Phenotypic, immunologic, and clinical characteristics of patients with nontuberculous mycobacterial lung disease in Korea

Beginning July 1, 2011 we prospectively recruited patients between 20 and 80 years of age at Seoul National University Hospital, Seoul, South Korea, who met the diagnostic criteria for NTM lung disease set forth by the American Thoracic Society [6]. Patients previously treated for NTM lung disease were excluded from this study. NTM patients with nodular BE types were included but those with upper lobe cavitary types were excluded. All patients provided written informed consent before enrollment. The study protocol was approved by the Institutional Review Board of Seoul National University Hospital. The clinical trial registration number is NCT 01616745 (www.ClinicalTrials.gov).

Beginning January 1, 2012 we began recruiting patients ≥ 20 years old diagnosed with BE in the absence of NTM infection (non-NTM) to serve as a control group. BE was diagnosed based on low dose computed tomography (CT) findings that included dilatation of an airway lumen, rendering it more than 1.5 times the width of a nearby vessel, lack of tapering of an airway toward the periphery, varicose constrictions along airways, and ballooned cysts at the end of a bronchus [7]. Two separate sputum mycobacterial cultures were performed to exclude patients with active NTM infections. The median interval between the two cultures was 24 months (interquartile range: 9–55 months).

Physical examinations were performed by board-certified physicians. Height and weight were measured by a team of two nurses.

Sputum was collected for bacterial and mycobacterial cultures. Samples of sputum were homogenized by incubation at 37°C for 15 min with an equal volume of 0.1% dithiothreitol (Sputolysin; Calbiochem Corp., San Diego, CA, USA). Homogenized sputum was sequentially diluted and placed in phosphate-buffered saline and plated on blood, chocolate, and MacConkey agar plates. Sputum isolates were classified as potential pathogens or as normal flora. Potential pathogens were Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and other gram-negative rods; other bacterial species were classified as normal flora [8].

Sputum and bronchial washing fluid were decontaminated with 4% sodium hydroxide, homogenized, and concentrated by centrifugation at 3000 × g for 20 min. The specimens were stained using the Ziehl–Nielsen method [6]. Concentrated specimens were cultured in 3% Ogawa medium and observed weekly for 9 weeks after inoculation. Following isolation of a suspected mycobacterial species, confirmation of NTM was performed by analyzing the sequences of three genes; 16S rRNA, rpoB and tuf. Polymerase chain reaction and subsequent sequencing were performed, and the resulting sequences were compared with the reference database using basic local alignment search tools. Mycobacterial species were identified using 16S rRNA sequences, using the algorithm described in Clinical and Laboratory Standards Institute guideline MM18-A [9].

Laboratory tests consisted of the following: leukocyte count including differential counts, hematocrit, hemoglobin, platelet count, total cholesterol, total protein, albumin, total and direct bilirubin, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, blood urea nitrogen, creatinine, electrolytes, erythrocyte sedimentation rate, C-reactive protein, fluorescent antinuclear antibody test (FANA), rheumatoid factor, serum immunoglobulins (IgG, IgA, IgM), and IFN-γ release assay (IGRA).

Pulmonary function tests, including forced expiratory volume at 1 second (FEV1), forced vital capacity (FVC), FEV1/FVC ratio, and diffusing capacity (DLCO) were performed. Simple posterior–anterior chest radiography, paranasal sinus radiography, and CT of the chest were carried out. Radiographic findings on CT scans were evaluated with regard to the presence of cavitations, nodule, and bronchiectasis. The anatomical distributions were also analyzed. Lesions were classified as showing either upper lobe cavitary disease or nodular bronchiectatic disease by radiographic type. When the disease did not belong to either the upper lobe cavitary form or the nodular bronchiectatic form, it was categorized as unclassifiable. The extent of bronchiectasis was scored in each of the six lobes (right upper lobe, right middle lobe, right lower lobe, upper division of the left upper lobe, lingular division of the left upper lobe, left lower lobe) according to the proportion of lung involvement. Extent scores ranged from 0 to 18; 0 if < 25%, 1 if 25–49%, 2 if 50–74%, 3 if ≥ 75% [10]. Scoliosis was determined from the posterior–anterior chest radiograph. Pectus excavatum was determined from CT scans of the chest using the Haller index and defined as a Haller index greater than 3.5 [11].

All participants were asked to complete the St. George’s respiratory questionnaire and Hospital Anxiety and Depression Scale (HADS) questionnaire. The HADS is a 14 item questionnaire measuring levels of anxiety (HADS-A, seven items) and depression (HADS-D, seven items). Each item is scored from 0–3; a cut-off point of 8 out of 21 is suggested for both the anxiety and depression sections [12].

Baseline characteristics were summarized using descriptive statistics such as proportion, median, and interquartile range. Student’s t-tests and Mann–Whitney U-tests were used for comparison of continuous variables. Categorical variables were compared using chi-square or Fisher’s exact tests, as appropriate. A P-value of ≤ 0.05 was considered to indicate statistical significance. All statistical analyses were performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA).

A total of 127 patients with NTM lung disease were enrolled in the study, with eight patients withdrawing consent prior to study completion. Among them, 84 patients with nodular BE type NTM lung disease were included in the study. In addition, 50 patients with non-NTM BE were initially enrolled in the study; three patients withdrew consent. The median ages were 67 and 64 years for NTM lung disease and non-NTM BE groups, respectively (P = 0.16). Of the patients with NTM lung disease, 54 (64.3%) were female, compared with 29 (61.7%) in the non-NTM BE group. Among comorbidities, cancer was more common among the NTM lung disease group (21.4% vs. 6.4%, P = 0.03) (Table 1). Weight loss was more common among the NTM lung disease group although it failed to reach the conventional P-value of 0.05. (15.5% vs. 4.3%, P = 0.08) (Table 1).

Of the 84 patients with NTM lung disease, 47 (60.0%) were infected with species belonging to the Mycobacterium avium complex; 11 (13.1%) were infected with M. abscessus complex species. Multiple NTM species were isolated in 20 (23.8%) patients (Table 2). Bacterial colonization by non-mycobacterial species was observed in 17 (20.2%) and 12 (25.5%) of NTM lung disease and non-NTM BE patients, respectively (P = 1.00). The trend that P. aeruginosa was more commonly isolated from non-NTM BE patients (5 patients, 10.6%) than NTM patients (2 patients, 2.4%) was observed (P = 0.10) (Table 3).

No difference in height was observed between NTM lung disease and non-NTM BE patients (160.0 vs. 159.0 cm, P = 0.23). However, patients with NTM lung disease were of lower body weight than those with non-NTM BE (54.0 vs. 55.5 kg, P = 0.04); consequently, the body mass index (BMI) of NTM lung disease patients was also lower than that of non-NTM BE patients (BMI = 20.8 vs. 22.2 kg/m², P < 0.001). Scoliosis was more common among patients with NTM lung disease than those with non-NTM BE (23.8% vs. 8.5%, P = 0.04) (Table 4).

Positive rheumatoid factor was detected more frequently in patients with non-NTM BE than with NTM lung disease (23.4% vs. 6.0%, P = 0.01); however, differences in mean rheumatoid factor levels were not statistically significant. No differences were seen regarding the presence of FANA, nor in FANA titers. Serum immunoglobulin levels were also similar between the two groups. Likewise, the proportion of IGRA-positive patients were similar in NTM lung disease patients and non-NTM BE patients (45.2 vs. 55.3%, P = 0.28) (Table 5).

The extent of BE was greater in the non-NTM BE group than in nodular bronchiectatic NTM lung disease patients (4 vs. 3 points, P = 0.001). Tubular BE was most common in both groups (Table 6).

No differences in FVC were observed between NTM lung disease patients and non-NTM BE patients in terms of either absolute volume (2.8 vs. 2.7 L, P = 0.13) although percentage of predicted volume were smaller among non-NTM BE patients (94% vs. 87%, P = 0.02). Both absolute volume of FEV1 (2.1 vs. 1.8 L, P = 0.001) and percentage of predicted volume (101% vs. 86%, P = 0.001) were smaller among non-NTM BE patients. However, the proportion of patients who met criteria for chronic obstructive pulmonary disease was not different between two groups (13.6% vs. 23.9%, P = 0.15) (Table 7).

Median St. George’s respiratory questionnaire scores were similar between the two groups (19.5 vs.18.1, P = 0.34). Twenty patients (23.8%) with NTM lung disease and 10 patients (21.3%) with non-NTM BE reported anxiety (P = 0.83). Twenty-three 23 patients (27.4%) with NTM lung disease and 10 patients (21.3%) with non-NTM BE reported symptoms of depression (P = 0.53).