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Clinicopathologic characteristics of nontuberculous mycobacterial lung disease in Taiwan
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Diagnostic Microbiology and Infectious Disease 68 (2010) 228 – 235 www.elsevier.com/locate/diagmicrobio
Mycobacteriology
Clinicopathologic characteristics of nontuberculous mycobacterial lung disease in Taiwan Cheng-Hsiang Hsiaoa,b , Yi-Ting Lina , Chih-Cheng Laic , Po-Ren Hsuehd,⁎ a
Department of Pathology, National Taiwan University Hospital, National Taiwan University College of Medicine, Taipei 10063, Taiwan b National Taipei College of Nursing, Taipei 11219, Taiwan c Department of Intensive Care Medicine, Chi Mei Medical Center, Liouying, Tainan 73657, Taiwan d Departments of Internal Medicine and Laboratory Medicine, National Taiwan University College of Medicine, Taipei 10063, Taiwan Received 10 March 2010; accepted 10 June 2010
Abstract Taiwan is an endemic area for tuberculosis (TB), and the incidence of pulmonary infection caused by nontuberculous mycobacteria (NTMs) is also increasing. This study aims to investigate the clinicopathologic characteristics of patients with NTM lung disease during 1998 to 2007 at a medical center in Taiwan. The medical records of patients with confirmed NTM pulmonary infections who underwent open lung surgery in a medical center were reviewed. Twenty-four patients with confirmed NTM pulmonary infections were identified. These patients were histologically classified into 4 types: fibrocavitary/tuberculoid (n = 10), nodular bronchiectatic (n = 4), sarcoidal (n = 6), and other (n = 4). The fibrocavitary/tuberculoid type usually (90%) develops in the upper lobes of old patients with preexisting lung disease. Pulmonary TB (n = 7, 70%) was the major underlying disease before 2003. Nodular bronchiectatic type occurred mainly in the middle lobe of middle-aged women without preexisting lung disease. Sarcoidal type was usually associated with Mycobacterium avium complex infection and develops in middle-aged women. Immunoreactive bacilli were detected in 21 patients (87 %) by immunohistochemical staining using a polyclonal antibody against Mycobacterium tuberculosis and other mycobacterial species (M. avium-intracellulare, Mycobacterium phlei, and Mycobacterium parafortuitum), whereas conventional acid-fast staining was positive in only 21% of patients. In conclusion, TB was the major underlying disease in patients with NTM lung disease in Taiwan. The different histologic types of pulmonary NTM infection suggest each had a distinct pathogenesis. © 2010 Elsevier Inc. All rights reserved. Keywords: Nontuberculous mycobacteria; Tuberculosis; Clinicopathologic characteristics; Taiwan
1. Introduction Nontuberculous mycobacteria (NTMs) are widely distributed microorganisms of the environment that are frequently isolated from soil, tap water, and dust (Falkinham, 2002; Holland, 2001). These organisms can cause disease if host defenses are impaired or if skin or mucosal barriers are disrupted. Unlike Mycobacterium tuberculosis, NTMs do not transmit person-to-person (Field and Cowie, 2006). It was not until the 1950s that NTMs were identified as potential human pathogens (Lewis et al., 1959). Human disease associated
⁎ Corresponding author. Tel.: +886-2-23123456x65355; fax: +886-223224263. E-mail address: [email protected] (P.-R. Hsueh). 0732-8893/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2010.06.008
with NTM infection may present as pulmonary disease, lymphadenitis, cutaneous disease, or disseminated disease (Wolinsky, 1979). Among these, chronic pulmonary disease is the most common (Griffith et al., 2007). Diagnosis of NTM lung disease should be based on meeting the following criteria: 1) clinical pulmonary symptoms and characteristic radiographic opacities, nodules, or cavities; 2) at least 2 respiratory specimens or 1 bronchial washing/brushing sample being culture positive for the same NTM species; and 3) exclusion of other disorders, such as tuberculosis (TB) (Griffith et al., 2007). Lung disease associated with NTM infection commonly occurs in structural lung disease, such as chronic obstructive pulmonary disease (COPD), bronchiectasis, cystic fibrosis, and old TB (Griffith et al., 2007). In Taiwan, TB is still the major mycobacterial species complex isolated from respiratory specimens, but the incidence of
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NTM lung disease is increasing (Ding et al., 2006). Proportion of NTM among all mycobacterial isolations increased significantly from 32.3% to 49.8% from 2000 to 2008 at a medical center in Taiwan, and the incidence of diseases caused by NTM also increased remarkably from 2.7 to 10.2 per 100 000 inpatients and outpatients (Lai et al., 2010). Because the radiographic presentations of NTM pulmonary disease are often indistinguishable from those of pulmonary TB, clinicians and pathologists need to understand both its clinicopathologic manifestations and epidemiologic trends to facilitate correct diagnosis.
2. Materials and methods 2.1. Setting and NTM pulmonary diseases We reviewed the archive of National Taiwan University Hospital, Taipei, Taiwan, from 1998 to 2007 to identify immunocompetent patients who had undergone surgical resection of lung lesions without definite diagnosis. Patients whose culture results did not fulfill the diagnostic criteria for NTM lung disease proposed by the American Thoracic Society in 2007 were excluded (Griffith et al., 2007). A total of 24 patients, including 17 women and 7 men, were identified. Clinical, radiologic, and follow-up data were collected by chart review. Part of the clinical and microbiologic data of these patients was reported previously (Hsiao et al., 2010). 2.2. Processing of lung specimens Formalin-fixed and paraffin-embedded tissue blocks of resected lung specimens were retrieved from the department of pathology. Recut histologic sections with hematoxylin and eosin (H&E) staining were reviewed. Ziehl–Neelsen acid-fast staining for mycobacteria and Grocott's methenamine–silver nitrate staining for fungal organisms were also performed on the tissue sections. 2.3. Immunohistochemical staining For immunohistochemical (IHC), tissue sections were deparaffinized and hydrated. Steam heat antigen retrieval was performed with Trilogy retrieval buffer (Cell Marque Cooperation, Austin, TX) for 20 min. The slides were then incubated with primary antibodies against mycobacteria (1:1000 dilution; Biocare Medical, Concord, CA) at 4 °C overnight. The primary antibody, a purified rabbit polyclonal antibody against M. tuberculosis, is also reactive with other mycobacterial species, including: Mycobacterium aviumintracellulare, M. phlei, and Mycobacterium parafortuitum. The antigen was detected using an Envision G2 System/AP visualization kit (DakoCytomation, Denmark) according to the procedures provided by the manufacturer; specimens were then counterstained with hematoxylin. The intensity of immunoreactive mycobacteria present on the tissue sections was graded into 0 to III: in grade 0, no mycobacteria; grade I,
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rare (1–5) mycobacteria; grade II, moderate (5–10) mycobacteria; and grade III, numerous or clustering of mycobacteria were identified throughout all tissue section in oil immersion (1000×).
3. Results The histologic classification of the 24 patients with NTM lung disease was fibrocavitary/tuberculoid type in 10 patients, sarcoidal type in 6, nodular bronchiectatic type in 4, and other types in 4. Fibrocavitary/tuberculoid NTM lung disease was the most common type of pulmonary NTM infection for persons who had lung surgery in the first 5 years from 1998 to 2002 (7/8), and all the 7 patients with this type of NTM lung disease had old TB. In contrast, only 3 of the 12 patients with NTM lung disease in the last 5 years from 2003 to 2007 were fibrocavitary/tuberculoid NTM lung disease, and none of the 3 patients had TB. Clinical, microbiologic, and pathologic characteristics of the 24 patients are summarized in Table 1. All 10 patients in the fibrocavitary/tuberculoid group had TB-like lesions. These patients included 5 women and 5 men with ages ranging from 41 to 77 years (mean of 52.6 years for females and 62.6 years for males). All but 1 lesion occurred in the upper lung lobe, and the roentgenograms showed fibronodular lesions with occasional calcification. Eight of the 10 patients with fibrocavitary/tuberculoid type had chronic pulmonary disease, 7 had old TB, and 1 had COPD. All of the 7 patients with underlying old TB developed NTM lung disease before 2002. Five patients with fibrocavitary/tuberculoid type had a positive M. avium-intracellulare complex (MAC) culture from the sputum or surgical specimens. Mycobacterium chelonae was isolated from the sputum of case 7 and Mycobacterium fortuitum from the sputum of case 10. The remaining 3 patients in this group had mixed NTM infections. Histologic examination revealed that all 10 patients with the fibrocavitary/tuberculoid type had prominent caseous necrosis. A well-formed fibrotic cavity was found in 4 of these patients. Mild-to-moderate bronchiectasis was also noted in 6 of the 10 patients. IHC staining clearly demonstrated variable mycobacteria within the caseous necrosis of all 10 patients. In contrast, conventional acidfast staining demonstrated mycobacteria in only 3 of these patients (Fig. 1). The nodular bronchiectatic type was characterized by the presence of bronchiectasis, peribronchial chronic inflammation, and small noncaseating granulomas without evident caseous necrosis. The 4 patients in this group included 3 women and 1 man (mean age of 50.5 years). All of lung lesions occurred in the right middle lobe demonstrated as bronchiectasis with peribronchial and alveolar infiltration by computed tomography (CT). The 3 female patients presented with recurrent hemoptysis without underlying pulmonary disease, and the only male patient had both COPD and old
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Table 1 Clinical and IHC findings in 24 patients with different histologic types of NTM pulmonary infectiona No. Sex/age (year)
Involvement of lung lobes
Underlying disease
IHC stain resultsb
Acid-fast stainc
I. Fibrocavitary/tuberculoid type 1. F/41 MAC (×1 tissue)
RUL
Old TB
Caseous necrosis, fibrotic cavity Old TB Caseous necrosis, bronchiectasis, sarcoidal granuloma Old TB Caseous necrosis, bronchiectasis Old TB Caseous necrosis, fibrotic cavity Gout Caseous necrosis, bronchiectasis Old TB Caseous necrosis, bronchiectasis, fibrotic cavity Old TB Caseous necrosis, sarcoidal granuloma, fibrotic cavity COPD, Caseous necrosis, CAD mild bronchiectasis Hyperlipidemia, Caseous necrosis, sarcoidal gout granuloma Old TB, COPD, Caseous necrosis, DM, gout bronchiectasis
II
−
2.
F/46
MAC (×1 tissue)
RUL
I
−
3.
F/46
MAC (×2 sputum, ×2 abscess)
4.
F/64
Mycobacterium scrofulaceum (×1 tissue)
LUL, LLL RUL
I
−
I
−
5.
M/59
MAC (×2 sputum)
RUL
II
−
6.
F/66
MAC (×3 sputum)
RUL, RLL
II
−
7.
M/45
M. chelonae (×3 sputum)
RUL, LLL
II
−
8.
M/74
RML
9.
M/58
MAC (×4 sputum), M. abscessus (×1 sputum) MAC (×1 sputum), M. kansasii (×1 sputum, ×1 bronchial washing) M. fortuitum (×4 sputum), Mycobacterium gordonae (×1 sputum)
III
+
I
+
III
+
RML RML
Thyroid cancer Nil
Bronchiectasis, granuloma Bronchiectasis
I I
− −
RML
Nil
Bronchiectasis
I
−
RML
COPD, old TB
Bronchiectasis, granuloma
0
−
III. Sarcoidal type 1. F/51 Mycobacterium marinum (×1 tissue)
RLL
Nil
I
−
2.
F/67
MAC (×1 tissue)
RUL
Hypertension
0
−
3.
F/52
Both lungs
Nil
I
−
4.
F/69
MAC (×3 sputum, ×1 tissue), M. abscessus (×2 sputum) MAC (×1 sputum, ×1 tissue)
RML
BAC, CAD
II
+
5.
F/63
MAC (×1 tissue)
LUL , RML
Ovarian cancer
I
−
6.
F/47
MAC (×1 sputum, ×1 tissue, ×1 bronchial washing)
LUL
Nil
Sarcoidal granuloma, mild bronchiectasis Sarcoidal granuloma, mild bronchiectasis Sarcoidal granuloma, mild bronchiectasis Sarcoidal granuloma, focal necrosis Sarcoidal granuloma, mild bronchiectasis Sarcoidal granuloma, focal necrosis
I
−
IV. Others 1. F/38
MAC (×1 tissue)
RLL
M/32
M. kansasii (×1 tissue)
LLL
Dilated bronchus, interstitial I fibrosis, granuloma Dilated bronchus, granuloma I
−
2.
−
3.
F/54
Emphysema, collapse
II
+
4.
F/66
MAC (×2 bronchial washing, ×1 sputum, RML, RUL ×3 pleural effusion, ×2 blood, ×1 tissue) M. abscessus (×3 sputum), nonchromogen LUL, RUL NTM (×1 bronchial washing)
Intralobar sequestration Intralobar sequestration Systemic MAC infection Adenocarcinoma
Adenocarcinoma
0
−
10. M/77
Culture results (times and site of positive culture)
II. Bronchiectatic type 1. F/55 MAC (×1 sputum, ×1 tissue) 2. F/25 M. abscessus (×17 sputum), M. fortuitum (×1 sputum) 3. F/48 MAC (×13 sputum), M. kansasii (×1 sputum) photochromogen NTM (×3 sputum) 4. M/74 M. abscessus (×10 sputum), M. fortuitum (×2 sputum), MAC (×1 sputum)
RUL, RML LUL, LLL
Histologic change
BAC = bronchioloalveolar carcinoma; F = female; M = male; RUL = right upper lobe; LUL = left upper lobe; LLL = left lower lobe; RLL = bright lower lobe; RML = right middle lobe; CAD = coronary artery disease; TB = tuberculosis; COPD = chronic obstructive pulmonary disease; DM = diabetes mellitus. a Part of the clinical and microbiologic data of these patients was reported previously (Hsiao et al., 2010). b The intensity of immunoreactive mycobacteria present on the tissue sections was graded into 0 to III. In grade 0, no mycobacteria; grade I, 1 to 5 mycobacteria; grade II, 5 to 10 mycobacteria; and grade III, numerous or clustering of mycobacteria were identified throughout all tissue section in oil immersion (1000×). c “+” indicates acid-fast bacilli were found; “−” indicates acid-fast bacill were not visible.
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Fig. 1. Caseous necrosis with fibrocavitary/tuberculoid NTM infection. (A) A fibrotic cavity with residual caseous necrosis in the cavity wall (1×, H&E). (B) A well-formed caseous necrosis was present in the pulmonary parenchyma (100×, H&E). (C) Focal metaplastic squamous epithelium lined the cavity wall (100×, H&E). (D) IHC staining demonstrated numerous mycobacteria in the caseous necrosis (1000×, IHC stain).
Fig. 2. Nodular bronchiectatic NTM lung disease. (A) Marked bronchiectasis with peribronchial chronic inflammation (100×, H&E). (B) Focal granuloma formation (400×, H&E). (C) IHC staining revealed scant bacilli (arrow) in the lumen of the bronchus (400×, IHC stain). (D) Detailed morphology of the mycobacteria (arrow) under oil immersion (1000×, IHC stain).
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Fig. 3. Sarcoidal NTM lung disease. (A) Bronchocentric noncaseating granuloma in the pulmonary parenchyma (100×, H&E). (B) A confluent noncaseating granulomatous nodule was present (100×, H&E). (C) A small focus of necrosis in the center of the granuloma (200×, H&E). (D) A small number of immunoreactive bacilli (arrows) were identified in the necrotic center (1000×, IHC stain).
TB. Two of the 4 patients in this group had MAC isolates, and the other 2 had Mycobacterium abscessus isolates. No acid-fast positive mycobacteria were found in the pulmonary lesions, but rare immunoreactive bacilli were noted within the fibrinopurulent exudates in the dilated bronchial lumen of 3 patients (Fig. 2). All 6 of the patients with sarcoidal type were female, and their ages ranged from 47 to 69 years (mean of 59.7 years). All these patients presented as cough, dyspnea, chest discomfort, or hemoptysis. Mild decrease of vital capacity was also noted in 2 of the 6 patients by spirometry test. All the 6 patients were characterized by the presence of multiple
nodules or patchy opacities in the lung fields revealed by CT. Pathologically, all the 6 patients with sarcoidal type NTM lung disease had bronchiolocentric or perivascular sarcoidal granulomas with occasional confluent granulomatous nodules. No evident caseous necrosis was seen, but focal mild necrosis in the center of the granuloma was found in 2 patients. Occasional multinucleated giant cells were noted within the granuloma. Mild bronchiectasis was also found in 4 patients. No underlying pulmonary disease or preferential lobar involvement was noted, and hilar lymph nodes were free from sarcoidal granuloma. MAC was isolated from respiratory specimens in all but 1 patient in the sarcoidal
Fig. 4. Intrapulmonary sequestration with NTM infection. (A) Cystic dilatation of the bronchus with occasional granulomas (arrows) (100×, H&E). (B) noncaseating granulomas with multinucleated giant cells (200×, H&E).
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group. IHC staining revealed small numbers of bacteria within the small necrotic center (Fig. 3). No specific pathologic changes characteristic of the fibrocavitary/tuberculoid type, bronchiectatic type, or sarcoidal type were found in the remaining 4 patients. These patients were therefore broadly categorized as having other histologic types. Two of these patients (cases 1 and 2) had intrapulmonary sequestration, which was characterized by the presence of cystic dilated bronchi containing mucus or purulent material. Occasional noncaseating granulomas were present in the sequestrated lung (Fig. 4). Scant immunoreactive bacteria were noted in the exudates of the dilated bronchus. One (case 3) of the patients had a disseminated MAC infection, and small numbers of mycobacteria were found within the alveolar spaces on both IHC and Ziehl–Neelsen staining. Primary pulmonary adenocarcinoma was diagnosed in 1 patient (case 4). M. abscessus was cultivated from this patient's sputum repeatedly during the following 2 years, but no specific inflammation or mycobacteria were found. The clinicopathologic and IHC manifestations of the 24 patients are summarized in Table 1.
4. Discussion Human disease associated with NTM infection can be classified into 4 distinct clinical syndromes: pulmonary disease, lymphadenitis, cutaneous disease, and disseminated disease (Wolinsky, 1979). Among these, chronic pulmonary disease is the most common (Griffith et al., 2007). Three types of NTM lung disease (i.e., fibrocavitary/tuberculoid lung disease, nodular bronchiectasis, and hypersensitive pneumonitis) have been described (Field and Cowie, 2006). Fibrocavitary NTM lung disease tends to occur in the upper lobe of old patients with preexisting lung disease, such as COPD (Glassroth, 2008; Johnson et al., 2008). Ten of the 24 patients in this study had well-formed caseous necrosis with various necrotic cavities compatible with fibrocavitary/ tuberculoid NTM lung disease. TB was the most common underlying pulmonary disease (7 patients) in this study. Only 4 patients developed a thin-walled fibrotic cavity thought to be characteristic of fibrocavitary NTM lung disease. Residual caseous necrotic material and a focal squamous epithelial lining were seen along the cavity wall of these patients. These histologic findings suggest that the original caseous necrosis may have eroded the airway, and expectoration of caseous material through the airway led to the formation of fibrocavitary lesions. This process is very similar to that in TB (Fujita et al., 2002). Nodular bronchiectasis is the second most common type of pulmonary NTM infection in the present work. The incidence of this type of NTM lung disease has increased in recent years (Falkinham, 2003; Okumura et al., 2008). The increased proportion of bronchiectatic NTM lung infections may be due to the increased number of examinations of
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patients with lung disease by high-resolution CT and the decreased number of patients with preexisting lung disease (Fowler et al., 2006). Nodular bronchiectatic NTM infection usually occurs in the right middle lobe or left lingular segment of middle-aged women without underlying lung disease (Ilowite et al., 2008). Nodular bronchiectatic type NTM infection was found in 4 patients in this study, and all of them had right middle lobe involvement. Three of these patients were middle-aged women without underlying pulmonary disease. The association between NTM and bronchiectasis remains unclear. In 1998, Wallace et al. analyzed their heterogeneity using pulsed-field gel electrophoresis and seroagglutination among the repeated MAC isolates recovered from patients with chronic lung disease. They found that patients with nodular bronchiectasis have polyclonal MAC infections, whereas patients with cavitary lesions in the upper lobes are usually infected with only a single strain. In our study, numerous bacteria were demonstrated in tuberculoid NTM infection, but only scanty bacteria were identified in bronchiectatic NTM infection by IHC staining. This observation is consistent with findings by Wallace et al. and indicates that the patients with tuberculoid NTM infection have monoclonal proliferation, but the patients with nodular bronchiectasis have multiple and/or repeated NTM infections. In 1997, some patients developed sarcoidal granulomatous lung diseases after exposure to hot tubs that were contaminated with MAC, and the disease was termed hot tub lung (Embil et al., 1997; Kahana et al., 1997). In this study, 6 patients presented with multiple sarcoidal granulomas and occasional confluent nodules. Radiologically, all 6 patients had multiple nodules or patches infiltrations in the lung fields. The histologic changes in these 6 patients were also similar to pulmonary sarcoidosis, but no mediastinal or hilar lymph node involvement was found. All of these patients were middle- to old-aged women without underlying pulmonary disease. MAC was isolated from the respiratory specimens in all but one of these patients. Clinical, microbiologic, radiologic, and histologic manifestations of these patients are consistent with the patients with hot tub lung. Although no definite history of hot tub exposure in the 6 patients was recorded, the possibility of hot spring exposure cannot be ruled out because most Taiwanese like to enjoy hot spring all year round. The pathogenesis of hot tub lung is still unknown. Embil et al. (1997) reported 5 cases of hot tub lung and ascribed the disease to the hypersensitivity reaction due to the short duration between the exacerbation of the symptoms and the hot tub exposure and spontaneous recovery of all patients with cessation of the hot tub use. Histologically, hypersensitivity pneumonia is characterized by the triad of cellular bronchiolitis, patchy chronic interstitial pneumonia, and scattered vague granulomas (Travis et al., 2002). In a study of 10 cases with hot tub lung (Khoor et al., 2001) and in our study, the granulomas were more prominent than in hypersensitivity pneumonia, and no evident interstitial inflammation or
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cellular bronchiolitis is seen. Thus, the pathogenesis of hot tub lung needs to be further clarified. Although most patients with NTM lung disease in this study could be categorized as fibrocavitary/tuberculoid type, bronchiectatic type, or sarcoidal type according to their histologic manifestations, the histology of 4 patients did not fit into these 3 standard categories. Intralobar sequestration was found in 2 of these patients. Intralobar sequestration is thought to be an acquired lesion of inflammatory origin that follows obstruction of the tracheobronchial tree with secondary development of the systemic arterial supply (Rosati and Leslie, 2005; Travis et al., 2002). Despite its frequent association with bacterial infections, pulmonary sequestration associated with mycobacteria infection has only rarely been reported (Lin et al., 2005; Shiota et al., 2002; Yatera et al., 2005). In the 2 patients with pulmonary sequestration in this study, few immunoreactive bacteria were identified within the dilated bronchus by IHC staining. The location and amount of mycobacteria in the 2 patients was similar to that in bronchiectatic NTM lung infection. Therefore, pulmonary sequestration may also be a risk factor for NTM infection. This hypothesis is also based on the fact that most patients with mycobacterial-infected pulmonary sequestration were cured by surgical resection alone and did not require further antibiotic treatment. IHC staining using polyclonal antibody against M. tuberculosis has been confirmed to be more sensitive than conventional acid-fast staining in detecting the mycobacteria of pulmonary TB (Gutierrez and Garcia, 1993; Ulrichs et al., 2005). The polyclonal antibody is reactive with not only M. tuberculosis but also several NTM species. In this study, immunoreactive bacilli were identified in 87% of lung tissues of 24 patients with pulmonary NTM disease. However, acid-fast positive bacilli were identified in only 5 (21%) of these patients. Our results were in accordance with findings reported previously. Our study has 1 major limitation. Only patients who underwent surgical intervention for diagnosis of unknown lung lesions were enrolled in our study. The patients who received CT-guided biopsy or bronchoscopic biopsy for diagnosis of unknown lung lesions were excluded; therefore, there may be selection bias and the study size is limited. However, the larger size of specimens obtained from surgery would provide the physicians much more information than the smaller samples that were obtained from other diagnostic methods. Thus, the pathologists would make accurate diagnosis based on the findings from surgical samples. In summary, NTM lung diseases generally presents as fibrocavitary/tuberculoid type, bronchiectatic type, or sarcoidal type each of which suggests a different pathogenesis. TB is the most common underlying disease in patients with NTM lung disease in Taiwan, but its significance has decreased in recent years. IHC staining using primary antibody against M. tuberculosis is useful in detecting myobacteria causing NTM infection.
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Diagnostic Microbiology and Infectious Disease 68 (2010) 228 – 235 www.elsevier.com/locate/diagmicrobio
Mycobacteriology
Clinicopathologic characteristics of nontuberculous mycobacterial lung disease in Taiwan Cheng-Hsiang Hsiaoa,b , Yi-Ting Lina , Chih-Cheng Laic , Po-Ren Hsuehd,⁎ a
Department of Pathology, National Taiwan University Hospital, National Taiwan University College of Medicine, Taipei 10063, Taiwan b National Taipei College of Nursing, Taipei 11219, Taiwan c Department of Intensive Care Medicine, Chi Mei Medical Center, Liouying, Tainan 73657, Taiwan d Departments of Internal Medicine and Laboratory Medicine, National Taiwan University College of Medicine, Taipei 10063, Taiwan Received 10 March 2010; accepted 10 June 2010
Abstract Taiwan is an endemic area for tuberculosis (TB), and the incidence of pulmonary infection caused by nontuberculous mycobacteria (NTMs) is also increasing. This study aims to investigate the clinicopathologic characteristics of patients with NTM lung disease during 1998 to 2007 at a medical center in Taiwan. The medical records of patients with confirmed NTM pulmonary infections who underwent open lung surgery in a medical center were reviewed. Twenty-four patients with confirmed NTM pulmonary infections were identified. These patients were histologically classified into 4 types: fibrocavitary/tuberculoid (n = 10), nodular bronchiectatic (n = 4), sarcoidal (n = 6), and other (n = 4). The fibrocavitary/tuberculoid type usually (90%) develops in the upper lobes of old patients with preexisting lung disease. Pulmonary TB (n = 7, 70%) was the major underlying disease before 2003. Nodular bronchiectatic type occurred mainly in the middle lobe of middle-aged women without preexisting lung disease. Sarcoidal type was usually associated with Mycobacterium avium complex infection and develops in middle-aged women. Immunoreactive bacilli were detected in 21 patients (87 %) by immunohistochemical staining using a polyclonal antibody against Mycobacterium tuberculosis and other mycobacterial species (M. avium-intracellulare, Mycobacterium phlei, and Mycobacterium parafortuitum), whereas conventional acid-fast staining was positive in only 21% of patients. In conclusion, TB was the major underlying disease in patients with NTM lung disease in Taiwan. The different histologic types of pulmonary NTM infection suggest each had a distinct pathogenesis. © 2010 Elsevier Inc. All rights reserved. Keywords: Nontuberculous mycobacteria; Tuberculosis; Clinicopathologic characteristics; Taiwan
1. Introduction Nontuberculous mycobacteria (NTMs) are widely distributed microorganisms of the environment that are frequently isolated from soil, tap water, and dust (Falkinham, 2002; Holland, 2001). These organisms can cause disease if host defenses are impaired or if skin or mucosal barriers are disrupted. Unlike Mycobacterium tuberculosis, NTMs do not transmit person-to-person (Field and Cowie, 2006). It was not until the 1950s that NTMs were identified as potential human pathogens (Lewis et al., 1959). Human disease associated
⁎ Corresponding author. Tel.: +886-2-23123456x65355; fax: +886-223224263. E-mail address: [email protected] (P.-R. Hsueh). 0732-8893/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2010.06.008
with NTM infection may present as pulmonary disease, lymphadenitis, cutaneous disease, or disseminated disease (Wolinsky, 1979). Among these, chronic pulmonary disease is the most common (Griffith et al., 2007). Diagnosis of NTM lung disease should be based on meeting the following criteria: 1) clinical pulmonary symptoms and characteristic radiographic opacities, nodules, or cavities; 2) at least 2 respiratory specimens or 1 bronchial washing/brushing sample being culture positive for the same NTM species; and 3) exclusion of other disorders, such as tuberculosis (TB) (Griffith et al., 2007). Lung disease associated with NTM infection commonly occurs in structural lung disease, such as chronic obstructive pulmonary disease (COPD), bronchiectasis, cystic fibrosis, and old TB (Griffith et al., 2007). In Taiwan, TB is still the major mycobacterial species complex isolated from respiratory specimens, but the incidence of
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NTM lung disease is increasing (Ding et al., 2006). Proportion of NTM among all mycobacterial isolations increased significantly from 32.3% to 49.8% from 2000 to 2008 at a medical center in Taiwan, and the incidence of diseases caused by NTM also increased remarkably from 2.7 to 10.2 per 100 000 inpatients and outpatients (Lai et al., 2010). Because the radiographic presentations of NTM pulmonary disease are often indistinguishable from those of pulmonary TB, clinicians and pathologists need to understand both its clinicopathologic manifestations and epidemiologic trends to facilitate correct diagnosis.
2. Materials and methods 2.1. Setting and NTM pulmonary diseases We reviewed the archive of National Taiwan University Hospital, Taipei, Taiwan, from 1998 to 2007 to identify immunocompetent patients who had undergone surgical resection of lung lesions without definite diagnosis. Patients whose culture results did not fulfill the diagnostic criteria for NTM lung disease proposed by the American Thoracic Society in 2007 were excluded (Griffith et al., 2007). A total of 24 patients, including 17 women and 7 men, were identified. Clinical, radiologic, and follow-up data were collected by chart review. Part of the clinical and microbiologic data of these patients was reported previously (Hsiao et al., 2010). 2.2. Processing of lung specimens Formalin-fixed and paraffin-embedded tissue blocks of resected lung specimens were retrieved from the department of pathology. Recut histologic sections with hematoxylin and eosin (H&E) staining were reviewed. Ziehl–Neelsen acid-fast staining for mycobacteria and Grocott's methenamine–silver nitrate staining for fungal organisms were also performed on the tissue sections. 2.3. Immunohistochemical staining For immunohistochemical (IHC), tissue sections were deparaffinized and hydrated. Steam heat antigen retrieval was performed with Trilogy retrieval buffer (Cell Marque Cooperation, Austin, TX) for 20 min. The slides were then incubated with primary antibodies against mycobacteria (1:1000 dilution; Biocare Medical, Concord, CA) at 4 °C overnight. The primary antibody, a purified rabbit polyclonal antibody against M. tuberculosis, is also reactive with other mycobacterial species, including: Mycobacterium aviumintracellulare, M. phlei, and Mycobacterium parafortuitum. The antigen was detected using an Envision G2 System/AP visualization kit (DakoCytomation, Denmark) according to the procedures provided by the manufacturer; specimens were then counterstained with hematoxylin. The intensity of immunoreactive mycobacteria present on the tissue sections was graded into 0 to III: in grade 0, no mycobacteria; grade I,
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rare (1–5) mycobacteria; grade II, moderate (5–10) mycobacteria; and grade III, numerous or clustering of mycobacteria were identified throughout all tissue section in oil immersion (1000×).
3. Results The histologic classification of the 24 patients with NTM lung disease was fibrocavitary/tuberculoid type in 10 patients, sarcoidal type in 6, nodular bronchiectatic type in 4, and other types in 4. Fibrocavitary/tuberculoid NTM lung disease was the most common type of pulmonary NTM infection for persons who had lung surgery in the first 5 years from 1998 to 2002 (7/8), and all the 7 patients with this type of NTM lung disease had old TB. In contrast, only 3 of the 12 patients with NTM lung disease in the last 5 years from 2003 to 2007 were fibrocavitary/tuberculoid NTM lung disease, and none of the 3 patients had TB. Clinical, microbiologic, and pathologic characteristics of the 24 patients are summarized in Table 1. All 10 patients in the fibrocavitary/tuberculoid group had TB-like lesions. These patients included 5 women and 5 men with ages ranging from 41 to 77 years (mean of 52.6 years for females and 62.6 years for males). All but 1 lesion occurred in the upper lung lobe, and the roentgenograms showed fibronodular lesions with occasional calcification. Eight of the 10 patients with fibrocavitary/tuberculoid type had chronic pulmonary disease, 7 had old TB, and 1 had COPD. All of the 7 patients with underlying old TB developed NTM lung disease before 2002. Five patients with fibrocavitary/tuberculoid type had a positive M. avium-intracellulare complex (MAC) culture from the sputum or surgical specimens. Mycobacterium chelonae was isolated from the sputum of case 7 and Mycobacterium fortuitum from the sputum of case 10. The remaining 3 patients in this group had mixed NTM infections. Histologic examination revealed that all 10 patients with the fibrocavitary/tuberculoid type had prominent caseous necrosis. A well-formed fibrotic cavity was found in 4 of these patients. Mild-to-moderate bronchiectasis was also noted in 6 of the 10 patients. IHC staining clearly demonstrated variable mycobacteria within the caseous necrosis of all 10 patients. In contrast, conventional acidfast staining demonstrated mycobacteria in only 3 of these patients (Fig. 1). The nodular bronchiectatic type was characterized by the presence of bronchiectasis, peribronchial chronic inflammation, and small noncaseating granulomas without evident caseous necrosis. The 4 patients in this group included 3 women and 1 man (mean age of 50.5 years). All of lung lesions occurred in the right middle lobe demonstrated as bronchiectasis with peribronchial and alveolar infiltration by computed tomography (CT). The 3 female patients presented with recurrent hemoptysis without underlying pulmonary disease, and the only male patient had both COPD and old
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Table 1 Clinical and IHC findings in 24 patients with different histologic types of NTM pulmonary infectiona No. Sex/age (year)
Involvement of lung lobes
Underlying disease
IHC stain resultsb
Acid-fast stainc
I. Fibrocavitary/tuberculoid type 1. F/41 MAC (×1 tissue)
RUL
Old TB
Caseous necrosis, fibrotic cavity Old TB Caseous necrosis, bronchiectasis, sarcoidal granuloma Old TB Caseous necrosis, bronchiectasis Old TB Caseous necrosis, fibrotic cavity Gout Caseous necrosis, bronchiectasis Old TB Caseous necrosis, bronchiectasis, fibrotic cavity Old TB Caseous necrosis, sarcoidal granuloma, fibrotic cavity COPD, Caseous necrosis, CAD mild bronchiectasis Hyperlipidemia, Caseous necrosis, sarcoidal gout granuloma Old TB, COPD, Caseous necrosis, DM, gout bronchiectasis
II
−
2.
F/46
MAC (×1 tissue)
RUL
I
−
3.
F/46
MAC (×2 sputum, ×2 abscess)
4.
F/64
Mycobacterium scrofulaceum (×1 tissue)
LUL, LLL RUL
I
−
I
−
5.
M/59
MAC (×2 sputum)
RUL
II
−
6.
F/66
MAC (×3 sputum)
RUL, RLL
II
−
7.
M/45
M. chelonae (×3 sputum)
RUL, LLL
II
−
8.
M/74
RML
9.
M/58
MAC (×4 sputum), M. abscessus (×1 sputum) MAC (×1 sputum), M. kansasii (×1 sputum, ×1 bronchial washing) M. fortuitum (×4 sputum), Mycobacterium gordonae (×1 sputum)
III
+
I
+
III
+
RML RML
Thyroid cancer Nil
Bronchiectasis, granuloma Bronchiectasis
I I
− −
RML
Nil
Bronchiectasis
I
−
RML
COPD, old TB
Bronchiectasis, granuloma
0
−
III. Sarcoidal type 1. F/51 Mycobacterium marinum (×1 tissue)
RLL
Nil
I
−
2.
F/67
MAC (×1 tissue)
RUL
Hypertension
0
−
3.
F/52
Both lungs
Nil
I
−
4.
F/69
MAC (×3 sputum, ×1 tissue), M. abscessus (×2 sputum) MAC (×1 sputum, ×1 tissue)
RML
BAC, CAD
II
+
5.
F/63
MAC (×1 tissue)
LUL , RML
Ovarian cancer
I
−
6.
F/47
MAC (×1 sputum, ×1 tissue, ×1 bronchial washing)
LUL
Nil
Sarcoidal granuloma, mild bronchiectasis Sarcoidal granuloma, mild bronchiectasis Sarcoidal granuloma, mild bronchiectasis Sarcoidal granuloma, focal necrosis Sarcoidal granuloma, mild bronchiectasis Sarcoidal granuloma, focal necrosis
I
−
IV. Others 1. F/38
MAC (×1 tissue)
RLL
M/32
M. kansasii (×1 tissue)
LLL
Dilated bronchus, interstitial I fibrosis, granuloma Dilated bronchus, granuloma I
−
2.
−
3.
F/54
Emphysema, collapse
II
+
4.
F/66
MAC (×2 bronchial washing, ×1 sputum, RML, RUL ×3 pleural effusion, ×2 blood, ×1 tissue) M. abscessus (×3 sputum), nonchromogen LUL, RUL NTM (×1 bronchial washing)
Intralobar sequestration Intralobar sequestration Systemic MAC infection Adenocarcinoma
Adenocarcinoma
0
−
10. M/77
Culture results (times and site of positive culture)
II. Bronchiectatic type 1. F/55 MAC (×1 sputum, ×1 tissue) 2. F/25 M. abscessus (×17 sputum), M. fortuitum (×1 sputum) 3. F/48 MAC (×13 sputum), M. kansasii (×1 sputum) photochromogen NTM (×3 sputum) 4. M/74 M. abscessus (×10 sputum), M. fortuitum (×2 sputum), MAC (×1 sputum)
RUL, RML LUL, LLL
Histologic change
BAC = bronchioloalveolar carcinoma; F = female; M = male; RUL = right upper lobe; LUL = left upper lobe; LLL = left lower lobe; RLL = bright lower lobe; RML = right middle lobe; CAD = coronary artery disease; TB = tuberculosis; COPD = chronic obstructive pulmonary disease; DM = diabetes mellitus. a Part of the clinical and microbiologic data of these patients was reported previously (Hsiao et al., 2010). b The intensity of immunoreactive mycobacteria present on the tissue sections was graded into 0 to III. In grade 0, no mycobacteria; grade I, 1 to 5 mycobacteria; grade II, 5 to 10 mycobacteria; and grade III, numerous or clustering of mycobacteria were identified throughout all tissue section in oil immersion (1000×). c “+” indicates acid-fast bacilli were found; “−” indicates acid-fast bacill were not visible.
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Fig. 1. Caseous necrosis with fibrocavitary/tuberculoid NTM infection. (A) A fibrotic cavity with residual caseous necrosis in the cavity wall (1×, H&E). (B) A well-formed caseous necrosis was present in the pulmonary parenchyma (100×, H&E). (C) Focal metaplastic squamous epithelium lined the cavity wall (100×, H&E). (D) IHC staining demonstrated numerous mycobacteria in the caseous necrosis (1000×, IHC stain).
Fig. 2. Nodular bronchiectatic NTM lung disease. (A) Marked bronchiectasis with peribronchial chronic inflammation (100×, H&E). (B) Focal granuloma formation (400×, H&E). (C) IHC staining revealed scant bacilli (arrow) in the lumen of the bronchus (400×, IHC stain). (D) Detailed morphology of the mycobacteria (arrow) under oil immersion (1000×, IHC stain).
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Fig. 3. Sarcoidal NTM lung disease. (A) Bronchocentric noncaseating granuloma in the pulmonary parenchyma (100×, H&E). (B) A confluent noncaseating granulomatous nodule was present (100×, H&E). (C) A small focus of necrosis in the center of the granuloma (200×, H&E). (D) A small number of immunoreactive bacilli (arrows) were identified in the necrotic center (1000×, IHC stain).
TB. Two of the 4 patients in this group had MAC isolates, and the other 2 had Mycobacterium abscessus isolates. No acid-fast positive mycobacteria were found in the pulmonary lesions, but rare immunoreactive bacilli were noted within the fibrinopurulent exudates in the dilated bronchial lumen of 3 patients (Fig. 2). All 6 of the patients with sarcoidal type were female, and their ages ranged from 47 to 69 years (mean of 59.7 years). All these patients presented as cough, dyspnea, chest discomfort, or hemoptysis. Mild decrease of vital capacity was also noted in 2 of the 6 patients by spirometry test. All the 6 patients were characterized by the presence of multiple
nodules or patchy opacities in the lung fields revealed by CT. Pathologically, all the 6 patients with sarcoidal type NTM lung disease had bronchiolocentric or perivascular sarcoidal granulomas with occasional confluent granulomatous nodules. No evident caseous necrosis was seen, but focal mild necrosis in the center of the granuloma was found in 2 patients. Occasional multinucleated giant cells were noted within the granuloma. Mild bronchiectasis was also found in 4 patients. No underlying pulmonary disease or preferential lobar involvement was noted, and hilar lymph nodes were free from sarcoidal granuloma. MAC was isolated from respiratory specimens in all but 1 patient in the sarcoidal
Fig. 4. Intrapulmonary sequestration with NTM infection. (A) Cystic dilatation of the bronchus with occasional granulomas (arrows) (100×, H&E). (B) noncaseating granulomas with multinucleated giant cells (200×, H&E).
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group. IHC staining revealed small numbers of bacteria within the small necrotic center (Fig. 3). No specific pathologic changes characteristic of the fibrocavitary/tuberculoid type, bronchiectatic type, or sarcoidal type were found in the remaining 4 patients. These patients were therefore broadly categorized as having other histologic types. Two of these patients (cases 1 and 2) had intrapulmonary sequestration, which was characterized by the presence of cystic dilated bronchi containing mucus or purulent material. Occasional noncaseating granulomas were present in the sequestrated lung (Fig. 4). Scant immunoreactive bacteria were noted in the exudates of the dilated bronchus. One (case 3) of the patients had a disseminated MAC infection, and small numbers of mycobacteria were found within the alveolar spaces on both IHC and Ziehl–Neelsen staining. Primary pulmonary adenocarcinoma was diagnosed in 1 patient (case 4). M. abscessus was cultivated from this patient's sputum repeatedly during the following 2 years, but no specific inflammation or mycobacteria were found. The clinicopathologic and IHC manifestations of the 24 patients are summarized in Table 1.
4. Discussion Human disease associated with NTM infection can be classified into 4 distinct clinical syndromes: pulmonary disease, lymphadenitis, cutaneous disease, and disseminated disease (Wolinsky, 1979). Among these, chronic pulmonary disease is the most common (Griffith et al., 2007). Three types of NTM lung disease (i.e., fibrocavitary/tuberculoid lung disease, nodular bronchiectasis, and hypersensitive pneumonitis) have been described (Field and Cowie, 2006). Fibrocavitary NTM lung disease tends to occur in the upper lobe of old patients with preexisting lung disease, such as COPD (Glassroth, 2008; Johnson et al., 2008). Ten of the 24 patients in this study had well-formed caseous necrosis with various necrotic cavities compatible with fibrocavitary/ tuberculoid NTM lung disease. TB was the most common underlying pulmonary disease (7 patients) in this study. Only 4 patients developed a thin-walled fibrotic cavity thought to be characteristic of fibrocavitary NTM lung disease. Residual caseous necrotic material and a focal squamous epithelial lining were seen along the cavity wall of these patients. These histologic findings suggest that the original caseous necrosis may have eroded the airway, and expectoration of caseous material through the airway led to the formation of fibrocavitary lesions. This process is very similar to that in TB (Fujita et al., 2002). Nodular bronchiectasis is the second most common type of pulmonary NTM infection in the present work. The incidence of this type of NTM lung disease has increased in recent years (Falkinham, 2003; Okumura et al., 2008). The increased proportion of bronchiectatic NTM lung infections may be due to the increased number of examinations of
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patients with lung disease by high-resolution CT and the decreased number of patients with preexisting lung disease (Fowler et al., 2006). Nodular bronchiectatic NTM infection usually occurs in the right middle lobe or left lingular segment of middle-aged women without underlying lung disease (Ilowite et al., 2008). Nodular bronchiectatic type NTM infection was found in 4 patients in this study, and all of them had right middle lobe involvement. Three of these patients were middle-aged women without underlying pulmonary disease. The association between NTM and bronchiectasis remains unclear. In 1998, Wallace et al. analyzed their heterogeneity using pulsed-field gel electrophoresis and seroagglutination among the repeated MAC isolates recovered from patients with chronic lung disease. They found that patients with nodular bronchiectasis have polyclonal MAC infections, whereas patients with cavitary lesions in the upper lobes are usually infected with only a single strain. In our study, numerous bacteria were demonstrated in tuberculoid NTM infection, but only scanty bacteria were identified in bronchiectatic NTM infection by IHC staining. This observation is consistent with findings by Wallace et al. and indicates that the patients with tuberculoid NTM infection have monoclonal proliferation, but the patients with nodular bronchiectasis have multiple and/or repeated NTM infections. In 1997, some patients developed sarcoidal granulomatous lung diseases after exposure to hot tubs that were contaminated with MAC, and the disease was termed hot tub lung (Embil et al., 1997; Kahana et al., 1997). In this study, 6 patients presented with multiple sarcoidal granulomas and occasional confluent nodules. Radiologically, all 6 patients had multiple nodules or patches infiltrations in the lung fields. The histologic changes in these 6 patients were also similar to pulmonary sarcoidosis, but no mediastinal or hilar lymph node involvement was found. All of these patients were middle- to old-aged women without underlying pulmonary disease. MAC was isolated from the respiratory specimens in all but one of these patients. Clinical, microbiologic, radiologic, and histologic manifestations of these patients are consistent with the patients with hot tub lung. Although no definite history of hot tub exposure in the 6 patients was recorded, the possibility of hot spring exposure cannot be ruled out because most Taiwanese like to enjoy hot spring all year round. The pathogenesis of hot tub lung is still unknown. Embil et al. (1997) reported 5 cases of hot tub lung and ascribed the disease to the hypersensitivity reaction due to the short duration between the exacerbation of the symptoms and the hot tub exposure and spontaneous recovery of all patients with cessation of the hot tub use. Histologically, hypersensitivity pneumonia is characterized by the triad of cellular bronchiolitis, patchy chronic interstitial pneumonia, and scattered vague granulomas (Travis et al., 2002). In a study of 10 cases with hot tub lung (Khoor et al., 2001) and in our study, the granulomas were more prominent than in hypersensitivity pneumonia, and no evident interstitial inflammation or
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cellular bronchiolitis is seen. Thus, the pathogenesis of hot tub lung needs to be further clarified. Although most patients with NTM lung disease in this study could be categorized as fibrocavitary/tuberculoid type, bronchiectatic type, or sarcoidal type according to their histologic manifestations, the histology of 4 patients did not fit into these 3 standard categories. Intralobar sequestration was found in 2 of these patients. Intralobar sequestration is thought to be an acquired lesion of inflammatory origin that follows obstruction of the tracheobronchial tree with secondary development of the systemic arterial supply (Rosati and Leslie, 2005; Travis et al., 2002). Despite its frequent association with bacterial infections, pulmonary sequestration associated with mycobacteria infection has only rarely been reported (Lin et al., 2005; Shiota et al., 2002; Yatera et al., 2005). In the 2 patients with pulmonary sequestration in this study, few immunoreactive bacteria were identified within the dilated bronchus by IHC staining. The location and amount of mycobacteria in the 2 patients was similar to that in bronchiectatic NTM lung infection. Therefore, pulmonary sequestration may also be a risk factor for NTM infection. This hypothesis is also based on the fact that most patients with mycobacterial-infected pulmonary sequestration were cured by surgical resection alone and did not require further antibiotic treatment. IHC staining using polyclonal antibody against M. tuberculosis has been confirmed to be more sensitive than conventional acid-fast staining in detecting the mycobacteria of pulmonary TB (Gutierrez and Garcia, 1993; Ulrichs et al., 2005). The polyclonal antibody is reactive with not only M. tuberculosis but also several NTM species. In this study, immunoreactive bacilli were identified in 87% of lung tissues of 24 patients with pulmonary NTM disease. However, acid-fast positive bacilli were identified in only 5 (21%) of these patients. Our results were in accordance with findings reported previously. Our study has 1 major limitation. Only patients who underwent surgical intervention for diagnosis of unknown lung lesions were enrolled in our study. The patients who received CT-guided biopsy or bronchoscopic biopsy for diagnosis of unknown lung lesions were excluded; therefore, there may be selection bias and the study size is limited. However, the larger size of specimens obtained from surgery would provide the physicians much more information than the smaller samples that were obtained from other diagnostic methods. Thus, the pathologists would make accurate diagnosis based on the findings from surgical samples. In summary, NTM lung diseases generally presents as fibrocavitary/tuberculoid type, bronchiectatic type, or sarcoidal type each of which suggests a different pathogenesis. TB is the most common underlying disease in patients with NTM lung disease in Taiwan, but its significance has decreased in recent years. IHC staining using primary antibody against M. tuberculosis is useful in detecting myobacteria causing NTM infection.
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