilson R, Loebinger MR. The antimicrobial susceptibility of non-tuberculous mycobacteria. J Infect. 2016;72(3): 324-331.
18. Nash KA, Brown-Elliott BA, Wallace RJJ. 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(4):1367-1376.
28. Obregon-Henao A, Arnett KA, HenaoTamayo M, et al. Susceptibility of Mycobacterium abscessus to antimycobacterial drugs in preclinical models. Antimicrob Agents Chemother. 2015;59(11):6904-6912.
5. Esther CR, Henry MM, Molina PL, Leigh MW. Nontuberculous mycobacterial infection in young children with cystic fibrosis. Pediatr Pulmonol. 2005;40(1):39-44. 6. Pierre-Audigier C, Ferroni A, SermetGaudelus I, et al. Age-related prevalence and distribution of nontuberculous mycobacterial species among patients with cystic fibrosis. J Clin Microbiol. 2005;43(7):3467-3470. 7. Sermet-Gaudelus I, Bourgeois M, Le, Pierre-Audigier C, et al. Mycobacterium abscessus and children with cystic fibrosis. Emerg Infect Dis. 2003;9(12):1587-1591. 8. Qvist T, Pressler T, Høiby N, Katzenstein TL. Shifting paradigms of nontuberculous mycobacteria in cystic fibrosis. Respir Res. 2014;15:41.
19. Bryant JM, Grogono DM, Greaves D, et al. Whole-genome sequencing to identify transmission of Mycobacterium abscessus between patients with cystic fibrosis: a retrospective cohort study. Lancet. 2013;381(9877):1551-1560.
9. Esther CRJ, Esserman DA, Gilligan P, Kerr A, Noone PG. Chronic Mycobacterium abscessus infection and lung function decline in cystic fibrosis. J Cyst Fibros. 2010;9(2):117-123.
20. Bryant JM, Grogono DM, RodriguezRincon D, et al. Emergence and spread of a human-transmissible multidrugresistant nontuberculous mycobacterium. Science. 2016;354(6313):751-757.
10. Sanguinetti M, Ardito F, Fiscarelli E, et al. Fatal pulmonary infection due to multidrug-resistant Mycobacterium abscessus in a patient with cystic fibrosis. J Clin Microbiol. 2001;39(2):816-819. 11. Chalermskulrat W, Sood N, Neuringer IP, et al. Non-tuberculous mycobacteria in end stage cystic fibrosis: implications for lung transplantation. Thorax. 2006;61(6): 507-513. 12. Martiniano SL, Sontag MK, Daley CL, Nick JA, Sagel SD. Clinical significance of a first positive nontuberculous mycobacteria culture in cystic fibrosis. Ann Am Thorac Soc. 2014;11(1):36-44. 13. Adékambi T, Drancourt M. Dissection of phylogenetic relationships among 19 rapidly growing Mycobacterium species by 16S rRNA, hsp65, sodA, recA and rpoB gene sequencing. Int J Syst Evol Microbiol. 2004;54(Pt 6):2095-2105. 14. Adékambi T, Berger P, Raoult D, Drancourt M. rpoB gene sequence-based characterization of emerging nontuberculous mycobacteria with descriptions of Mycobacterium bolletii sp. nov., Mycobacterium phocaicum sp. nov. and Mycobacterium aubagnense sp. nov.
21. Adekambi T, Drancourt M. Mycobacterium bolletii respiratory infections. Emerg Infect Dis. 2009;15(2): 302-305. 22. Bastian S, Veziris N, Roux A-L, et al. Assessment of clarithromycin susceptibility in strains belonging to the Mycobacterium abscessus group by erm(41) and rrl sequencing. Antimicrob Agents Chemother. 2011;55(2):775-781. 23. Floto RA, Olivier KN, Saiman L, et al. US Cystic Fibrosis Foundation and European Cystic Fibrosis Society consensus recommendations for the management of non-tuberculous mycobacteria in individuals with cystic fibrosis. Thorax. 2016;71(suppl 1):i1-i22. 24. Griffith DE, Aksamit T, BrownElliott BA, et al. ATS Mycobacterial Diseases Subcommittee; American Thoracic Society; Infectious Diseases Society of America. An official ATS/ IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med. 2007;175(4):367-416. 25. Leao SC, Tortoli E, Euzeby JP, Garcia MJ. Proposal that
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29. Lerat I, Cambau E, Roth Dit Bettoni R, et al. In vivo evaluation of antibiotic activity against Mycobacterium abscessus. J Infect Dis. 2014;209(6):905-912. 30. Lobo LJ, Chang LC, Esther CRJ, Gilligan PH, Tulu Z, Noone PG. Lung transplant outcomes in cystic fibrosis patients with pre-operative Mycobacterium abscessus respiratory infections. Clin Transplant. 2013;27(4): 523-529.
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31. Qvist T, Pressler T, Thomsen VO, Skov M, Iversen M, Katzenstein TL. Nontuberculous mycobacterial disease is not a contraindication to lung transplantation in patients with cystic fibrosis: a retrospective analysis in a Danish patient population. Transplant Proc. 2013;45(1):342-345. 32. Macheras E, Konjek J, Roux A-L, et al. Multilocus sequence typing scheme for the Mycobacterium abscessus complex. Res Microbiol. 2014;165(2):82-90.
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33. Falkinham JO III. Surrounded by mycobacteria: nontuberculous mycobacteria in the human environment. J Appl Microbiol. 2009;107(2):356-367.
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34. Sapriel G, Konjek J, Orgeur M, et al. Genome-wide mosaicism within Mycobacterium abscessus: evolutionary and epidemiological implications. BMC Genomics. 2016;17(1):118.
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35. Brown-Elliott BA, Vasireddy S, Vasireddy R, et al. Utility of sequencing the erm(41) gene in isolates of Mycobacterium abscessus subsp. abscessus with low and intermediate clarithromycin MICs. J Clin Microbiol. 2015;53(4):12111215.
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Mycobacterium bolletii Lung Disease in Cystic Fibrosis
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Virginie Rollet-Cohen, MD; Anne-Laure Roux, MD; Muriel Le Bourgeois, MD; Guillaume Sapriel, PhD;
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The cystic fibrosis (CF) pathogen, Mycobacterium abscessus complex, covers three subspecies: M. abscessus, M. massiliense, and M. bolletii. There are no clinical outcome data concerning M. bolletii. Our aim was to characterize M. bolletii lung infections in patients with CF. BACKGROUND:
17 18 19 20 21
We included patients with M. bolletii lung infection recorded between 1994 and 2012 in France. Data were collected from the CF registry, medical records, and questionnaires submitted to the CF primary physician. Strains were typed by multilocus sequence typing analysis.
METHODS:
22 23 24 25
RESULTS: Fifteen cases were identified in nine CF centers. Nine patients (60%) presented with nontuberculous mycobacterial pulmonary disease. Follow-up of 13 patients showed a trend to more rapid decline in FEV1 in the first year of colonization (–9.4%; SD 19.3) in comparison with noninfected control subjects (–2.3%; SD 12.1) (P ¼ .16). Twelve patients were treated, and 11 received oral macrolides. Treatment-induced eradication occurred in five patients (41.7%). Four patients died (26.7%), including one patient with fatal nontuberculous mycobacterial pulmonary disease. Inducible macrolide resistance was demonstrated in all strains. Patients always harbored unique strains.
26 27 28 29
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Our study reports the largest study cohort of CF patients infected with M. bolletii. M. bolletii infection affects both children and young adults, is most often symptomatic, and may be fatal. Macrolide-based therapies have poor effectiveness. There is no evidence of patient-to-patient transmission. CHEST 2019; -(-):---
CONCLUSIONS:
35 36 37 38 39
clarithromycin; cystic fibrosis; erm41; macrolide resistance; multilocus sequence typing analysis; Mycobacterium bolletii; Mycobacterium abscessus; Mycobacterium massiliense; nontuberculous mycobacteria KEY WORDS:
40 41 42
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Q2 ABBREVIATIONS:
ATS = American Thoracic Society; CF = cystic fibrosis; MABSC = Mycobacterium abscessus complex; MLST = multilocus sequence typing; NTM = nontuberculous mycobacteria; NTMPD = nontuberculous mycobacterial pulmonary disease AFFILIATIONS: From the Centre de Ressources et de Compétences pour la Mucoviscidose, Pneumologie et Allergologie Pédiatriques (Drs Rollet-Cohen, Le Bourgeois, and Sermet-Gaudelus), Hôpital Necker Enfants Malades, Paris, France; UMR 1173 (Drs Roux, Sapriel, Heym, and Gaillard), UFR des Sciences de la Santé Simone Veil, Université de Versailles Saint-Quentin-en-Yvelines, Montigny-le-Bretonneux, France; the Laboratoire de Microbiologie (Drs Roux, Heym, and
Gaillard), Hôpital Ambroise Paré, Boulogne, France; the Atelier de Bioinformatique (Dr Sapriel), Institut Systématique Evolution Biodiversité, UMR 7205, Paris, France; the Département de Biostatistiques (Drs El Bahri and Jais), Hôpital Necker Enfants Malades, Paris, France; the Laboratoire de Bactériologie (Drs Mougari and Raskine), Hôpitaux Lariboisière-Saint Louis, Paris, France; the Centre National de Référence des Mycobactéries et de la Résistance des Mycobactéries aux Antituberculeux (Drs Mougari and Raskine), Laboratoire de Bactériologie, Hôpitaux Universitaires Pitié-Salpetrière, Paris, France; and Sorbonne Université (Dr Véziris), Centre d’Immunologie et des Maladies Infectieuses, and Centre National de Référence des
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Mycobacterium abscessus complex (MABSC) is a rapidly growing, nontuberculous mycobacterium (NTM) that is increasingly recognized as a respiratory pathogen in people with cystic fibrosis (CF).1–4 MABSC may infect patients with CF at any age, but infection peaks between 11 and 15 years of age,5–7 with a prevalence from 3% to 6%.1,3,6–8 MABSC infection has been associated with a decline in lung function,7,9 severe pseudotuberculous lung disease, and fatal dissemination.10,11 However, MABSC can also transiently, intermittently, or permanently colonize the lungs of patients with CF without causing pulmonary disease.12 MABSC is a single species comprising three “subspecies”: M. abscessus, M. massiliense, and M. bolletii.13,14 Previous studies have focused on M. abscessus and M. massiliense, which are responsible for > 90% of MABSC cases in CF.15,16 Major clinical differences have been reported between these two subspecies. M. abscessus and M. massiliense target different CF subpopulations, with young and malnourished subjects being particularly susceptible to M. massiliense.17 Infections linked to M. abscessus are
more severe than M. massiliense and generally refractory to clarithromycin-based combination therapies, due to the expression of a functional erm41 gene that causes macrolide resistance (“T28 erm41 genotype”) in most strains. M. massiliense carries a nonfunctional, truncated erm41 gene and is susceptible to macrolides,18 inducing therefore more frequent success in eradication. Finally, patient-to-patient transmission in CF centers has exclusively been reported for M. massiliense.19,20 It is still unknown whether M. bolletii displays a specific profile for infection and therapeutic susceptibility in CF. Very few cases of M. bolletii lung disease have been reported and exclusively in non-CF adult patients.21 M. bolletii was described as resistant to clarithromycin, consistent with the expression of functional erm41 in this species.15,21,22 In this study, we present the largest cohort of CF patients infected with M. bolletii ever published and analyze the characteristics and outcome of 15 cases of M. bolletii lung infection in patients with CF, in association with comprehensive strain characterization.
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166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190
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Methods Study Population We included all M. bolletii lung infection cases recorded between 1994 and 2012 in the French CF Registry. Cases were defined according to the American Thoracic Society (ATS) bacteriologic criteria for NTM lung infection (ie, positive culture results from at least two separate expectorated sputum samples).23,24 NTM pulmonary disease (NTMPD) was defined according to the ATS/Infectious Diseases Society of America (IDSA) 2007 statement.24 NTM eradication was defined by at least three negative cultures for M. bolletii over at least 1 year. Clinical and radiologic data were collected from medical records and the French CF Registry with the permission of an international review board. All the patients enrolled in the French CF Registry had given informed consent for data use.
multicenter database created in 2008 (PHRC P100201), according to age categories, sex, Pseudomonas aeruginosa colonization, and FEV1 at entry in the study. We focused on the first year of colonization to determine whether the colonization was a risk factor for immediate degradation. Bacteriologic Methods All strains were sent to the National Reference Center (NRC) for Mycobacteria and cryoconserved at –80 C. rpoB-based identification, erm41 genotyping, and drug susceptibility testing were performed by the NRC (see the online article). Genetic analysis of strains was performed by the microbiology laboratory of Ambroise Paré Hospital, using multilocus sequence typing (MLST) (see the online article).
193 194 195 196 197 198 199 200 201 202 203 204
Statistical Methods
205
Qualitative variables were analyzed by c2 test or Fisher exact test, and continuous variables by Student t test. Statistical significance was accepted for P < .05.
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DFEV1 (FEV1 change over 1 year) between the infected patients and
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the control group was compared using a linear mixed model (LME4 package of R v3.4; R Foundation) to take into account the paired design and the variable number of control subjects per stratum. Significance was defined as p < .05.
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To investigate the impact of M. bolletii on the evolution of respiratory function, colonized patients were matched with NTM-negative control subjects issued from the French CF Registry and a prospective
Mycobactéries et de la Résistance des Mycobactéries aux Antituberculeux, Département de Bactériologie, AP-HP, Hôpitaux Universitaires de l’Est Parisien, Paris, France. 157 Drs Rollet-Cohen and Roux contributed equally to this manuscript. 158 FUNDING/SUPPORT: This work was supported by the association 159 Q3 Q4 Vaincre la Mucoviscidose. 160 CORRESPONDENCE TO: Isabelle Sermet-Gaudelus, MD, Centre de 161 Ressources et de Compétences pour la Mucoviscidose, Pneumologie et Allergologie Pédiatriques, Hôpital Necker Enfants Malades, Paris 162 Q5 75014, France; e-mail: [email protected] 163 Copyright Ó 2019 Published by Elsevier Inc under license from the 164 American College of Chest Physicians. 165 DOI: https://doi.org/10.1016/j.chest.2019.03.019 156
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Results
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Cases Included
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Fifteen M. bolletii lung infection cases were identified between 1994 and 2012. All cases fulfilled the ATS bacteriologic criteria for NTM lung infection, and nine
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TABLE 1
] Demographic Characteristics of Patients at Patients (n ¼ 15)
Characteristics Sex ratio, men/women, No. (%)
6/9 (0.67)
Age, y, mean (SD)
15.9 (10.7)
227
F508del/F508dela: No. (%)
228
BMI, kg/m2, mean (SD)
229
FEV1, % predicted, mean (SD)
230
Pancreatic insufficiencya
231 232 233 234 235 236 237 238 239 240
Treated ABPA
7 (46.7) 17.5 (2.6) (n ¼ 14)
243
13 (86.7)
a
3 (20.0)
Low-dose azithromycina
6 (42.8) (n ¼ 14)
Staphylococcus aureus colonizationa,b
12 (80.0)
Pseudomonas aeruginosa colonizationa,b
11 (73.3)
ABPA ¼ allergic bronchopulmonary aspergillosis. a Number/total number evaluated (%). b At least one positive sample within the previous year.
(60.0%) had NTM-PD. The mean follow-up was 7.8 years (2-19 years).
245
Patient Characteristics
246
Demographic characteristics of patients at the first isolation of M. bolletii are presented in Table 1. Nine of 15 (60%) were younger than 15 years. Six patients (40%) had a negative BMI z-score value. Two patients (5 and 36 years old) received a diagnosis of CF during the search for an underlying cause of M. bolletii infection.
248 249 250 251 252 253 254
Clinical and Radiologic Presentation
255
Clinical and radiologic findings are presented in Table 2. Eleven patients (73%) were symptomatic at the first isolation of M. bolletii. CT scan modifications were
256 257 258 259 260
TABLE 2
] Clinical and Radiologic Presentation at First
261 262 263 264 265 266
Isolation of Mycobacterium bolletii
Presentation
No. (%)
Clinical signs and symptoms Any
11 (73.3)
Respiratory deterioration
11 (73.3)
267
Weight loss
7 (46.7)
268
Fever
4 (26.7)
269
CT scan abnormalities
270
Any
9 (60.0)
271
Tree-in-bud
5 (33.3)
272
Consolidation
5 (33.3)
Nodules
4 (26.7)
Cavities
3 (20.0)
273 274 275
278 279 280
0
281 282 283
–20 NTM-negative control subjects n = 32
M. bolletii infected patients n = 13
Figure 1 – FEV1 follow-up 1 year after the first isolation of Mycobacterium bolletii. Absolute change in FEV1 (% predicted) in 13 patients infected with M. bolletii and 32 NTM-negative control subjects with cystic fibrosis. Each rectangle spans the first quartile to the third quartile (the interquartile range). Median and minimum/maximum range are shown. NTM ¼ nontuberculous mycobacteria.
284 285 286 287 288 289
Q21
290 291
Q16
292 293
244
247
277
+20
71.4 (19.6)
241 242
276
First Isolation of Mycobacterium bolletii
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print & web 4C=FPO
222
FEV1 change (%)
221
observed in nine patients (60%). Worsening of respiratory symptoms was defined by breathlessness, increased cough and/or sputum production. CT scan abnormalities involved at least two lobes in all patients. Two or more abnormalities were associated in four patients (27%), most frequently consolidations and nodules associated with cavities.
297 298 299 300 301 303
Follow-up of respiratory function was not available for two patients. The 13 remaining patients were compared with 32 CF control subjects negative for NTM. Infected patients presented a trend to a more rapid decline in FEV1 (–9.4%; SD 19.3) during the first year of colonization in comparison with the control subjects (–2.3%; SD 12.1) (delta estimate, 6.54; SD 4.6) (Fig 1). Q9 The difference was, however, not significant (P ¼ .16).
304 305 306 307 308 309 310 311 312 313
Response to Antimycobacterial Treatment
Twelve patients were treated with antimycobacterial combination therapies during the follow-up period. Treatment was implemented after a mean (SD) delay of 21 (28.3) months after the first isolation of M. bolletii, and the mean (SD) duration of treatment was 15 (24.8) months. Three patients (20%) had “spontaneous” eradication of M. bolletii from their sputum and were not treated. All treated patients received combination therapies based on oral macrolides (clarithromycin, nine patients; clarithromycin followed by azithromycin, two patients), except one patient, who was treated with moxifloxacin, ethambutol, and amikacin. Drugs combined with macrolides were inhaled (n ¼ 7) and/or intravenous amikacin (n ¼ 7), ethambutol (n ¼ 4), linezolid (n ¼ 3),
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Evolution of Respiratory Function
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314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330
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TABLE 3
] Comparison of Patients With or Without Eradication of Mycobacterium bolletii
332 333 334
Sex ratio, men/women
335
Age, y, mean (SD)
336
BMI, kg/m2, mean (SD)
337
F508del/F508dela
338
FEV1, % predicted, mean (SD)
339 340 341
Pseudomonas aeruginosa colonization, No. (%) a
NTM pulmonary disease, No. (%)
342
Months before antimycobacterial treatment, mean (SD)
343
Months of antimycobacterial treatment, mean (SD)
344 345
Eradication (n ¼ 5)
No Eradication (n ¼ 7)
1/4
4/3
.29
9.6 (6.4)
21.4 (12.8)
.09
P Value
387 388 389 390
.5
391
4 (80.0)
2 (28.6)
.24
392
65.4 (17.1)
73.4 (23.8)
.54
393 394
16.5 (2.7)
a
386
17.4 (1.5)
4 (80.0)
5 (71.4)
1
2 (40.0)
6 (85.7)
.22
25.7 (36.0)
19.7 (25.0)
.77
10 (4.3)
31.9 (35.1)
.26
395 396 397 398 399
NTM ¼ nontuberculous mycobacteria. a At least one positive sample within the previous year.
400
346
401
347
402
348 349 350 351 352 353 354 355 356 357 358 359
moxifloxacin (n ¼ 2), tigecycline (n ¼ 2), cefoxitin (n ¼ 2), imipenem (n ¼ 2), ciprofloxacin (n ¼ 1), and trimethoprim/sulfamethoxazole (n ¼ 1). Total eradication of M. bolletii following treatment was obtained in five of 12 patients (42%), vs three of 15 nontreated patients (20%) who underwent spontaneous eradication. This difference was not significant (eTable 1). Although no factor was significantly associated with eradication, patients who experienced eradication tended to be younger (mean [SD] age, 9.6 [6.4] vs 21.4 [12.8] years; not significant) (Table 3).
360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385
Mortality
Four of the 15 patients (27%) died during the study period. Two patients died following lung transplant. One patient was determined to have end-stage CF disease after being excluded for lung transplant because of repeated positive cultures under antimycobacterial treatment. The last patient was a young boy who developed severe M. bolletii lung disease treated with clarithromycin and imipenem in association with intravenous and inhaled amikacin. The patient died at 9 years of age with respiratory insufficiency and blood cultures positive for M. bolletii. Lung Transplant
Four patients underwent lung transplant during the study period. M. bolletii was eradicated in three of those for at least 12 months before transplant. The last patient was transplanted while being treated for M. bolletii lung disease and remained NTM positive after lung transplant without receiving any antimycobacterial treatment. Two of the lung transplant recipients were still alive 2 and 4 years after transplant, including the patient who failed to culture convert; the other two
4 Original Research
patients died because of organ rejection at 8 and 9 months posttransplant.
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Drug Susceptibility Testing Results
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Inducible macrolide resistance was observed in all strains, with minimal inhibitory concentrations $ 16 mg/L after extended incubation (e-Table 2). Sequencing of erm41 showed that all strains displayed a “T28” genotype, including the two M. bolletii/ M. massiliense “mosaic” strains identified by MLST analysis. The proportions of strains susceptible to amikacin, linezolid, and tigecycline were, respectively, 10 of 12 (83.3%), 5 of 12 (41.7%), and 10 of 10 (100%); 2 of 12 (16.7%) and 7 of 12 (58.3%) of the strains were susceptible and intermediate in response to cefoxitin, respectively; no strain showed susceptibility to moxifloxacin (e-Table 2).
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Genetic Analysis of Strains
MLST analysis of the M. bolletii strains isolated from the 15 patients showed that each patient harbored a Q10 sequence type (ST) that differed from all other STs; there was no genetic proximity among the strains isolated from patients attending the same CF center (Fig 2). Although harboring M. bolletii rpoB and erm41 sequences, the strains from two patients appeared to be closer to M. massiliense (Fig 3). These “mosaic” M. bolletii/M. massiliense strains were eradicated (one without treatment, and the other after antimycobacterial course).
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Discussion
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This study, based on the analysis of 15 cases of M. bolletii infection, is the largest series published to date, even outside the context of CF. Surprisingly,
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Figure 2 – Minimum spanning tree (MST) of Mycobacterium bolletii population. MST was generated from MLST data, using PHYLOViZ (a platformindependent Java tool; http://www.phyloviz.net/). Red circles, NTM-PD strains; green circles, non-NTM-PD strains. MLST ¼ multilocus sequence typing; NTM-PD ¼ nontuberculous mycobacterial pulmonary disease.
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although M. bolletii is considered to be genetically closer to M. massiliense,25 we found that the main characteristics of infection with M. bolletii were close to those of M. abscessus infection.25 As for M. abscessus, M. bolletii was isolated not only from children and adolescents, but also from young adults. Moreover, 60% of our patients presented with NTM-PD, as assessed by ATS/IDSA criteria.24 We reanalyzed the data from our previous study to classify cases according to the criteria used in this study.23,24 This analysis yielded figures of 39% (9/23) of NTM-PD for M. abscessus and 7% (1/14) for M. massiliense. Infection severity is thus similar for M. bolletii and M. abscessus. This is also supported by the fact that patients infected with M. bolletii underwent a trend to more rapid decline in
lung function by almost 10% in contrast to control subjects whose FEV1 declined by 2.3%, a change similar to reports from European and US registries.26 Although the difference from the control group was not significant because of the low number of patients, this difference is highly relevant and supports the initiation of an eradication therapy as early as possible. In contrast, M. massiliense infections were much less severe and more likely to be treated successfully than infections with M. bolletii or M. abscessus. This supports the hypothesis that M. massiliense is a much less pathogenic member among MABSCs, at least in the context of CF.17 Q11 Indeed, our two cases of infection with a mosaic strain consisting mostly of M. massiliense were eradicated, even spontaneously in one case.
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Figure 3 – Multiple correspondence analysis (MCA) of Mycobacterium bolletii population. Each ST was projected onto the first two axes of MCA, based on the MLST allele profile. Red dots represent the 15 strains included in the present study. *The two mosaic strains. MLST ¼ multilocus sequence typing; ST ¼ sequence type.
Our study clearly demonstrates the inefficacy of macrolides for treating M. bolletii infections. This finding is consistent with previous studies showing that M. bolletii consistently displays inducible resistance to macrolides, due to the presence of a genotype T28 erm41 gene.18,22 This genotype is similar to that of almost all M. abscessus strains.22,28,29 Indeed, we found a bacteriologic success rate (total eradication from the sputum) on treatment in 40% of patients infected with M. bolletii, a figure not significantly different from the spontaneous eradication rate of 20%. This rate is close to the eradication pattern of M. abscessus.17 Indeed, in our previous cohort study, macrolide-based therapies were also ineffective against M. abscessus (eradication rate, 25% with treatment vs 33.3% without; P ¼ .65), and highly effective against M. massiliense (eradication rate, 85.7% with treatment vs 28.6% without; P ¼ .03). M. bolletii isolates tested in our study showed patterns of susceptibility to amikacin, cefoxitin, linezolid, tigecycline, and moxifloxacin similar to those reported for MABSC isolates.27 This suggests that antibiotic
6 Original Research
regimens recommended for treatment of MABSC-PD23 should also be used for M. bolletii pulmonary disease.
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The consensus of the US CF Foundation and the European CF Society clarifies the course of action in cases of lung transplant, a problem that has been the subject of several studies in CF and non-CF patients.30,31 The largest and most comprehensive study, carried out by the University of North Carolina Chapel Hill between 1990 and 2003, showed that MABSC infection before transplant was a significant risk factor for the recurrence of NTM after transplant, with a significant impact on morbidity but not mortality.11 In the current consensus, it is strongly recommended not to exclude patients with current or previous respiratory tract samples positive for NTM from the transplant program.23 Our observations are consistent with these recommendations. The two transplant patients who died were NTM negative for over 1 year. The cause of death was severe lung graft rejection, whereas the sole patient who remained
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positive for M. bolletii after lung transplantation did not develop any complications linked to NTM infection.
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MLST genetic analysis showed that the strains of M. bolletii were extremely diverse. Each patient, including those monitored at the same center, carried a unique strain. This is consistent with most of the molecular typing studies carried out to date in the context of CF32 and supports the hypothesis of patient contamination from diverse environmental sources.33 This eliminates the possibility of cross-transmission between patients or from a common environmental source within the hospital. However, this result must be interpreted with caution. Indeed, two studies using whole-genome sequencing identified outbreaks of M. massiliense cases in a UK CF center, suggesting that person-to person transmission of MABSC strains may occur.19,20
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MLST analysis also confirmed the existence of mosaic strains of MABSC, resulting from horizontal genetic transfers involving chromosomal regions of various sizes between the three subspecies, probably allowing them to adapt to their environmental niches.34 We currently have no data concerning the relationship of these processes to differences in pathogenicity in humans. However, the existence of these mosaic strains poses two practical problems. The first is related to the possible presence of the rpoB gene (or of any other target gene used for the diagnosis of subspecies) in the transferred region, leading, for example, to a mosaic strain with an
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“M. massiliense” genetic background. In contrast, carrying the rpoB gene of M. bolletii, as in our series, leads to an “erroneous” diagnosis of M. bolletii infection. The other issue is the prediction of strain sensitivity to macrolides. Indeed, genetic transfers between subspecies of MABSC may involve the erm41 gene, resulting in the replacement of the endogenous gene with a gene from a different subspecies. For any MABSC strain, it is therefore prudent (1) to sequence the erm41 gene to ensure consistency between the genotype found and the type of subspecies; and (2) to determine macrolide sensitivity in vitro, which can detect both inducible resistance involving erm41 and high-level acquired resistance due to mutations of the rrl gene (23S rDNA gene).18,22,35
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Conclusion This study is the largest, to date, that specifically addresses lung infection due to M. bolletii in the setting of CF. It shows that M. bolletii lung infection shares a number of similarities with M. abscessus infection such as a rapid decline in respiratory function and inefficiency of macrolides, due to the T28 erm41 genotype. This is in contrast to the high success rate of eradication obtained against M. massiliense, which carries a nonfunctional erm41 gene. The erm41 genotype should be determined before any treatment, due to the possibility of horizontal genetic transfers within MABSC.
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Acknowledgments Author contributions: Guarantor of the article: I. S.-G. had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis, including and especially any adverse effects. I. S.-G. and J.-L. G. designed the study, participated in acquisition of the data, analyzed the results, and wrote the manuscript. V. R.-C., A.-L. R., and M. L. B. participated in the design of the study, analyzed the results, and participated in the writing of manuscript. G. S., E. Cambau, A. Ferroni, B. H., F. M., L. R., and N. V. performed the bacteriological study, analyzed the results, and participated in the writing of the manuscript. M. E. B. and J.-P. J. performed the statistical analysis and participated in the writing of the manuscript. P. R. Burgel, T. Gendry, H. Corvol, L. Couderc, D. Grenet, A. Haloun, V. Houdouin, F. Huet, S. Marchand, I. Pin Isabelle, and S. Quetant provided medical records and reviewed the manuscript. L. Lemonnier provided French CF Registry data. A. Edelman analyzed the results and
participated in the writing of the manuscript.
analysis of the data, or the preparation of the manuscript.
Financial/nonfinancial disclosures: The authors have reported to CHEST the following: I. S.-G. has no relevant conflict with the data included in this article. She declares the following funding from the Vertex Pharmaceuticals Innovation Award funding program in 2016 (Use of Nasal Epithelial Cells to Investigate the Relationship Between CF Mutation, Clinical Phenotype and CFTR Dysfunction in Cystic Fibrosis) and 2017 (ATP12A as a Novel Therapeutic Target in Cystic Fibrosis Lung Diseases). She was PI in clinical trials for Vertex Pharmaceuticals VX-809-103, VX-809-109, VX-661-108, and VX-661-109. She took part on scientific advisory boards organized by Vertex Pharmaceuticals in June 2017, September 2017, October 2017, and June 2018. None declared (V. R.-N., A.-L. R., M. L. B., G. S., M. E. B., J.-P. J., B. H., F. M., L. R., N. V., J.-L. G.). Role of sponsors: The sponsor had no role in the design of the study, the collection and
Other contributions: The authors thank all the clinicians for their contributions: PierreRégis Burgel, MD; Thierry Gendry, MD; Harriet Corvol, MD; Laure Couderc, MD; Dominique Grenet, MD; Alain Haloun, MD; Véronique Houdouin, MD; Frédéric Huet, MD; Sophie Marchand, MD; Isabelle Pin, MD; Lydie Lemonnier, PhD; Sébastien Quetant, MD; Emmanuelle Cambau, MD; and Agnès Ferroni, MD.
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Additional information: The e-Appendix and e-Tables can be found in the Supplemental Materials section of the online article.
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35. Brown-Elliott BA, Vasireddy S, Vasireddy R, et al. Utility of sequencing the erm(41) gene in isolates of Mycobacterium abscessus subsp. abscessus with low and intermediate clarithromycin MICs. J Clin Microbiol. 2015;53(4):12111215.
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