International Journal of Infectious Diseases 98 (2020) 33–40

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International Journal of Infectious Diseases

journal homepage: www.elsevier.com/locate/ijid

Different clinical features of patients with pulmonary

disease caused by various Mycobacterium
avium–intracellulare complex subspecies and
antimicrobial susceptibility
Chia-Ling Changa , Lun-Che Chenb , Chong-Jen Yua , Po-Ren Hsueha,c , Jung-Yien Chiena,*
a
Department of Internal Medicine, National Taiwan University Hospital, National Taiwan University College of Medicine, Taipei, Taiwan
b
Department of Internal Medicine, National Taiwan University Hospital Hsinchu Biomedical Science Park Branch, National Taiwan University College of
Medicine, Hsinchu, Taiwan
c
Department of Laboratory Medicine, National Taiwan University Hospital, National Taiwan University College of Medicine, Taipei, Taiwan

A R T I C L E I N F O A B S T R A C T

Article history: Objectives: Characteristics of the Mycobacterium avium–intracellulare complex pulmonary disease
Received 5 May 2020 (MAC-PD) caused by distinct subspecies remain uncertain.
Received in revised form 3 June 2020 Methods: This study was conducted from 2013–2015 in three hospitals in Taiwan.
Accepted 5 June 2020
Results: Among the 144 patients with MAC-PD, 57 (39.6%), 37 (25.7%), 37 (25.7%), and 13 (9.0%) were
infected with Mycobacterium intracellulare subspecies intracellulare (MIsI), Mycobacterium avium
Keywords: subspecies hominissuis (MAsH), Mycobacterium intracellulare subspecies chimaera (MIsC), and others,
Mycobacterium avium–intracellulare
respectively. Patients with MAsH-PD were younger (p = 0.010) with higher human immunodeficiency
complex
Mycobacterium intracellulare subspecies
virus infection rates (27.0%, 0.0%, 0.0%, and 7.7% for MAsH-PD, MIsC-PD, MIsI-PD, and others, respectively;
intracellulare p < 0.001). Twenty-two (15.3%) patients reported spontaneous culture-negative conversion, but 15
Mycobacterium avium subspecies (10.4%) and 33 (22.9%) patients developed radiographic progression and unfavorable outcomes,
hominissuis especially MAsH-PD. The susceptibility rates to clarithromycin and inhaled amikacin were both 98.6%.
Mycobacterium intracellulare subspecies MAsH demonstrated the lowest rate of resistance to moxifloxacin (66.7%, 97.3%, 89.1%, and 92.3% for
chimaera MAsH-PD, MIsC-PD, MIsI-PD, and others, respectively; p = 0.001) and MIsI isolates had the highest rate of
Pulmonary disease resistance to intravenous amikacin (25%, 13.5%, 38.2%, and 15.4% for MAsH-PD, MIsC-PD, MIsI-PD, and
others, respectively; p = 0.024).
Conclusions: Pulmonary disease caused by distinct MAC subspecies had different outcomes and drug
susceptibility. The local prevalence of species needs to be monitored.
© 2020 The Author(s). Published by Elsevier Ltd on behalf of International Society for Infectious Diseases.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-
nd/4.0/).

Introduction Mycobacterium avium–intracellulare complex (MAC) is the most

Nontuberculous mycobacteria (NTM) are ubiquitous in the
environment and commonly found in water and soil (Falkinham,
2015). They can cause pulmonary and extra-pulmonary disease
(Haworth et al., 2017); the incidence of nontuberculous mycobac-
terial pulmonary disease (NTM-PD) has been increasing in many
industrialized countries such as North America, Europe, Australia
and East Asia (Japan, South Korea and Taiwan) (Huang et al., 2018;
Prevots and Marras, 2015; Yu et al., 2019). Among the NTM-PDs,
* Corresponding author at: Department of Internal Medicine, National Taiwan
University Hospital, 7 Chung Shan South Rd, Taipei, Taiwan.
E-mail address: jychien@ntu.edu.tw (J.-Y. Chien).
common pathogen (Chien et al., 2014; Furuuchi et al., 2019;
Zweijpfenning et al., 2018). Recently, using a novel genome-based
taxonomic classification method, a new nomenclature of myco-
bacterial species was proposed (Tortoli et al., 2019) and the major
causative species in MAC were renamed as Mycobacterium
intracellulare subspecies intracellulare (MIsI), Mycobacterium intra-
cellulare subspecies chimaera (MIsC) and Mycobacterium avium
subspecies hominissuis (MAsH) (Daley, 2017; Haworth et al., 2017).
However, data regarding the diverse virulence and drug suscepti-
bility of distinct MAC species are still lacking (Huang et al., 1999).
Therefore, this study was performed to investigate the character-
istics of pulmonary disease caused by different MAC species, risk
factors for disease progression, and drug susceptibility of distinct
MAC species among untreated MAC-PD patients.

https://doi.org/10.1016/j.ijid.2020.06.019
1201-9712/© 2020 The Author(s). Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
34 C.-L. Chang et al. / International Journal of Infectious Diseases 98 (2020) 33–40

Methods Smear microscopy and culture

Setting, participants and data collection
This retrospective cohort study was conducted at three
hospitals in Taiwan. Between January 2013 and December
2015, all consecutive patients diagnosed with MAC-PD,
according to the 2007 American Thoracic Society/Infectious
Diseases Society of America guideline (Griffith et al., 2007),
were enrolled. They were followed-up for 2 years or until they
started anti-MAC treatment. The diagnostic criteria included
clinical and microbiologic sections. The clinical section
required pulmonary symptoms with evident lesions on the
chest radiograph or high-resolution computed tomography
(CT) scan and exclusion of other diagnoses. The microbiological
criteria included positive culture results from at least two
sputum cultures of the same NTM species or at least one
positive bronchial wash/lavage culture or compatible myco-
bacterium histopathological findings with at least one positive
NTM culture, from either sputum or bronchoscopy specimen.
The timing of starting anti-MAC treatment was judged by
clinical physicians according to the severity of MAC-PD, the
risk of progressive MAC-PD and the presence of comorbidity
(Haworth et al., 2017). This study was approved by the
Institutional Review Board of the National Taiwan University
Hospital (201608039RINA).
Smear microscopy and mycobacterium culture were performed
as previously described (Chien et al., 2015). Each respiratory
specimen was first processed by adding an equal volume of NaOH-
citrate-N-acetyl-L-cysteine at room temperature for 15 min. After
centrifugation, the precipitate was resuspended in 1 mL of
phosphate-buffered saline. The smears were performed using
the Ziehl-Nielsen stain. All isolates were cultured on BACTEC MGIT
tube (Becton-Dickinson, Sparks, USA) and the Lowenstein-Jensen
Medium slant (Becton-Dickinson) media before further testing.
Identification of MAC species and subspecies
MAC isolates were first identified using conventional biochem-
ical methods. The rpoB gene and internal transcribed spacer (ITS)
region of the 16S-23S ribosomal RNA (rRNA) gene were further
sequenced to identify the subspecies of the MAC isolates. The
primers used for rpoB gene sequencing were 5ʹ-GGCAAGGT-
CACCCCGAAGGG-3ʹ and 5ʹ-AGCGGCTGCTGGGTGATCATC-3ʹ, and
the primers used for sequencing the 16S-23S rRNA ITS region were
5ʹ-TTGTACACACCGCCCGTCA-3ʹ and 5ʹ-TCTCGATGCCAAGGCATC-
CACC-3ʹ. Sequence comparisons were performed using Basic Local
Alignment Search Tool (BLAST) analysis utilizing sequences from
the National Center for Biotechnology Information database. The
identification of species was further confirmed by amplification

Table 1
Clinical characteristics of 144 patients with Mycobacterium avium–intracellulare complex pulmonary disease.

All (n = 144) MAsH (n = 37) MIsC (n = 37) MIsI (n = 57) Others (n = 13) P value

Male 64 (44.4%) 20 (54.1%) 15 (40.5%) 24 (42.1%) 5 (38.5%) 0.588

Age, years 70.8 (24–100) 61.1 (24–88) 73.1 (40–100) 71.8 (27–99) 67.3 (26–91) 0.010
BMI, kg/m2 20.1 (9.5–34.7) 20.1 (13.2–34.7) 19.8 (14.2–26.1) 20.3 (12.2–30.7) 20.4 (9.5–23.4) 0.665
Smoker 21/94 (22.3%) 6/24 (25.0%) 7/27 (25.9%) 7/36 (19.4%) 1/7 (14.3%) 0.865
Comorbidities
Asthma 12 (8.3%) 4 (10.8%) 1 (2.7%) 7 (12.3%) 0 0.243
COPD 25 (17.4%) 3 (8.1%) 11 (29.7%) 10 (17.5%) 1 (7.7%) 0.072
Prior pulmonary tuberculosis 24 (16.7%) 3 (8.1%) 10 (27.0%) 8 (14.0%) 3 (23.1%) 0.140
Lung cancer 14 (9.7%) 5 (13.5%) 1 (2.7%) 6 (10.5%) 2 (15.4%) 0.362
Hypertension 32 (22.2%) 4 (10.8%) 11 (29.7%) 14 (24.6%) 3 (23.1%) 0.243
CAD 5 (3.5%) 3 (8.1%) 1 (2.7%) 1 (1.8%) 0 0.333
Transplantation 2 (1.4%) 0 0 1 (1.8%) 1 (7.7%) 0.182
Immunosuppressants 20 (13.9%) 4 (10.8%) 3 (8.1%) 10 (17.5%) 3 (23.1%) 0.410
HIV 11 (7.6%) 10 (27.0%) 0 0 1 (7.7%) <0.001
Type 2 DM 15 (10.4%) 2 (5.4%) 7 (18.9%) 6 (10.5%) 0 0.146
GERD 11 (7.6%) 2 (5.4%) 6 (16.2%) 3 (5.3%) 0 0.130
RA 1 (0.01%) 0 1 (2.7%) 0 0 0.405

Sputum acid-fast smear n = 131 n = 37 n = 30 n = 51 n = 13

Positive 45 (34.4%) 15 (40.5%) 8 (26.7%) 18 (35.3%) 4 (30.8%) 0.680
Negative-conversion
Duration (days) 208.0 (26–357) 335.0 (333–357) 82.0 44.5 (26–63) 0.117
6-month 3 (6.7%) 0 (0.0%) 1 (12.5%) 2 (11.1%) 0 (0.0%) 0.500
1-year 6 (13.3%) 3 (20.0%) 1 (12.5%) 2 (11.1%) 0 0.735
2-year 6 (13.3%) 3 (20.0%) 1 (12.5%) 2 (11.1%) 0 0.735
Sputum culture
Negative-conversion
Duration (days) 208.5 (5–637) 264.0 (5–673) 218.5 (11–375) 84.5 (26–638) – 0.781
6-month 11 (7.6%) 2 (5.4%) 4 (10.8%) 5 (8.8%) 0 0.579
1-year 16 (11.1%) 4 (10.8%) 7 (18.9%) 5 (8.8%) 0 0.238
2-year 22 (15.3%) 6 (16.2%) 8 (21.6%) 8 (14.0%) 0 0.310
Radiographic findings
Fibro-cavity 18 (12.5%) 5 (13.5%) 1 (2.7%) 10 (17.5%) 2 (15.4%) 0.192
Nodular bronchiectasis 108 (75.0%) 30 (81.1%) 28 (75.7%) 40 (70.2%) 10 (76.9%)
Mixed type 18 (12.5%) 2 (5.4%) 8 (21.6%) 7 (12.3%) 1 (7.7%)
2-year radiographic progression 15 (10.4%) 7 (18.9%) 2 (5.4%) 5 (8.8%) 1 (7.7%) 0.248
2-year mortality 22 (15.3%) 8 (21.6%) 6 (16.2%) 6 (10.5%) 2 (15.4%) 0.538
2-year unfavorable outcomes 33 (22.9%) 12 (32.4%) 7 (18.9%) 11 (19.3%) 3 (23.1%) 0.631

Data were presented as No. (%) or median (range).

Abbreviations: MIsC, Mycobacterium intracellulare subspecies chimaera; MAsH, Mycobacterium avium subspecies hominissuis; MIsI, Mycobacterium intracellulare subspecies
intracellulare; BMI, body mass index; COPD, chronic obstructive pulmonary disease; CAD, coronary artery disease; HIV, human immunodeficiency virus; DM, diabetes
mellitus; GERD, gastroesophageal reflux disease; RA, rheumatoid arthritis.
 C.-L. Chang et al. / International Journal of Infectious Diseases 98 (2020) 33–40 35

et al., 1971). Briefly, each lung was divided into three areas and
each area was rated on a 4-point scale of 0–3 for the extent of
infiltrates, with a maximum radiographic score of 18. Higher
scores implied a greater extent of disease involvement. The
negative smear/culture conversion was defined as three conse-
cutive negative smears/cultures during at least a 3-month period
(Wallace et al., 1996). Radiographic progression was defined as an
increase in radiographic score by at least 1 point. An unfavorable
outcome was defined as presentation of either radiographic
progression or mortality.

Antimicrobial susceptibility testing

Figure 1. The prevalence of pulmonary disease caused by Mycobacterium avium
subspecies hominissuis (MAsH), Mycobacterium intracellulare subspecies chimaera
(MIsC), M. intracellulare subspecies intracellulare (MIsI), and other species of M.
avium complex 2013–2015.
and sequencing of the heat-shock protein 65 gene (hsp65) (Boyle
et al., 2015). In addition, the recently revised nomenclature of
mycobacterial species was used, which was based on genome-
based analysis (Tortoli et al., 2019).
Radiographic severity and outcome evaluation
Chest radiographs and high-resolution CT scans were
reviewed by one trained chest specialists and one thoracic
radiologist blinded to the clinical data. When their interpretations
differed, the image was further evaluated by a third blinded
pulmonologist. The disease extent assessed on the initial and
follow-up image was interpreted as a radiographic score (Snider
The first NTM isolate from a patient with NTM-PD was used for
antimicrobial susceptibility testing. The minimum inhibitory
concentrations (MICs) of the isolates were determined using the
SLOMYCO Sensititre1 panel (TREK Diagnostic Systems, Cleveland,
OH, USA) (Babady et al., 2010) and the susceptible (S), intermediate
(I) or resistant (R) isolates were determined according to the 2018
Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI,
2018). Susceptibility testing of mycobacteria, Nocardia spp., and
other aerobic actinomycetes was performed according to the CLSI
(3rd ed, CLSI Standard Document, M62). The breakpoints used
were 8 mg/mL (S), 16 mg/mL (I) and 32 mg/mL (R) for
clarithromycin; 16 mg/mL (S), 32 mg/mL (I) and 64 mg/mL (R)
for intravenous amikacin; 64 mg/mL (S) and 128 mg/mL (R) for
inhaled liposomal-amikacin; 1 mg/mL (S), 2 mg/mL (I) and 4 mg/
mL (R) for moxifloxacin; 8 mg/mL (S), 16 mg/mL (I) and 32 mg/
mL (R) for linezolid. The MIC50 and the MIC90 were defined as the
minimum concentration required to inhibit 50% and 90% of the
isolates, respectively.

Table 2
Factors predicting 2-year sputum culture-negative conversion among patients with Mycobacterium avium–intracellulare complex pulmonary disease.

Univariate analysis Multivariate analysis

HR 95% confidence P value aHR 95% confidence P value

interval interval
Species
MAsH 1.00
MIsC 1.41 0.49–4.05 0.529
MIsI 0.87 0.30–2.50 0.791
Others – – –
Demographic variables
Male 1.07 0.46–2.48 0.876
Age 0.99 0.96–1.02 0.390
BMI 0.92 0.81–1.04 0.171 0.93 0.81–1.07 0.329
Smoker 0.82 0.24–2.89 0.761
Comorbidities
Asthma 3.76 1.38–10.20 0.009 2.00 0.52–7.74 0.316
COPD 0.46 0.11–1.98 0.298
Prior pulmonary tuberculosis 0.22 0.03–1.67 0.144 0.00 0.00–> 100.00 0.972
Lung cancer 0.98 0.23–4.19 0.978
Hypertension 0.55 0.16–1.86 0.337
CAD 0.05 0–>100.00 0.540
Transplantation 4.69 0.63–35.00 0.132 1.13 0.13–9.94 0.909
Immunosuppressants 2.58 1.01–6.58 0.048 1.92 0.57–6.51 0.294
HIV 0.58 0.08–4.34 0.599
Type 2 DM 0.04 0.00–13.57 0.281
GERD 2.08 0.62–7.03 0.239
RA 0.05 0.00–>100.00 0.785
Sputum acid-fast smear
Positive 0.20 0.05–0.85 0.029 0.12 0.02–0.94 0.043
6-month negative-conversion 1.00 0.01–>100 1.000
Radiographic findings
Fibro-cavity 1.00
Nodular bronchiectasis 1.66 0.39–7.13 0.495
Mixed type 0.49 0.04–5.38 0.558
Initial radiographic score 0.88 0.79–0.98 0.020 0.98 0.85–1.12 0.732

Abbreviations: HR, hazard ratio; MIsC, Mycobacterium intracellulare subspecies chimaera; MAsH, Mycobacterium avium subspecies hominissuis; MIsI, Mycobacterium
intracellulare subspecies intracellulare; BMI, body mass index; COPD, chronic obstructive pulmonary disease; CAD, coronary artery disease; HIV, human immunodeficiency
virus; DM, diabetes mellitus; GERD, gastroesophageal reflux disease; RA, rheumatoid arthritis.
36 C.-L. Chang et al. / International Journal of Infectious Diseases 98 (2020) 33–40

Statistical analysis
Statistical analyses were performed using SPSS 25.0 (IBM Corp.,
Armonk, NY, USA). Continuous variables were reported as the
median (range). The between-group differences with continuous
variables were compared using the Kruskal-Wallis H test.
Categorical variables were expressed as numbers (percentage).
The difference between categorical variables was compared using
the Chi-square test. Time-to-event was assessed using the Cox
proportional hazards analysis. Variables with a p < 0.20 in the
univariate analysis were introduced into the multivariate analysis.
Outcomes between MAsH and non-MAsH isolates were estimated
using the Kaplan-Meier analysis and compared with the log-rank
test. Statistical tests were two-sided and significance was set at
p < 0.05.
Results
A total of 144 patients with MAC-PD were enrolled; 44.4%
(64/144) were males. The median age was 70.8 years and the
median body mass index (BMI) was 20.1 kg/m 2 . Among the 131
patients with available initial sputum acid-fast smear results,
45 (34.4%) were positive and the overall smear-negative
conversion rates were 6.7% (3/45), 13.3% (6/45) and 13.3%
(6/45) at 6 months, 1 year and 2 years from diagnosis,
respectively. Furthermore, 22 (15.3%) patients died and 33
(22.9%) received anti-MAC treatment due to the progression of
MAC-PD during the study period.
Table 1 reveals that the most common causative pathogen was
MIsI (n = 57, 39.6%), followed by MAsH (n = 37, 25.7%), MIsC (n = 37,
25.7%), and other species (n = 13, 9.0%), including Mycobacterium
colombiense (n = 5, 3.5%), Mycobacterium marseillense (n = 5, 3.5%),
and Mycobacterium timonense (n = 3, 2.0%). In 2013 and 2014, MIsI
showed the highest prevalence; however, MAsH was the most
common pathogen in 2015 (Figure 1). A downtrend in the
prevalence of MIsC was observed from 2013 through 2015, but
the prevalence of other species fluctuated. Patients with MAsH-PD
were the youngest (median age: 61.1, 73.1, 71.8, and 67.3 years for
MAsH-PD, MIsC-PD, MIsI-PD, and others, respectively; p = 0.01)
and most commonly presented with an HIV infection (27.0%, 0.0%,
0.0%, and 7.7% for MAsH-PD, MIsC-PD, MIsI-PD, and others,
respectively; p < 0.001). Among 11 patients with HIV infection,
the median CD4 count and HIV viral load were 27.5 (0–487) and
24,500 (0–374,000), respectively, and nine (81.8%) had CD4 count
<200 cells/mm3. The prevalence of other demographic variables,
comorbidities, sputum smear and culture results, radiographic
findings, treatment, and mortality were consistent among patients
infected by different species.
Twenty-two (15.3%) patients demonstrated spontaneous nega-
tive culture conversion during the 2-year follow-up (Table 1).
Based on the univariate Cox proportional hazard analysis, asthma
(HR = 3.76, 95% CI = 1.38–10.20, p = 0.009) and immunosuppressant
therapy (HR = 2.58, 95% CI = 1.01–6.58, p = 0.048) were associated
with higher 2-year spontaneous negative culture conversion rates;
however, positive initial acid-fast smear (HR = 0.20, 95% CI = 0.05–
0.85, p = 0.029) was a risk factor of persistently positive sputum
cultures. Using multivariate Cox analysis, a positive initial acid-fast
smear was the only independent predictor (HR = 0.12, 95%
CI = 0.02–0.94, p = 0.043) for persistently positive sputum cultures
during the 2-year follow-up period (Table 2).

Table 3
Factors predicting the 2-year radiographic progression among patients with Mycobacterium avium–intracellulare complex pulmonary disease.

Univariate analysis Multivariate analysis

HR 95% confidence P value aHR 95% confidence P value

interval interval
Species
MAsH 1.00 1.00
MIsC 0.21 0.04–0.99 0.049 0.10 0.01–0.98 0.048
MIsI 0.37 0.12–1.18 0.093 0.23 0.05–1.17 0.760
Others 0.36 0.04–2.93 0.339 0.22 0.02–3.28 0.274
Demographic variables
Male 6.16 1.74–21.85 0.005 4.19 0.93–18.92 0.062
Age 0.98 0.94–1.01 0.193 1.01 0.97–1.05 0.736
BMI 0.88 0.76–1.01 0.059 0.82 0.67–1.01 0.066
Smoker 1.37 0.36–5.16 0.643
Comorbidities
Asthma 2.50 0.71–8.87 0.156 1.60 0.19–13.46 0.667
COPD 0.85 0.24–3.00 0.795
Prior pulmonary tuberculosis 1.59 0.45–5.66 0.474
Lung cancer 0.04 0.00–>100.00 0.440
Hypertension 0.51 0.11–2.25 0.371
CAD 4.25 0.56–32.60 0.164 6.19 0.17–>100.00 0.321
Transplantation 4.93 0.65–37.63 0.124 13.72 0.83–>100.00 0.068
Immunosuppressants 1.38 0.39–4.89 0.618
HIV 2.58 0.34–19.77 0.362
Type 2 DM 0.04 0.00–43.65 0.370
GERD 2.82 0.79–10.06 0.111 0.88 0.10–7.51 0.903
RA 0.05 0.00–>100.00 0.771
Laboratory evaluation
Initial positive acid-fast smear 3.05 1.07–8.72 0.038 3.38 0.83–13.77 0.089
6-month smear negative- conversion 0.04 0.00–>100.00 0.608
6-month culture negative-conversion 0.75 0.09–5.73 0.784
Radiographic findings
Fibro-cavity 1.00
Nodular bronchiectasis 0.45 0.12–1.63 0.221
Mixed type 0.56 0.09–3.36 0.527
Initial radiographic score 0.96 0.85–1.08 0.478

Abbreviations: HR, hazard ratio; MIsC, Mycobacterium intracellulare subspecies chimaera; MAsH, Mycobacterium avium subspecies hominissuis; MIsI, Mycobacterium
intracellulare subspecies intracellulare; BMI, body mass index; COPD, chronic obstructive pulmonary disease; CAD, coronary artery disease; HIV, human immunodeficiency
virus; DM, diabetes mellitus; GERD, gastroesophageal reflux disease; RA, rheumatoid arthritis.
 C.-L. Chang et al. / International Journal of Infectious Diseases 98 (2020) 33–40
The percentage of radiographic progression during the 2-year
follow-up was 10.4% (15/144). Being male (HR = 6.16, 95% CI = 1.74–
21.85, p = 0.005) and having a positive initial acid-fast smear
(HR = 3.05, 95% CI = 1.07–8.72, p = 0.038) were risk factors associ-
ated with the 2-year radiographic progression. Additionally, MIsC-
PD (HR = 0.21, 95% CI = 0.04–0.99, p = 0.049) demonstrated a lower
2-year radiographic progression than MAsH-PD. The multivariate
Cox proportional hazard analysis indicated that MIsC (HR = 0.10;
95% CI = 0.01–0.98, p = 0.048) presented less radiographic progres-
sion than MAsH (Table 3). The Kaplan-Meier analysis revealed that
patients with MAsH-PD presented higher radiographic progression
rates than those with non-MAsH-PD (log-rank test p = 0.017)
(Figure 2A).
Overall, 33 (22.9%) patients demonstrated unfavorable out-
comes during the 2-year follow-up period (Table 1). In the
univariate Cox proportional hazard analysis, coronary artery
disease (CAD) (HR = 6.30, 95% CI = 2.19–18.12, p = 0.001) and being
male (HR = 3.35, 95% CI = 1.59–7.05, p = 0.001) were risk factors for
the 2-year unfavorable outcomes. The multivariate Cox propor-
tional hazard analysis demonstrated that comorbidity with CAD
(HR = 18.42, 95% CI = 4.00–84.70, p < 0.001), being male (HR = 2.51,
95% CI = 1.03–6.12, p = 0.043), older age (HR = 1.04, 95% CI = 1.01–
1.08, p = 0.032), and lower BMI (HR = 0.85, 95% CI = 0.76–0.94,
p = 0.003) were independent risk factors for unfavorable outcomes.
More importantly, MIsC-PD (HR = 0.22; 95% CI = 0.06–0.77,
Figure 2. Probability of radiographic progression (Panel A) and unfavorable outcomes (Panel B) among patients with Mycobacterium avium subspecies hominissuis (MAsH)
pulmonary disease and non-MAsH pulmonary disease during the 2-year follow-up.
37
p = 0.018) showed less unfavorable outcomes than MAsH-PD
(Table 4). The Kaplan-Meier analysis further demonstrated that
patients with MAsH-PD had increased risk for unfavorable
outcomes than those with non-MAsH-PD (log-rank test
p = 0.046) (Figure 2B).
Table 5 shows that the susceptibility rates of all isolates to
clarithromycin and inhaled amikacin were 98.6%. Based on
breakpoints proposed by the 2018 CLSI (CLSI, 2018), 37 (26.2%)
isolates were resistant to intravenous amikacin, and 121 (85.5%)
and 129 (91.5%) were resistant to moxifloxacin and linezolid,
respectively. Compared with other species MAsH isolates demon-
strated the lowest resistance rate (66.7%, p = 0.001) and the highest
intermediate susceptibility rate (33.3%, p = 0.001) to moxifloxacin.
MIsI isolates reported the highest (38.2%, p = 0.024) resistance and
MIsC had the highest (59.5%, p = 0.024) intermediate susceptibility
to intravenous amikacin compared with other species. The
antimicrobial susceptibilities of different species were similar
for clarithromycin, amikacin (inhalation) and linezolid (Table 5).
Discussion
In this MAC-PD cohort, the most common pathogen was MIsI
(n = 57, 39.6%), followed by MAsH (n = 37, 25.7%) and MIsC (n = 37,
25.7%). This finding indicated that the most common causative
organism of MAC-PD was either Mycobacterium avium or
38 C.-L. Chang et al. / International Journal of Infectious Diseases 98 (2020) 33–40

Table 4
Factors predicting 2-year unfavorable outcome among patients with Mycobacterium avium–intracellulare complex pulmonary disease.

Univariate analysis Multivariate analysis

HR 95% confidence P value aHR 95% confidence P value

interval interval
Species
MAsH 1.00 1.00
MIsC 0.45 0.18–1.14 0.092 0.22 0.06–0.77 0.018
MIsI 0.49 0.22–1.12 0.090 0.42 0.16–1.12 0.083
Others 0.63 0.18–2.23 0.473 1.14 0.29–4.47 0.854
Demographic variables
Male 3.35 1.59–7.05 0.001 2.51 1.03–6.12 0.043
Age 1.03 0.99–1.06 0.090 1.04 1.01–1.08 0.032
BMI 0.93 0.84–1.02 0.123 0.85 0.76–0.94 0.003
Smoker 1.48 0.65–3.39 0.349
Comorbidities
Asthma 1.41 0.50–4.02 0.517
COPD 0.96 0.42–2.21 0.919
Prior TB 1.33 0.55–3.22 0.530
Lung cancer 1.37 0.48–3.90 0.558
Hypertension 1.25 0.58–2.69 0.567
CAD 6.30 2.19–18.12 0.001 18.42 4.00–84.70 < 0.001
Transplantation 2.09 0.29–15.34 0.467
Immunosuppressants 1.00 0.38–2.58 0.993
HIV 2.93 0.89–9.70 0.079 3.21 0.56–18.27 0.189
Type 2 DM 0.79 0.24–2.59 0.698
GERD 1.14 0.35–3.76 0.824
RA 0.05 0.00–100.00 0.683
Laboratory evaluation
Initial positive acid-fast smear 2.02 0.99–4.13 0.055 1.47 0.64–3.37 0.361
6-month smear negative- conversion 0.97 0.13–7.45 0.973
6-month culture negative-conversion 2.37 0.98–5.74 0.057 1.87 0.62–5.59 0.265
Radiographic findings
Fibro-cavity 1.00
Nodular bronchiectasis 0.91 0.32–2.61 0.859
Mixed type 0.64 0.14–2.87 0.563
Initial radiographic score 1.04 0.96–1.13 0.341

Abbreviations: HR, hazard ratio; MIsC, Mycobacterium intracellulare subspecies chimaera; MAsH, Mycobacterium avium subspecies hominissuis; MIsI, Mycobacterium
intracellulare subspecies intracellulare; BMI, body mass index; COPD, chronic obstructive pulmonary disease; TB, Mycobacterium tuberculosis; CAD, coronary artery disease;
HIV, human immunodeficiency virus; DM, diabetes mellitus; GERD, gastroesophageal reflux disease; RA, rheumatoid arthritis.

Mycobacterium intracellulare but not Mycobacterium chimaera.
Consistent with results observed in a Brazilian cohort (Carneiro
et al., 2018), the current study observed that MAsH was the most
prevalent pathogen among patients with an HIV infection (27%, 0%,
0%, and 7.7% for MAsH, MIsI, MIsC, and others, respectively;
p < 0.001). It found that 15.3% (22/144 patients) of MAC-PD
patients demonstrated a 2-year spontaneous culture-negative
conversion. Furthermore, a positive initial sputum smear was the
only predictor for persistently positive sputum culture. In a Korean
cohort, Kwon et al. observed that 52.2% of patients with non-
cavitary nodular bronchiectatic untreated MAC-PD presented
spontaneous culture-negative conversion (Kwon et al., 2019). In
addition, Hwang et al. documented that 51.6% of patients with
untreated non-HIV MAC-PD presented spontaneous culture-
negative conversion (Hwang et al., 2017), and younger age, higher
BMI, positive acid-fast smear, and transient anti-tuberculosis
treatment were predictors for negative conversion (Hwang et al.,
2017). In a previous Taiwanese study, investigators reported that
among the 126 non-HIV infection patients with untreated MAC-
PD, 40.2% demonstrated spontaneous culture-negative conversion,
and low BMI, radiographic nodular bronchiectasis and higher acid-
fast stain grades were predictors for microbiological persistence
(Pan et al., 2017). In the current study, the 2-year culture-negative
conversion rates were 14.3% and 15.4% among lung cancer and
non-lung cancer groups, but 9.1% and 15.8% in HIV and non-HIV
groups, respectively. This implied that HIV patients with MAC-PD
tended to have less spontaneous culture-negative conversion. In
addition, it found that asthma and immunosuppressant therapy
usage were associated with spontaneous culture-negative
conversion in univariate analysis, but they were not independent
factors in multivariate analysis. Further randomized controlled
studies are warranted to clarify the impact of asthma and
immunosuppressant therapy on NTM disease.
In the current cohort, 10.4% (15/144 patients) of MAC-PD
patients showed radiographic progression during the 2-year
follow-up. In a recent study by Gochi et al. in Japan, 41.2% nodular
bronchiectatic MAC-PD patients demonstrated radiographic dete-
rioration during the median follow-up period of 5 years (Gochi
et al., 2015) and the presence of idiopathic pulmonary fibrosis,
hemoglobin <11.3 mg/dL, C-reactive protein levels >1.0 mg/dL, and
cavity presence were regarded as risk factors (Gochi et al., 2015).
Moreover, in the current study, 22 (15.3%) patients died and 33
(22.9%) patients had unfavorable outcomes during the 2-year
follow-up. Hayashi et al. demonstrated that the 5-year all-cause
and MAC-specific mortality rates were 23.9% and 5.4%, respectively
(Hayashi et al., 2012). Furthermore, being male, older age, presence
of comorbidities, non-nodular bronchiectatic radiographic pattern,
BMI < 18.5 kg/m2, anemia, hypoalbuminemia, and an erythrocyte
sedimentation rate 50 mm/h were reported as predictors for all-
cause mortality (Hayashi et al., 2012). In the current study, the all-
cause mortality rate was similar to rates reported in a meta-
analysis, where the 5-year all-cause mortality of MAC-PD was 27%
(range 10–48%). Furthermore, reports from Asia have indicated
lower mortality rates (Asia 19%, Europe 35% and North America
33%) (Diel et al., 2018).
The current study observed that patients with MAsH-PD
demonstrated a worse radiographic presentation and increased
unfavorable outcomes than those with non-MAsH-PD. Boyle et al.
 C.-L. Chang et al. / International Journal of Infectious Diseases 98 (2020) 33–40 39

Table 5
Antimicrobial susceptibilities and minimum inhibitory concentrations of Mycobacterium avium–intracellulare complex isolates to 13 antimicrobial agents.

Antimicrobial susceptibilities All (n = 141) MAsH (n = 36) MIsC (n = 37) MIsI (n = 55) Others (n = 13) P value

Clarithromycin MIC50 4 4 4 4 2
MIC90 8 8 4 8 4
S 139 (98.6%) 34 (94.4%) 37 (100.0%) 55 (100.0%) 13 (100.0%) 0.433
I 1 (0.7%) 1 (2.8%) 0 (0.0%) 0 (0.0%) 0 (0.0%)
R 1 (0.7%) 1 (2.8%) 0 (0.0%) 0 (0.0%) 0 (0.0%)
Moxifloxacin MIC50 8 8 8 8 4
MIC90 >8 >8 8 8 >8
S 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0.001
I 20 (14.2%) 12 (33.3%) 1 (2.7%) 6 (10.9%) 1 (7.7%)
R 121 (85.8%) 24 (66.7%) 36 (97.3%) 49 (89.1%) 12 (92.3%)
Amikacin MIC50 32 32 32 32 16
MIC90 64 64 64 64 32
Intravenous S 46 (32.6%) 10 (27.8%) 10 (27.0%) 19 (34.5%) 7 (53.8%) 0.024
I 58 (41.1%) 17 (47.2%) 22 (59.5%) 15 (27.3%) 4 (30.8%)
R 37 (26.2%) 9 (25.0%) 5 (13.5%) 21 (38.2%) 2 (15.4%)
Inhalation S 139 (98.6%) 36 (100.0%) 37 (100.0%) 53 (96.4%) 13 (100.0%) 0.366
R 2 (1.4%) 0 (0.0%) 0 (0.0%) 2 (3.6%) 0 (0.0%)
Linezolid MIC50 64 64 64 64 32
MIC90 >64 >64 >64 64 64
S 6 (4.3%) 1 (2.8%) 2 (5.4%) 3 (5.5%) 0 (0.0%) 0.937
I 6 (4.3%) 2 (5.6%) 1 (2.7%) 2 (3.6%) 1 (7.7%)
R 129 (91.5%) 33 (91.7%) 25 (91.9%) 50 (90.9%) 12 (92.3%)
Rifampin MIC50 >8 >8 >8 8 8
MIC90 >8 >8 >8 >8 >8
Rifabutin MIC50 4 4 4 4 2
MIC90 4 4 4 4 4
Ethambutol MIC50 8 8 8 4 8
MIC90 16 16 16 16 16
Ethionamide MIC50 5 2.5 5 10 5
MIC90 >20 20 >20 >20 >20
Isoniazid MIC50 8 4 4 >8 >8
MIC90 >8 >8 >8 >8 >8
Ciprofloxacin MIC50 >16 16 >16 >16 >16
MIC90 >16 >16 >16 >16 >16
Streptomycin MIC50 64 64 64 64 32
MIC90 >64 >64 >64 >64 64
Doxycycline MIC50 >16 >16 >16 >16 >16
MIC90 >16 >16 >16 >16 >16
TMP/SMX MIC50 >8/152 8/152 >8/152 >8/152 >8/152
MIC90 >8/152 >8/152 >8/152 >8/152 >8/152

Data were presented as No. (%).

Abbreviations: MIsC, Mycobacterium intracellulare subspecies chimaera; MAsH, Mycobacterium avium subspecies hominissuis; MIsI, Mycobacterium intracellulare subspecies
intracellulare; MIC, minimum inhibitory concentration; S, susceptible; I, intermediate; R, resistant.

reported that patients with M. avium and M. chimaera PD were
more likely to relapse than those with M. intracellulare PD (Boyle
et al., 2015). However, Hwang et al. indicated that non-HIV patients
infected with M. intracellulare present a more progressive disease
than those with the M. avium infection (Hwang et al., 2017).
Another study by Koh et al. reported that patients in Korea with M.
intracellulare PD have a worse presentation and poor disease
prognosis than those with M. avium PD (Koh et al., 2012). Notably,
the virulence of NTM is mediated by the cell membrane and/or cell
wall lipids, which results in inactivation of the cathelicidin
antibacterial peptide (Honda et al., 2015). The virulence of different
MAC species and different isolates of the same species tend to vary
(Honda et al., 2019). Among the various MAC species, the diversity
of the lipid structure could contribute to the distinct virulence of
MAC in different geographical settings.
The suggested first-line treatment for MAC-PD is a combination
of macrolide, rifampin and ethambutol (Haworth et al., 2017). In
the current study, the susceptibility rates of all MAC species to
clarithromycin and inhaled amikacin were 98.6% for both;
however, the drug resistance rates were 32.6%, 85.5% and 91.5%
for intravenous amikacin, moxifloxacin and linezolid, respectively.
Consistent with the current findings, previous studies have
indicated that clarithromycin demonstrates the best antimicrobial
activity against common MAC species (Cho et al., 2018; Mok et al.,
2019; Wei et al., 2015; Zhao et al., 2014). Reportedly, some studies
have indicated low moxifloxacin and linezolid resistance rates in
M. avium (Wei et al., 2015). However, most studies have reported
higher moxifloxacin and linezolid resistance rates, which are
similar to the current findings among the MAC isolates (Mok et al.,
2019; Zhao et al., 2014). Compared with other species, it was
observed that MAsH isolates demonstrated the lowest rate of
resistance to moxifloxacin and, although 26.2% of MAC isolates
were resistant to intravenous amikacin, most MAC isolates (98.6%)
were still susceptible to inhaled amikacin.
There were several limitations to the current study. First, it was
a retrospective cohort study. All data were retrospectively
collected and some data were missing. Second, the follow-up
period was 2 years, which may have led to an underestimation of
the spontaneous sputum negative-conversion rate and radio-
graphic progression rate. Third, because few patients were treated
and there was a high variation in anti-MAC treatment regimens, it
could not evaluate the treatment response and focused on the
natural course of untreated MAC-PD.
In conclusion, among patients with MAC-PD, it was observed
that the 2-year spontaneous negative culture conversion rate was
15.3%, the 2-year radiographic progression rate was 10.4%, and
22.9% of patients demonstrated unfavorable outcomes. Compared
with those with MAsH-PD, patients with non-MAsH-PD developed
40 C.-L. Chang et al. / International Journal of Infectious Diseases 98 (2020) 33–40
less radiographic progression and fewer unfavorable outcomes.
Although all MAC species demonstrated high susceptibility rates to
clarithromycin and inhaled amikacin, MAsH demonstrated the
lowest resistance rates to moxifloxacin and MIsI showed the
highest rate of resistance to intravenous amikacin in this study.
Monitoring the local prevalence of specific species/subspecies
causing MAC-PD is crucial to promote efficacious treatment.
Author contributions
J.Y.C. assumed responsibility for the content of the manuscript,
including the data and analysis. C.L.C. and L.C.C. had full access to
all study data and assumed responsibility for the integrity of the
data and the accuracy of the data analysis. C.L.C., L.C.C., C.J.Y, J.Y.C.,
and P.R.H. substantially contributed to the study design, data
analysis and interpretation, and the writing of the manuscript.
Funding
This study was supported by funding from the Ministry of
Science and Technology, Taiwan (MOST 107-2314-B-002 -211).
Conflict of interest
None.
Ethics approval
The Institutional Review Board of the National Taiwan
University Hospital (201608039RINA) approved this study.
References
Babady NE, Hall L, Abbenyi AT, Eisberner JJ, Brown-Elliott BA, Pratt CJ, et al.
Evaluation of Mycobacterium avium complex clarithromycin susceptibility
testing using SLOMYCO Sensititre panels and JustOne strips. J Clin Microbiol
2010;48(5):1749–52.
Boyle DP, Zembower TR, Reddy S, Qi C. Comparison of clinical features, virulence,
and relapse among Mycobacterium avium complex species. Am J Respir Crit Care
Med 2015;191(11):1310–7.
Carneiro MDS, Nunes LS, David SMM, Dias CF, Barth AL, Unis G. Nontuberculous
mycobacterial lung disease in a high tuberculosis incidence setting in Brazil. J
Bras Pneumol 2018;44(2):106–11.
Chien JY, Chang TC, Chiu WY, Yu CJ, Hsueh PR. Performance assessment of the
BluePoint MycoID Plus Kit for identification of Mycobacterium tuberculosis,
including rifampin- and isoniazid-resistant isolates, and nontuberculous
Mycobacteria. PLoS One 2015;10(5)e0125016.
Chien JY, Lai CC, Sheng WH, Yu CJ, Hsueh PR. Pulmonary infection and colonization
with nontuberculous mycobacteria, Taiwan, 2000–2012. Emerg Infect Dis
2014;20(8):1382–5.
Cho EH, Huh HJ, Song DJ, Moon SM, Lee SH, Shin SY, et al. Differences in drug
susceptibility pattern between Mycobacterium avium and Mycobacterium
intracellulare isolated in respiratory specimens. J Infect Chemother 2018;24
(4):315–8.
CLSI. Performance Standards for Susceptibility Testing of Mycobacteria, Nocardia
spp., and Other Aerobic Actinomycetes. CLSI supplement M62. 1st ed. Wayne,
PA: Clinical and Laboratory Standards Institute; 2018.
Daley CL. Mycobacterium avium complex disease. Microbiol Spectr 2017;5(2).
Diel R, Lipman M, Hoefsloot W. High mortality in patients with Mycobacterium
avium complex lung disease: a systematic review. BMC Infect Dis 2018;18
(1):206.
Falkinham 3rd JO. Environmental sources of nontuberculous mycobacteria. Clin
Chest Med 2015;36(1):35–41.
Furuuchi K, Morimoto K, Yoshiyama T, Tanaka Y, Fujiwara K, Okumura M, et al.
Interrelational changes in the epidemiology and clinical features of non-
tuberculous mycobacterial pulmonary disease and tuberculosis in a referral
hospital in Japan. Respir Med 2019;152:74–80.
Gochi M, Takayanagi N, Kanauchi T, Ishiguro T, Yanagisawa T, Sugita Y. Retrospective
study of the predictors of mortality and radiographic deterioration in 782
patients with nodular/bronchiectatic Mycobacterium avium complex lung
disease. BMJ Open 2015;5(8)e008058.
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 non-
tuberculous mycobacterial diseases. Am J Respir Crit Care Med 2007;175
(4):367–416.
Haworth CS, Banks J, Capstick T, Fisher AJ, Gorsuch T, Laurenson IF, et al. British
Thoracic Society guidelines for the management of non-tuberculous mycobac-
terial pulmonary disease (NTM-PD). Thorax 2017;72(Suppl 2):ii1–ii64.
Hayashi M, Takayanagi N, Kanauchi T, Miyahara Y, Yanagisawa T, Sugita Y. Prognostic
factors of 634 HIV-negative patients with Mycobacterium avium complex lung
disease. Am J Respir Crit Care Med 2012;185(5):575–83.
Honda JR, Hess T, Carlson R, Kandasamy P, Nieto Ramirez LM, Norton GJ, et al.
Nontuberculous mycobacteria show differential infectivity and use phospho-
lipids to antagonize LL-37. Am J Respir Cell Mol Biol 2019;.
Honda JR, Hess T, Malcolm KC, Ovrutsky AR, Bai X, Irani VR, et al. Pathogenic
nontuberculous mycobacteria resist and inactivate cathelicidin: implication of a
novel role for polar mycobacterial lipids. PLoS One 2015;10(5)e0126994.
Huang CC, Wu MF, Chen HC, Huang WC. In vitro activity of aminoglycosides,
clofazimine, d-cycloserine and dapsone against 83 Mycobacterium avium
complex clinical isolates. J Microbiol Immunol Infect 2018;51(5):636–43.
Huang JH, Kao PN, Adi V, Ruoss SJ. Mycobacterium avium-intracellulare pulmonary
infection in HIV-negative patients without preexisting lung disease: diagnostic
and management limitations. Chest 1999;115(4):1033–40.
Hwang JA, Kim S, Jo KW, Shim TS. Natural history of Mycobacterium avium complex
lung disease in untreated patients with stable course. Eur Respir J 2017;49(3).
Koh WJ, Jeong BH, Jeon K, Lee NY, Lee KS, Woo SY, et al. Clinical significance of the
differentiation between Mycobacterium avium and Mycobacterium intracellulare
in M avium complex lung disease. Chest 2012;142(6):1482–8.
Kwon BS, Lee JH, Koh Y, Kim WS, Song JW, Oh YM, et al. The natural history of non–
cavitary nodular bronchiectatic Mycobacterium avium complex lung disease.
Respir Med 2019;150:45–50.
Mok S, Hannan MM, Nolke L, Stapleton P, O’Sullivan N, Murphy P, et al. Antimicrobial
susceptibility of clinical and environmental Mycobacterium chimaera isolates.
Antimicrob Agents Chemother 2019;63(9).
Pan SW, Shu CC, Feng JY, Wang JY, Chan YJ, Yu CJ, et al. Microbiological persistence in
patients with Mycobacterium avium complex lung disease: the predictors and
the impact on radiographic progression. Clin Infect Dis 2017;65(6):927–34.
Prevots DR, Marras TK. Epidemiology of human pulmonary infection with
nontuberculous mycobacteria: a review. Clin Chest Med 2015;36(1):13–34.
Snider GL, Doctor L, Demas TA, Shaw AR. Obstructive airway disease in patients with
treated pulmonary tuberculosis. Am Rev Respir Dis 1971;103(5):625–40.
Tortoli E, Meehan CJ, Grottola A, Fregni Serpini G, Fabio A, Trovato A, et al. Genome-
based taxonomic revision detects a number of synonymous taxa in the genus
Mycobacterium. Infect Genet Evol 2019;75:103983.
Wallace Jr. RJ, Brown BA, Griffith DE, Girard WM, Murphy DT. Clarithromycin
regimens for pulmonary Mycobacterium avium complex. The first 50 patients.
Am J Respir Crit Care Med 1996;153(6 Pt 1):1766–72.
Wei G, Huang M, Wang G, Huo F, Dong L, Li Y, et al. Antimicrobial susceptibility
testing and genotyping of Mycobacterium avium isolates of two tertiary
tuberculosis designated hospital, China. Infect Genet Evol 2015;36:141–6.
Yu HH, Hu TC, Lee NC, Chien YH, Yang YH, Hwu WL, et al. Mycobacterium abscessus
infection in a boy with X-linked anhidrotic ectodermal dysplasia, immunodefi-
ciency. J Microbiol Immunol Infect 2019;52(3):504–6.
Zhao X, Wang Y, Pang Y. Antimicrobial susceptibility and molecular characterization
of Mycobacterium intracellulare in China. Infect Genet Evol 2014;27:332–8.
Zweijpfenning SMH, Ingen JV, Hoefsloot W. Geographic distribution of non-
tuberculous mycobacteria isolated from clinical specimens: a systematic
review. Semin Respir Crit Care Med 2018;39(3):336–42.