Nontuberculous mycobacteria isolated during the treatment of pulmonary tuberculosis

Respiratory Medicine (2009) 103, 1936e1940 available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/rmed Nontuberculous mycobacteria isolated during the treatment of pulmonary tuberculosis Hee-Jung Jun a, Kyeongman Jeon a, Sang-Won Um a, O Jung Kwon a, Nam Yong Lee b, Won-Jung Koh a, * a Division of Pulmonary and Critical Care Medicine, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwon-dong, Gangnam-gu, Seoul 135-710, Republic of Korea b Department of Laboratory Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea Received 31 January 2009; accepted 25 May 2009 Available online 2 July 2009 KEYWORDS Atypical mycobacteria; Pulmonary tuberculosis; Lung diseases Summary Background: The significance of nontuberculous mycobacteria (NTM) isolated from a patient during therapy for pulmonary tuberculosis (TB) is uncertain. We investigated the frequency and clinical significance of NTM isolated from patients receiving anti-tb treatment. Methods: We conducted a retrospective cohort study of all patients with culture-confirmed pulmonary TB, and identified patients with respiratory cultures positive for NTM during therapy for pulmonary TB. Results: From January 2003 to December 2005, 958 patients were diagnosed with cultureconfirmed pulmonary TB. NTM were isolated from 113 specimens in 68 (7.1%) patients during anti-tb treatment. The most frequently isolated NTM species were Mycobacterium abscessus (n Z 35, 31%), Mycobacterium fortuitum (n Z 17, 15%), Mycobacterium avium complex (n Z 9, 8%), and Mycobacterium gordonae (n Z 9, 8%). Forty-eight (71%) patients had only one positive culture, while 20 (29%) had two or more positive cultures for NTM. Only two (3%) patients who had two or more positive culture after anti-tb treatment showed the same NTM species, which were M. abscessus. Conclusion: The isolation of NTM in patients with pulmonary TB is not uncommon during anti- TB treatment. However, this is likely the result of colonization, a transient infection, or specimen contamination. The co-existence of pulmonary TB and NTM lung disease may be rare but should be considered in patients with relatively virulent NTM species such as M. abscessus. ª 2009 Elsevier Ltd. All rights reserved. Abbreviations: NTM, nontuberculous mycobacteria; ATS, American Thoracic Society; IDSA, Infectious Diseases Society of America; TB, tuberculosis; AFB, acid-fast bacilli; HIV, human immunodeficiency virus; IQR, interquartile range. * Corresponding author. Tel.: þ82 2 3410 3429; fax: þ82 2 3410 6956. E-mail address: wjkoh@skku.edu (W.-J. Koh). 0954-6111/$ - see front matter ª 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.rmed.2009.05.025

NTM isolated during treatment of pulmonary TB 1937 Introduction Mycobacteria other than Mycobacterium tuberculosis complex and Mycobacterium leprae are collectively referred to as nontuberculous mycobacteria (NTM). 1e3 Human disease resulting from NTM infection is classified into four distinct clinical syndromes: pulmonary disease, lymphadenitis, cutaneous disease, and disseminated disease. Among these, chronic pulmonary disease is the most common localized clinical condition. 1e3 Studies have demonstrated that disease attributable to NTM is on the rise. NTM are responsible for an increasing proportion of mycobacterial diseases in many developed and developing countries. 4e8 NTM are ubiquitous organisms and are frequently isolated from environmental sources, including surface water, tap water, and soil. 1,2,9 Accordingly, the isolation of NTM species from a respiratory sample is insufficient evidence for the presence of NTM lung disease. Some patients are infected with NTM without evidence of pulmonary disease, and such infection may indicate colonization or transient infection. Therefore, the diagnosis of NTM lung disease must rely on clinical, radiographic, and microbiological criteria. The American Thoracic Society (ATS) issued diagnostic criteria for NTM lung disease in 1997, 1 and the ATS and the Infectious Diseases Society of America (IDSA) recently revised the criteria in 2007. 2 The guidelines state that the reasonable exclusion of other etiologies of pulmonary disease is a necessary clinical criterion, along with the presence of respiratory symptoms, compatible radiographic abnormalities, and bacteriological evidence of NTM. 2 Furthermore, the guidelines state that there are clinical problems not directly addressed by the diagnostic guidelines; one of these is the significance of NTM isolated from a patient during therapy for pulmonary tuberculosis (TB). To date, only a few reports of cases in Japan with concurrent TB and NTM infections have been published. 10e14 However, the frequency and clinical significance of NTM isolated from patients during treatment for pulmonary TB are not well studied. Therefore, we investigated the frequency and clinical significance of NTM isolated from patients receiving anti-tb treatment. Patients and methods Study subjects We conducted a retrospective cohort study of all patients with culture-confirmed pulmonary TB who had been diagnosed and treated at Samsung Medical Center from January 2003 through December 2005. Samsung Medical Center is a 1250-bed university-affiliated tertiary referral hospital in Seoul, South Korea. Among these patients, those with respiratory cultures positive for NTM during therapy for pulmonary TB were identified from a computer-assisted search of medical records. The Institutional Review Board at Samsung Medical Center approved the study protocol. Informed consent was waived because of the retrospective nature of the study. Smears, cultures, and species identification Generally, the patients with pulmonary TB were treated on an outpatient basis. Sputum smear and culture examinations were performed monthly for the first 2 months and then at 1- to 2-month intervals until the end of treatment. Sputum specimens were collected spontaneously, and tap water was not any part of the specimen processing. Clinical specimens were stained using the ZiehleNeelsen method, according to the ATS guidelines. 15 The results of smear microscopy were reported semi-quantitatively. A positive smear was defined as one with one or more acidfast bacilli (AFB) per 100 high-power fields. Sputum or bronchial wash specimens were decontaminated using the N-acetyl-L-cysteine 2% NaOH method, and the processed specimens were plated on LöwensteineJensen medium. Inoculated tubes were incubated at 37 C and then inspected weekly for 8 weeks. To distinguish between M. tuberculosis and NTM, all AFB isolates were assessed according to growth rates, colony morphology, and pigmentation, as well as with a commercial DNA probe (AMPLICO test; Roche Molecular Systems, Branchburg, NJ). NTM species were identified using a polymerase chain reaction and restriction length polymorphism method based on the rpob gene, as previously described. 6,16,17 Evaluation of clinical and radiographic findings The medical records of the patients were reviewed, with respect to the following information: age, gender, smoking history, underlying pulmonary and non-pulmonary illnesses, results of anti-human immunodeficiency virus (HIV) antibody, AFB smear status, number of positive NTM isolates, species of NTM organisms, and anti-tb treatment information. The extent of radiographic pulmonary TB disease was graded using the U.S. National Tuberculosis and Respiratory Disease Association scheme, which classifies disease into minimal, moderately advanced, and far-advanced disease. 18 Results Patients characteristics From January 2003 to December 2005, 958 patients were diagnosed with culture-confirmed pulmonary TB. Of these, 68 (7.1%) patients were identified as having positive culture results for NTM during anti-tb treatment. The median age of these 68 patients was 52.5 years (interquartile range [IQR], 35.5e64.0 years). Forty-two (62%) of the patients were men and 26 were women. Thirty (44%) patients had never smoked (Table 1). Twenty-three (34%) patients had a history of pulmonary TB. Nine (13%) patients had idiopathic pulmonary fibrosis, and six (9%) had bronchiectasis. Twenty-seven (40%) patients had underlying non-pulmonary disease, including diabetes mellitus, malignancies, chronic liver disease, and chronic kidney disease (Table 1). No patient was seropositive for antibodies to HIV or belonged to a high-risk group (i.e. homosexuals, injectable drug users, persons with hemophilia, or coagulation disorders, etc.).

1938 H.-J. Jun et al. Of the 68 patients, 44 (65%) had drug-susceptible TB, 15 (22%) had multidrug-resistant TB, and seven (10%) had any other drug-resistant TB. The results of drug susceptibility tests were not available for two patients. Forty-nine (72%) of the patients had moderately advanced or far-advanced radiographic disease (Table 1). The duration of the anti-tb treatment in the 68 patients was a median of 6.6 months (IQR, 6.0e16.8 months). Microbiological findings During anti-tb treatment in the 68 patients, 799 respiratory specimens were sent to the laboratory for mycobacterial culture, and 113 specimens were found to be positive for NTM. The median number of specimens sent for culture per patient was eight (IQR, 6e16), and the median number of cultures for NTM per patient was one (IQR, 1e2). The median interval from starting anti-tb treatment to the first positive culture for NTM was 2.8 months (IQR, 0.9e5.6 months). Forty-eight (71%) patients had only one positive culture, 13 (19%) patients had two, and seven (10%) patients had three or more cultures positive for NTM. Fifty-three (47%) of the 113 specimen cultures positive culture for NTM were negative on AFB stain, and 94 (83%) specimen cultures resulted in fewer than 50 colonies (Table 2). From the 113 specimens, 114 NTM were isolated and identified, as two different NTM species were isolated from one sputum specimen. In nine cases, M. tuberculosis and NTM were detected from the same sample on sputum culture. Among the 113 specimens, the most frequently isolated organisms were Mycobacterium abscessus (n Z 35, Table 1 Patients characteristics (n Z 68). Variable Value Age (years) 52.5 (35.5e64) Male/female 42 (62%)/26 (38%) Body mass 21.7 (19.6e24.0) index (kg/m 2 ) Underlying lung Previous pulmonary 23 (34%) disease tuberculosis Idiopathic pulmonary 9 (13%) fibrosis Bronchiectasis 6 (9%) Others 6 (9%) Underlying Diabetes mellitus 16 (24%) medical Malignancy 5 (7%) disease Chronic liver disease 3 (4%) Chronic kidney disease 2 (3%) Others 5 (7%) Drug susceptibility test Radiologic severity Drug-susceptible TB 44 (65%) Multidrug-resistant TB 15 (22%) Other drug-resistant TB 7 (10%) Not available 2 (3%) Minimal 19 (28%) Moderately advanced 39 (57%) Far advanced 10 (15%) Data are presented as the median (interquartile range) or number (%). Table 2 AFB smear and culture findings of specimens with cultures positive for NTM (n Z 113). Quantity reported Number (%) AFB smear results AFB culture results 31%), Mycobacterium fortuitum (n Z 17, 15%), Mycobacterium avium complex (n Z 9, 8%), and Mycobacterium gordonae (n Z 9, 8%) (Table 3). Among the 68 patients, the frequently isolated species were, in decreasing order, M. fortuitum (n Z 14, 21%), M. abscessus (n Z 13, 19%), M. avium complex (n Z 8, 12%), and M. gordonae (n Z 8, 12%), Mycobacterium terrae complex (n Z 3, 4%), and Mycobacterium chelonae (n Z 2, 3%). Follow-up culture findings No AFB seen 53 (47%) Doubtful 20 (18%) Rare (1þ) 17 (15%) Few (2þ) 11 (10%) Moderate (3þ) 8 (7%) Numerous (4þ) 4 (3%) Fewer than 50 colonies 94 (83%) 50e100 colonies 12 (11%) 100e200 colonies 2 (2%) 200e500 colonies 4 (3%) (almost confluence) >500 colonies (confluence) 1 (1%) Of the 68 patients, 65 had available follow-up specimens for AFB culture after the first positive NTM culture results. Of these 65 patients, 56 converted to negative cultures for NTM on follow-up sputum cultures during anti-tb treatment. Nine (13%) patients had repeated positive culture results for NTM after the completion of anti-tb treatment. The median duration of follow-up after-tb treatment was 23.9 months (IQR, 15.2e32.0 months) in these nine Table 3 Frequency of NTM isolates. Organisms Isolates (n Z 114) Patients (n Z 68) a Mycobacterium abscessus 35 (31%) 13 (19%) Mycobacterium fortuitum 17 (15%) 14 (21%) Mycobacterium avium complex 9 (8%) 8 (12%) Mycobacterium intracellulare 3 3 M. avium 6 5 Mycobacterium gordonae 9 (8%) 8 (12%) Mycobacterium terrae complex 3 (3%) 3 (4%) Mycobacterium chelonae 3 (3%) 2 (3%) Mycobacterium septicum 2 (2%) 2 (3%) Mycobacterium szulgai 1 (1%) 1 (1.5%) Mycobacterium aubagmense 1 (1%) 1 (1.5%) Mycobacterium nonchromogenicum 1 (1%) 1 (1.5%) Unclassified 10 (9%) 10 (15%) Contamination or loss of isolates 23 (20%) 22 (32%) a More than one NTM species were isolated from some patient, so the sum of frequency was over 68.

NTM isolated during treatment of pulmonary TB 1939 patients. Four patients showed negative NTM culture on the last sputum culture, and five patients had positive NTM culture on the last sputum culture. However, only two (3%) patients who had two or more positive culture results after anti-tb treatment grew the same NTM species with those which were isolated during anti-tb treatment. The NTM isolated from these two patients who had persistent positive cultures for NTM during and after anti- TB treatment was M. abscessus. Despite anti-tb treatment for culture-confirmed pulmonary TB, these two patients had respiratory symptoms and compatible radiographic findings (i.e. cavities or multiple bronchiolitis associated with bronchiectasis). M. abscessus was isolated more than five times from these two patients, during and after anti-tb treatment. Therefore, they could be diagnosed with M. abscessus lung disease. However, they did not have demonstrable radiographic progression. So, they were on the regular follow-up without antibiotic treatment for M. abscessus lung disease. The duration of follow-up after anti-tb treatment in these two patients was 32 and 51 months, respectively. Discussion NTM are ubiquitous environmental organisms. Patients with pre-existing lung damage are susceptible to NTM. Therefore, chronic obstructive pulmonary disease, pneumoconiosis, previous pulmonary TB, and bronchiectasis are all associated with NTM lung infection. 1,2 According to the guidelines, the diagnosis of NTM lung disease requires the exclusion of other disorders such as TB and fungal infection. 1,2 Therefore, the significance of NTM isolated from a patient with pulmonary TB or pulmonary fungal disease is uncertain. 2 Recently, NTM infection was reported to be related to Aspergillus-related disease including aspergilloma and chronic necrotizing pulmonary aspergillosis. 19,20 However, the frequency and clinical significance of NTM isolated from patients during treatment for pulmonary TB has not been well studied. Until now, only 10 cases of concurrent M. tuberculosis and NTM infections have been reported in Japan. 10e14 To our knowledge, this is the largest case series describing TB patients with cultures positive for NTM during their anti-tb treatment. We found that 7% of the patients with culture-confirmed pulmonary TB had positive cultures for NTM during anti-tb treatment. About two-thirds (44/68, 65%) of our patients had underlying lung disease other than the diagnosed TB, and 49 (72%) of the patients had moderately advanced or far-advanced pulmonary TB. Therefore, the isolation of NTM from patients with pulmonary TB was not uncommon during anti-tb treatment, at least in our study population. Given that NTM are ubiquitous in nature, positive cultures from human airways may indicate infection, colonization, or specimen contamination. 21 In our study, 71% (48/68) of the patients had a single positive culture for NTM during anti-tb treatment, and only 13% (9/68) had repeated positive cultures for NTM after completing anti-tb treatment. Moreover, only two (3%) patients had two or more positive culture results with the same NTM species after anti-tb treatment. AFB staining of the 113 specimens with cultures positive for NTM revealed that a large number (n Z 73, 65%) of them were smear-negative. In addition, the culture results showed that a large majority of the specimen (n Z 94, 83%) had fewer than 50 colonies. The clinical relevance of this finding is not clear, but it indicates that even when a specimen was positive for NTM, the bacterial burden was low. Interestingly, the most commonly isolated organism in our study, on a per-patient basis, was M. fortuitum, which is a low-virulence NTM. 16 In 18% (12/68) of the patients, NTM species that are usually contaminants, such as M. gordonae and M. terrae complex were isolated. 22 All of these findings suggest that an NTM isolated from a patient during therapy for pulmonary TB is usually the result of colonization (i.e., recovery of organisms more than once without demonstration of specific pulmonary disease), a transient infection (i.e., transient isolation of organisms with subsequent negative cultures and no evidence of disease progression), or specimen contamination, rather than clinically relevant pulmonary infection. Therefore, simple follow-up of sputum culture may be enough at 1- to 2-month intervals until the end of anti-tb treatment in most patients. In conclusion, the isolation of NTM from patients with pulmonary TB is not uncommon during anti-tb treatment. However, this usually represents colonization, a transient infection, or specimen contamination. The co-existence of pulmonary TB and NTM lung disease may be rare, but should be considered in patients with relatively virulent NTM species such as M. abscessus. Conflict of interest statement All authors have no financial or other potential conflicts of interest to disclose. Acknowledgements This work was supported by the Samsung Biomedical Research Institute grant (# SBRI C-A8-203-1). References 1. Wallace Jr RJ, Cook JL, Glassroth J, Griffith DE, Olivier KN, Gordin F. American Thoracic Society statement: diagnosis and treatment of disease caused by nontuberculous mycobacteria. Am J Respir Crit Care Med 1997;156:S1e25. 2. Griffith DE, Aksamit T, Brown-Elliott BA, et al. An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med 2007;175:367e416. 3. Piersimoni C, Scarparo C. Pulmonary infections associated with non-tuberculous mycobacteria in immunocompetent patients. Lancet Infect Dis 2008;8:323e34. 4. Martin-Casabona N, Bahrmand AR, Bennedsen J, et al. Nontuberculous mycobacteria: patterns of isolation. A multicountry retrospective survey. Int J Tuberc Lung Dis 2004;8: 1186e93. 5. Wickremasinghe M, Ozerovitch LJ, Davies G, et al. Nontuberculous mycobacteria in patients with bronchiectasis. Thorax 2005;60:1045e51.

1940 H.-J. Jun et al. 6. Koh WJ, Kwon OJ, Jeon K, et al. Clinical significance of nontuberculous mycobacteria isolated from respiratory specimens in Korea. Chest 2006;129:341e8. 7. Fowler SJ, French J, Screaton NJ, et al. Nontuberculous mycobacteria in bronchiectasis: prevalence and patient characteristics. Eur Respir J 2006;28:1204e10. 8. Dailloux M, Abalain ML, Laurain C, et al. Respiratory infections associated with nontuberculous mycobacteria in non-hiv patients. Eur Respir J 2006;28:1211e5. 9. Field SK, Cowie RL. Lung disease due to the more common nontuberculous mycobacteria. Chest 2006;129:1653e72. 10. Nakagawa Y, Ebihara M, Shimazu K. A case of mixed pulmonary infection with Mycobacterium tuberculosis and M. intracellulare. Kokyu 1997;16:1456e7. 11. Kobashi Y, Okimoto N, Hara Y, Matsushima T. A case of mixed pulmonary infection with Mycobacterium tuberculosis and M. intracellulare due to familial prevalence. Nihon Kokyuki Gakkai Zasshi 2002;40:35e9. 12. Yanagisawa T, Sugita H, Matsushima H, et al. Investigation of the cases that Mycobacterium tuberculosis and nontuberculous mycobacteriosis were detected simultaneously. Kekkaku 2002; 77:324. 13. Fujii S, Terashita K. Three cases of mixed infection with Mycobacterium tuberculosis and nontuberculous mycobacteriosis. Kekkaku 2003;78:412. 14. Takeda M, Ito W, Kobayashi N, et al. Co-existence of Mycobacterium tuberculosis and Mycobacterium intracellulare in one sputum sample. Intern Med 2008;47:1057e60. 15. American Thoracic Society. Diagnostic standards and classification of tuberculosis in adults and children. Am J Respir Crit Care Med 2000;161:1376e95. 16. Park S, Suh GY, Chung MP, et al. Clinical significance of Mycobacterium fortuitum isolated from respiratory specimens. Respir Med 2008;102:437e42. 17. Kim S, Park EM, Kwon OJ, et al. Direct application of the PCR restriction analysis method for identifying NTM species in AFB smear-positive respiratory specimens. Int J Tuberc Lung Dis 2008;12:1344e6. 18. Falk A, O Connor JB, Pratt PC, Webb WR, Wier JA, Wolinsky E. Classification of pulmonary tuberculosis. In: Diagnostic standards and classification of tuberculosis. 12th ed. New York: National Tuberculosis and Respiratory Disease Association; 1969. 19. Kobashi Y, Fukuda M, Yoshida K, Miyashita N, Niki Y, Oka M. Chronic necrotizing pulmonary aspergillosis as a complication of pulmonary Mycobacterium avium complex disease. Respirology 2006;11:809e13. 20. Kunst H, Wickremasinghe M, Wells A, Wilson R. Nontuberculous mycobacterial disease and Aspergillus-related lung disease in bronchiectasis. Eur Respir J 2006;28:352e7. 21. Catanzaro A. Diagnosis, differentiating colonization, infection, and disease. Clin Chest Med 2002;23:599e601. 22. Eckburg PB, Buadu EO, Stark P, Sarinas PS, Chitkara RK, Kuschner WG. Clinical and chest radiographic findings among persons with sputum culture positive for Mycobacterium gordonae: a review of 19 cases. Chest 2000;117:96e102.