Advertisement

Distribution and outcomes of infection of Mycobacterium avium complex species in cystic fibrosis

Open ArchivePublished:August 06, 2019DOI:https://doi.org/10.1016/j.jcf.2019.07.007

      Abstract

      Background

      The majority of nontuberculous mycobacterial (NTM) pulmonary infections in people with cystic fibrosis (CF) are caused by Mycobacterium avium complex (MAC) species. Data on MAC species distribution and outcomes of infection in CF are lacking.

      Methods

      This was a single center, retrospective study. MAC isolates had species identification with MLSA of rpoB and the 16S 23S ITS region. Clinical data were compared between species.

      Results

      Twenty-three people with CF and 57 MAC isolates were included. Infection with M. avium was the most common (65.2%). M. intracellulare was associated with higher rates of NTM disease, younger age, and steeper decline in lung function prior to infection.

      Conclusions

      We observed worse clinical outcomes in people with M. intracellulare infection relative to other MAC species. Further investigation of clinical outcomes of MAC infection among CF patients is warranted to better define the utility of species-level identification of MAC isolates in CF.

      Keywords

      1. Background

      Mycobacterium avium complex (MAC) accounts for ~70% of nontuberculous mycobacterial (NTM) pulmonary infections in people with CF [
      • Floto R.A.
      • Olivier K.N.
      • Saiman L.
      • Daley C.L.
      • Herrmann J.L.
      • Nick J.A.
      • 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.
      ,
      • Olivier K.N.
      • Weber D.J.
      • Wallace R.J.
      • Faiz A.R.
      • Lee J.H.
      • Zhang Y.
      • et al.
      Nontuberculous mycobacteria: I: Multicenter prevalence study in cystic fibrosis.
      ], and is the most common cause of NTM pulmonary infection in non-CF populations worldwide [
      • Griffith D.E.
      • Aksamit T.
      • Brown-Elliott B.A.
      • Catanzaro A.
      • Daley C.
      • Gordin F.
      • et al.
      An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases.
      ,
      • Hoefsloot W.
      • Van Ingen J.
      • Andrejak C.
      • Ängeby K.
      • Bauriaud R.
      • Bemer P.
      • et al.
      The geographic diversity of nontuberculous mycobacteria isolated from pulmonary samples: an NTM-NET collaborative study.
      ]. Within MAC, three species (Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium chimaera) account for the majority of MAC pulmonary infections worldwide, with species distribution varying by region [
      • Hoefsloot W.
      • Van Ingen J.
      • Andrejak C.
      • Ängeby K.
      • Bauriaud R.
      • Bemer P.
      • et al.
      The geographic diversity of nontuberculous mycobacteria isolated from pulmonary samples: an NTM-NET collaborative study.
      ,
      • Boyle D.P.
      • Zembower T.R.
      • Reddy S.
      • Qi C.
      Comparison of clinical features, virulence, and relapse among Mycobacterium avium complex species.
      ,
      • Kim S.Y.
      • Shin S.H.
      • Moon S.M.
      • Yang B.
      • Kim H.
      • Kwon O.J.
      • et al.
      Distribution and clinical significance of Mycobacterium avium complex species isolated from respiratory specimens.
      ]. Clinical laboratories do not routinely identify species within MAC [
      • Floto R.A.
      • Olivier K.N.
      • Saiman L.
      • Daley C.L.
      • Herrmann J.L.
      • Nick J.A.
      • 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.
      ,
      • Griffith D.E.
      • Aksamit T.
      • Brown-Elliott B.A.
      • Catanzaro A.
      • Daley C.
      • Gordin F.
      • et al.
      An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases.
      ], thus the clinical implications of MAC species identification are overall unclear.
      Certain studies in non-CF populations have suggested that clinical outcomes may vary between MAC species, and thus suggest clinical utility of MAC species identification. In non-CF populations, patients with M. chimaera infection have lower rates of smear-positive AFB cultures and lower rates of diagnosis of NTM lung disease (i.e., necessitating NTM-directed antibiotic treatment) compared to those with either M. avium or M. intracellulare infection [
      • Boyle D.P.
      • Zembower T.R.
      • Reddy S.
      • Qi C.
      Comparison of clinical features, virulence, and relapse among Mycobacterium avium complex species.
      ,
      • Kim S.Y.
      • Shin S.H.
      • Moon S.M.
      • Yang B.
      • Kim H.
      • Kwon O.J.
      • et al.
      Distribution and clinical significance of Mycobacterium avium complex species isolated from respiratory specimens.
      ]. In a single center study, M. intracellulare infection was associated with higher rates of smear-positive AFB cultures, higher rates of NTM lung disease, and lower rates of microbiologic response to NTM treatment compared to M. avium infection (though M. chimaera was not distinguished from M. intracellulare in this study) [
      • Koh W.J.
      • Jeong B.H.
      • Jeon K.
      • Lee N.Y.
      • Lee K.S.
      • Woo S.Y.
      • et al.
      Clinical significance of the differentiation between Mycobacterium avium and Mycobacterium intracellulare in M avium complex lung disease.
      ].
      In people with CF, the distribution of MAC species and differences in clinical outcomes associated with MAC species are unknown [
      • Martiniano S.L.
      • Sontag M.K.
      • Daley C.L.
      • Nick J.A.
      • Sagel S.D.
      Clinical significance of a first positive nontuberculous mycobacteria culture in cystic fibrosis.
      ]. The objectives of this single center, retrospective study were to (1) Identify the distribution of MAC species among CF patients, and (2) Compare patient demographics and clinical outcomes between MAC species.

      2. Methods

      2.1 Patients and clinical data

      With Michigan Medicine Institutional Review Board approval, CF patients with MAC infection between December 2013 and June 2018 were identified. Patients with a prior organ transplant or with Mycobacterium abscessus complex co-infection were excluded. Patient data were extracted from the medical records [
      • Hanauer D.A.
      EMERSE: the electronic medical record search engine.
      ]. Infections were categorized as transient (single positive MAC culture followed by at least one negative AFB culture), persistent (at least two positive MAC cultures), or NTM disease (defined as physician's decision to initiate antibiotics targeting MAC) [
      • Martiniano S.L.
      • Sontag M.K.
      • Daley C.L.
      • Nick J.A.
      • Sagel S.D.
      Clinical significance of a first positive nontuberculous mycobacteria culture in cystic fibrosis.
      ].

      2.2 Multilocus sequence analysis (MLSA) for MAC species identification

      MAC isolates were recovered from respiratory samples through routine clinical care, sub-cultured onto Middlebrook 7H11 agar, and DNA were extracted [
      • Liu L.
      • Coenye T.
      • Burns J.L.
      • Whitby P.W.
      • Stull T.L.
      • LiPuma J.J.
      Ribosomal DNA-directed PCR for identification of Achromobacter (Alcaligenes) xylosoxidans recovered from sputum samples from cystic fibrosis patients.
      ]. Bacterial lysates were used as templates for PCR targeting rpoB and the 16S 23S ITS regions [
      • Boyle D.P.
      • Zembower T.R.
      • Reddy S.
      • Qi C.
      Comparison of clinical features, virulence, and relapse among Mycobacterium avium complex species.
      ,
      • Frothingham R.
      • Wilson K.H.
      Sequence-based differentiation of strains in the Mycobacterium avium complex.
      ,
      • Ben Salah I.
      • Adékambi T.
      • Raoult D.
      • Drancourt M.
      rpoB sequence-based identification of Mycobacterium avium complex species.
      ]. Purified PCR products underwent Sanger sequencing. Sequences were edited (Chromas version 2.6.5, Technelysium Pty. Ltd), aligned to MAC reference genomes with Clustal W (MegAlign version 12.2.0, DNAStar, Madison, WI), and trimmed based on homology to a consensus sequence. Trimmed sequences (sequence length 1214 bp) were concatenated, aligned with Clustal V, and a dendrogram was produced using default MegAlign parameters to identify MAC species based on the closest reference strain.

      2.3 Statistics

      Analyses were performed using R (version 3.5.1) [
      • R Core Team
      ]. Age and lung function at MAC acquisition were compared between M. avium, M. intracellulare, and M. chimaera with one-way ANOVA. CFTR genotype, AFB smear results, CF pathogen data, and sex were compared between the four species with separate Fisher's exact tests. Longitudinal percent predicted FEV1 (ppFEV1) values were analyzed with a linear mixed effects model, controlling for patient age, with patient ID slope and intercept random effects using the “lmerTest” package in R [
      • Kuznetsova A.
      • Brockhoff P.B.
      • Christensen R.H.B.
      lmerTest package: tests in linear mixed effects models.
      ].

      3. Results

      3.1 MAC species distribution

      Twenty-three patients and 57 MAC isolates (mean 2.45 isolates/patient, range = 1–10) were included. Based on species identification of each patient's first isolate (Fig. 1), infection with M. avium was the most common (65.2% of patients), followed by M. intracellulare (17.4%), M. chimaera (13.1%), and M. timonense (4.3%). Fourteen patients had multiple isolates included, and 78.6% of these patients (11/14) had the same species on serial isolates.
      Fig. 1
      Fig. 1Distribution of MAC species. Dendrogram of clinical MAC isolates (n = 57) with reference strains (n = 6) based on rpoB and 16S 23S ITS MLSA. Clinical isolates are labelled by a patient identifier (A-W), and serial isolates within patients are numbered sequentially (1−10). Patients in red (patients B, D, and F) had more than one MAC species identified among serial isolates. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

      3.2 Clinical differences between MAC species

      In our cohort, patients with M. intracellulare infection had features of more aggressive lung disease, including higher rates of homozygous F508del genotype and lower rates of infection with Staphylococcus aureus (p < .05, Fisher's exact tests) (Table 1). Lung function (ppFEV1) was similar across the groups, even though patients with M. intracellulare infection were younger than the majority of patients with other MAC species.
      Table 1Patient demographics and clinical features at initial MAC infection.
      M. aviumM. intracellulareM. chimaeraM. timonensep-value
      No. patients (%) (n = 23)15 (65.2%)4 (17.4%)3 (13.1%)1 (4.3%)
      No. female (%)9/15 (60%)1/4 (25%)3/3 (100%)00.21
      Age in years (mean, range)31.4 (10.9–52)14.3 (5.8–21.3)32.6 (21.9–52.4)400.12
      ppFEV1 (mean, range)71.5% (30–80%)71.8% (45–98%)73.7% (47–105%)68%0.99
      No. ΔF508 homozygous6/15 (40%)4/4 (100%)01 (100%)0.01
      No. AFB smear positive5/15 (33.3%)1/4 (25%)000.84
      CF pathogens present within year prior to MAC acquisition:
      Pseudomonas aeruginosa8/15 (53.3%)3/4 (75%)2/3 (67%)1 (100%)0.89
      Staphylococcus aureus14/15 (93.3%)2/4 (50%)3/3 (100%)00.03
      Stenotrophomonas maltophilia6/15 (40%)1/4 (25%)1/3 (33%)01
      Burkholderia cepacia complex2/15 (13.3%)0001
      Aspergillus fumigatus9/15 (60%)3/4 (75%)1/3 (33%)00.54
      Patients included in outcomes analysis (n = 22)14431
      NTM disease4/14 (29%)3/4 (75%)00
      Persistent infection7/14 (50%)1/4 (25%)2/3 (67%)0
      Transient infection3/14 (21%)01/3 (33%)1/1 (100%)
      Twenty-two patients had clinical data and AFB cultures available for outcomes evaluation (Table 1). Seven of these patients (32%) were diagnosed with NTM disease, five had transient MAC infection (23%), and ten had persistent MAC infection (45%). M. intracellulare infection was associated with the highest rates of NTM disease in this cohort (75%). None of the patients with M. chimaera were diagnosed with NTM disease.
      Longitudinal lung function values (ppFEV1) (n = 217, mean of 4.93 values/patient, range = 2–23) were available preceding and following MAC acquisition for 21 patients (Fig. 2). Rates of ppFEV1 change were significantly different from zero for M. intracellulare infection (p = .02 and p = .002, respectively, linear mixed model), but not for the other species. All groups were associated with decreasing ppFEV1 preceding MAC acquisition, with the greatest decrease associated with M. intracellulare infection. Patients with M. intracellulare infection displayed increasing ppFEV1 following MAC acquisition, whereas those with M. chimaera continued to decrease, and those with M. avium remained stable.
      Fig. 2
      Fig. 2Lung function trends before and after infection based on MAC species. Shown are linear regression lines of longitudinal lung function measurements (ppFEV1) in relation to time of MAC acquisition (time 0). Lung function changes before and after acquisition of M. intracellulare (dotted line) were significantly different from zero (p = .02 and p = .002, respectively, linear mixed effects model).

      4. Discussion

      In this study of persons with CF and MAC pulmonary infection, the distribution of MAC species was similar to those in non-CF populations, with the majority of patients having M. avium infection [
      • Boyle D.P.
      • Zembower T.R.
      • Reddy S.
      • Qi C.
      Comparison of clinical features, virulence, and relapse among Mycobacterium avium complex species.
      ,
      • Kim S.Y.
      • Shin S.H.
      • Moon S.M.
      • Yang B.
      • Kim H.
      • Kwon O.J.
      • et al.
      Distribution and clinical significance of Mycobacterium avium complex species isolated from respiratory specimens.
      ,
      • Koh W.J.
      • Jeong B.H.
      • Jeon K.
      • Lee N.Y.
      • Lee K.S.
      • Woo S.Y.
      • et al.
      Clinical significance of the differentiation between Mycobacterium avium and Mycobacterium intracellulare in M avium complex lung disease.
      ]. We observed associations between M. intracellulare and worse clinical outcomes compared to other MAC species, including greater lung function decline prior to infection and higher rates of NTM disease. These findings suggest greater pathogenicity of M. intracellulare compared to other MAC species, and suggest that MAC species identification in CF may have utility in anticipating clinical course, similar to the known utility of subspecies identification within the M. abscessus complex [
      • Floto R.A.
      • Olivier K.N.
      • Saiman L.
      • Daley C.L.
      • Herrmann J.L.
      • Nick J.A.
      • 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.
      ].
      In addition to greater lung function decline prior to infection, patients with M. intracellulare infection had other features of more aggressive lung disease, and these likely collectively contributed to the increased rates of NTM disease diagnosis in this group (whether or not they were attributable to M. intracellulare directly). Similarly, lung function improvements following M. intracellulare infection may reflect the higher rates of treatment with MAC directed antibiotics in the M. intracellulare group, suggesting efficacy of MAC treatment. However, these improvements also likely reflect lung function improvements gained through the optimization of airway clearance, treatment of other CF pathogens (e.g., P. aeruginosa), and CF-related co-morbidities that typically occur post-NTM infection. These potential confounders in interpreting lung function changes relative to NTM disease diagnoses and response to NTM treatment highlight difficulties in determining causality in the setting of NTM infections.
      This was a single center, retrospective study limited by small sample size, particularly in the M. intracellulare and M. chimaera groups, and inability to account for all potential confounders of outcomes between the MAC species groups. We grouped patients based on the species of the first isolate available in our collection. Eight patients had MAC infection prior to the first available isolate, and thus may have had a different MAC species at the time of initial infection. The MSLA approach used provides MAC species level identification and was able to identify a minority of patients with different MAC species on serial isolates, consistent with a recent study of MAC species identified with whole genome sequencing [
      • Operario D.J.
      • Pholwat S.
      • Koeppel A.F.
      • Prorock A.
      • Bao Y.
      • Sol-Church K.
      • et al.
      Mycobacterium Avium complex diversity within lung disease as revealed by whole genome sequencing.
      ]. The MLSA method does not, however, provide strain-level resolution, and we were not able to determine if isolates of the same species within a patient represented single or multiple strains. Finally, we acknowledge the possibility that the different rates of NTM disease observed across the species could reflect patterns of environmental NTM, rather than differences in species virulence (e.g., if the hospital water supply has higher rates of M. avium or M. chimaera, this could lead to sputum sample contamination with these species rather than true NTM infection).
      In summary, we observed differences in the distribution of and clinical outcomes associated with different MAC species in this single center study of people with CF, including greater lung function decline and higher rates of NTM disease associated with M. intracellulare infection. There may be differences in pathogenicity between different species of MAC in CF pulmonary infections warranting further study. If these trends are borne out in analyses of larger numbers of patients, identification of MAC isolates to the species level would have bearing on the prognosis and management of MAC infection in CF.

      Declaration of Competing Interest

      The authors have no conflicts of interest.

      Acknowledgements

      This work was supported by funding to LJC from the National Institutes of Health (K23HL136934) and the Cystic Fibrosis Foundation (CAVERL17A0).

      References

        • Floto R.A.
        • Olivier K.N.
        • Saiman L.
        • Daley C.L.
        • Herrmann J.L.
        • Nick J.A.
        • 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: i1-22https://doi.org/10.1136/thoraxjnl-2015-207360
        • Olivier K.N.
        • Weber D.J.
        • Wallace R.J.
        • Faiz A.R.
        • Lee J.H.
        • Zhang Y.
        • et al.
        Nontuberculous mycobacteria: I: Multicenter prevalence study in cystic fibrosis.
        Am J Respir Crit Care Med. 2003; 167: 828-834https://doi.org/10.1164/rccm.200207-678OC
        • Griffith D.E.
        • Aksamit T.
        • Brown-Elliott B.A.
        • 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: 367-416https://doi.org/10.1164/rccm.200604-571ST
        • Hoefsloot W.
        • Van Ingen J.
        • Andrejak C.
        • Ängeby K.
        • Bauriaud R.
        • Bemer P.
        • et al.
        The geographic diversity of nontuberculous mycobacteria isolated from pulmonary samples: an NTM-NET collaborative study.
        Eur Respir J. 2013; 42: 1604-1613https://doi.org/10.1183/09031936.00149212
        • Boyle D.P.
        • Zembower T.R.
        • Reddy S.
        • Qi C.
        Comparison of clinical features, virulence, and relapse among Mycobacterium avium complex species.
        Am J Respir Crit Care Med. 2015; 191: 1310-1317https://doi.org/10.1164/rccm.201501-0067OC
        • Kim S.Y.
        • Shin S.H.
        • Moon S.M.
        • Yang B.
        • Kim H.
        • Kwon O.J.
        • et al.
        Distribution and clinical significance of Mycobacterium avium complex species isolated from respiratory specimens.
        Diagn Microbiol Infect Dis. 2017; 88: 125-137https://doi.org/10.1016/j.diagmicrobio.2017.02.017
        • Koh W.J.
        • Jeong B.H.
        • Jeon K.
        • Lee N.Y.
        • Lee K.S.
        • Woo S.Y.
        • et al.
        Clinical significance of the differentiation between Mycobacterium avium and Mycobacterium intracellulare in M avium complex lung disease.
        Chest. 2012; 142: 1482-1488https://doi.org/10.1378/chest.12-0494
        • Martiniano S.L.
        • Sontag M.K.
        • Daley C.L.
        • Nick J.A.
        • Sagel S.D.
        Clinical significance of a first positive nontuberculous mycobacteria culture in cystic fibrosis.
        Ann Am Thorac Soc. 2014; 11: 36-44https://doi.org/10.1513/AnnalsATS.201309-310OC
        • Hanauer D.A.
        EMERSE: the electronic medical record search engine.
        AMIA Annu Symp Proc. 2006; 331: 941
        • Liu L.
        • Coenye T.
        • Burns J.L.
        • Whitby P.W.
        • Stull T.L.
        • LiPuma J.J.
        Ribosomal DNA-directed PCR for identification of Achromobacter (Alcaligenes) xylosoxidans recovered from sputum samples from cystic fibrosis patients.
        J Clin Microbiol. 2002; 40: 1210-1213https://doi.org/10.1128/JCM.40.4.1210-1213.2002
        • Frothingham R.
        • Wilson K.H.
        Sequence-based differentiation of strains in the Mycobacterium avium complex.
        J Bacteriol. 1993; 175: 2818-2825https://doi.org/10.1128/jb.175.10.2818-2825.1993
        • Ben Salah I.
        • Adékambi T.
        • Raoult D.
        • Drancourt M.
        rpoB sequence-based identification of Mycobacterium avium complex species.
        Microbiology. 2008; 154: 3715-3723https://doi.org/10.1099/mic.0.2008/020164-0
        • R Core Team
        R: A language and environment for statistical computing. 2018
        • Kuznetsova A.
        • Brockhoff P.B.
        • Christensen R.H.B.
        lmerTest package: tests in linear mixed effects models.
        J Stat Softw. 2017; 82: 1-26https://doi.org/10.18637/jss.v082.i13
        • Operario D.J.
        • Pholwat S.
        • Koeppel A.F.
        • Prorock A.
        • Bao Y.
        • Sol-Church K.
        • et al.
        Mycobacterium Avium complex diversity within lung disease as revealed by whole genome sequencing.
        Am J Resp Crit Care Med. 2019; https://doi.org/10.1164/rccm.201903-0669LE