Clinical Significance of Nontuberculous Mycobacteria Isolated From Respiratory Specimens in a Chinese Tuberculosis Tertiary Care Center

The clinical relevance of non-tuberculous mycobacteria (NTM) has been reported to be different dramatically by species or by regions, however, no such evaluation has been performed in China.A retrospective study was performed in Beijing Chest Hospital. All the NTM strains isolated from respiratory specimens in the past 5 years, and patients’ clinical records (symptoms and radiographic information etc.) were investigated. The clinical relevance was evaluated according to the criteria recommended by the American Thoracic society. Totally 232 NTM strains were recruited, among them, M. intracellulare was the dominant species (40.5%), followed by M. abscessus (28.4%). 109 patients, with 185 total isolates, had full clinical records available for review. 84.4% (38/45), 85.7% (24/28%) and 63.6% (7/11) of patients with isolation of M. intracellulare, M. abscessus and M. kansasii, respectively, were categorized as definite NTM disease. Whereas all the 10 patients with isolation of M. gordonae were defined as unlikely NTM disease. The majority of NTMs isolates yielded from respiratory specimens in Beijing Chest Hospital were clinically significant, and M. intracellulare and M. abscessus was the dominated species of NTM lung disease. NTM lung infections demonstrated some specific chest radiograph characteristics.

In recent years, the frequency of nontuberculous mycobacteria (NTM) isolation from respiratory specimens, as well as the number of NTM infection patient, has increased rapidly in many countries [1][2][3][4][5] . Moreover, marked geographic variability was observed regarding both the prevalence of NTM lung disease and the mycobacterial species responsible for it 6 . Patients with NTM disease are often indistinguishable clinically or radiographically from patients infected with Mycobacterium tuberculosis [7][8][9][10] . However, the strategy of treatment and the prognosis of NTM disease can be significantly different from TB, resulting in different implications for public health.
In China, tuberculosis remains a serious public health problem and as a consequence, patients with positive outcomes of acid-fast bacilli (AFB) smear test or with isolation of mycobacteria by culture for sputum specimens have been routinely diagnosed as pulmonary tuberculosis and been administrated with anti-tuberculosis drugs 11 . The extent of misdiagnosis and inappropriate treatment of disease due to NTM infection in China lacks comprehensive understanding. On the other hand, NTM isolation does not affirmatively mean disease, so that the health providers have to stay alert for possible contamination or NTM colonization all the time. Thus the isolation of NTM species may pose a diagnostic difficulty for clinicians.
We performed an institution-based observational study for NTM lung disease in Beijing Chest Hospital (Beijing, China), and investigated the clinical, microbiological and radiographic information of possible NTM Scientific RepoRts | 6:36299 | DOI: 10.1038/srep36299 infection patients from 2010 to 2015. The purpose of the current study was to determine the isolation frequency of different NTM species from respiratory specimen over a 5-year period, and to evaluate their clinical significance.

Materials and Methods
Study subjects. All NTM strains isolated from respiratory specimens in Beijing Chest Hospital over a 5-year period from May 2010 to May 2015 were retrospectively analyzed. The medical records of patients from whom NTM strains isolated were retrospectively reviewed. Informed consent was obtained from all subjects included in the study. The protocols and procedures for the protection of human subjects were approved by the Ethics Committee of Beijing Chest Hospital. Furthermore, all the methods were carried out in accordance with the approved guidelines.
Mycobacterium cultures. Sputum and bronchial washing specimens were decontaminated using the N-acetyl-L-cysteine 2% NaOH method. Löwenstein-Jensen medium culture or liquid culture using MGIT 960 system (BD Biosciences, Sparks, MD, USA), or both methods were performed to recover mycobacteria. Mycobacterial growth in PNB-containing medium was used as a presumptive test for NTM screening.
Identification of NTM species. NTM isolates were identified to the species level by sequence alignment of 16S rDNA, 16-23S rRNA gene internal transcribed spacer (ITS), rpoB and hsp65 genes. Preparation of the PCR amplification and DNA sequencing were done as described previously 12 .       disease during antibiotic treatment. This strain was excluded from further analysis. The implicated NTM organisms are shown in Table 1.

Clinical and radiographic characteristics of patients with definite lung disease. Characteristics
of the patients in current study are summarized in Table 2. There were no significant differences in the sex, mean age, body mass index (BMI), presence of underlying disease and symptom among the three groups. Sputum AFB

Discussion
NTM have been implicated in an increasingly large proportion of pulmonary disease throughout the world, in both immunocompetent and immunocompromised hosts [16][17][18][19] . In many countries, MAC (including M. intracellulare and M. avium) are the most common NTM isolates. On the other hand, M. avium is predominant in North-and South America, M. intracellulare is most frequently isolated in Australia-Queensland, South Africa and some provinces in China 6,20-22 . Our study showed that M. intracellulare was the most common species of NTM isolation, followed by M. abscessus in Beijing Chest Hospital which is located in north of China.
It is believed that NTM disease is under-reported in the tuberculosis-endemic countries. The main reason could be the high burden of tuberculosis which attracts bulk of the attention of clinicians. In our knowledge, this is the first research to document the clinical significance of NTM isolates from respiratory specimens in China, a high TB burden country. The major findings of this study are as following: (1) NTM strains isolated from respiratory specimens demonstrated very good relation with lung infections (90.9%). (2) M. intracellulare was the most common NTM species (40.5%) isolated from clinical respiratory specimens, and it was also the most common pathogen (52.8%) of NTM lung disease in our institution. Although in some areas M. intracellulare is less likely to cause NTM lung disease 23 , our data demonstrated that M. intracellulare is the predominant bacteria for NTM lung disease in Beijing Chest Hospital. (3) M. abscessus was the second most commonly isolated species (28.4%) and represented the second most common pathogen (33.3%). Since M. intracellulare and M. abscessus lung diseases had the most poor prognosis among all the NTM lung diseases, according to our outcomes, the NTM lung disease treatment will be very challenging in China as those two species account for the majority of NTM lung diseases 13,24 .
Although the incidence of pulmonary disease caused by M. kansasii is the second most common (approximately 20%) type of NTM pulmonary disease in Japan and USA 25,26 , it is uncommon in our institution. However, in Shanghai, another industrialized city in the south of China, M. kansasii is the most frequently identified isolate, accounting for 45% of all NTM isolates 20 . It seems that geographic distribution of NTM species is significantly different in a large country like China.
Among the definite, probable and unlikely NTM lung disease, there were no significant differences in the sex, mean age, BMI, presence of underlying disease and symptoms, but fewer patients with unlikely NTM lung disease were sputum AFB smear positive. It is possible that unlikely NTM lung diseases have less bacterial load, however with a more sensitive method, such as liquid culture, the chances to recover it can increase. 8 out of the 10 unlikely NTM infection cases had isolations recovered by MGIT960 only, which was consistent with this assumption. At a hospital in USA, the dramatic increase of NTM isolation was attributed to the more sensitive liquid culture technique, which led to less clinical significance of NTM isolations 27 . All the 10 unlikely NTM lung disease cases had the isolations of M. gordonae in this study, which warns clinicians to interpret NTM recovery by MGIT960 systems with cautions, especially for M. gordonae.
Compared with reports from other countries 14,15,23 , nearly 66.1% of the patients with NTM isolations were diagnosed as definite NTM disease in this assay, which is much higher than those of most other studies. Furthermore, we speculated that some more patients with possible NTM lung disease due to less bacterial evidence would fulfill the definite NTM lung disease criteria during the follow-up process. We attributed the tight relationship of NTM isolation with lung infection mainly to more severe cases enrolled in the study. Our institution is the top tuberculosis referral center in China and has admitted various patients with serious symptoms transferred from other hospitals. In addition, another contributing factor may be that clinicians in our hospital were very knowledgeable of NTM disease, hence they regularly prescribed repeated mycobacterium culture for suspected NTM disease patients. The only species we did not find good clinical significance was M. fortuitum. A study in Japan also found that among 26 patients with a minimum of two M. fortuitum isolations, none showed clinical aggravation during the follow-up period 28 . It is likely that colonization or contamination during culture happens frequently with this species.
NTM lung infection may have some specific image characteristics for a CT scan. Among patients with definite NTM lung disease, fewer cases have unclassifiable form for radiographic features compared with patients with probable and unlikely NTM disease. Based on our research, cases without typical nodular bronchiectatic and upper lobe cavitary forms are less likely to be related to NTM lung disease. The position of nodular bronchiectatic lesion demonstrated species-specific characteristics. M. intracellulare lung diseases were more likely to be implicated in the lingular segment and the right middle lobe in contrast with M. abscessus lung diseases.

Conclusions
In conclusion, a substantial proportion (90.9%) of patients, from whom NTM isolates were recovered, exhibited definite or probable NTM lung disease in our institution. The most common etiologies of NTM lung disease included M. intracellulare and M. abscessus. NTM lung infections frequently demonstrated the upper lobe cavitary form or nodular bronchiectatic form for chest radiograph examination. However, as NTM only accounted for 2.6% of all the mycobacterial isolations in our hospital according to previous work 29 , the clinical significance in other areas of China with high percentages of NTM isolation and different species constitutions might be different with our conclusions, but more investigations need be done to prove this assumption.