Clinical Characteristics and Antimicrobial Susceptibility of Mycobacterium intracellulare and Mycobacterium abscessus Pulmonary Diseases: A Retrospective Study

The incidence of nontuberculous mycobacteria (NTM) diseases is increasing every year. The present study was performed to investigate the clinical characteristics, CT findings, and drug susceptibility test (DST) results of patients diagnosed with M. intracellulare or M. abscessus nontuberculous mycobacterial pulmonary disease (NTMPD). This retrospective study included patients diagnosed with NTMPD due to M. intracellulare or M. abscessus for the first time at Anhui Chest Hospital between 01/2019 and 12/2021. The patients were grouped as M. intracellulare-NTMPD group or M. abscessus-NTMPD group. Clinical features, imaging data and DST data, were collected. Patients with M. intracellulare infection had a higher rate of acid-fast smears (66.1% vs. 45.2%, P=0.032) and a higher rate of cavitation based on pulmonary imaging (49.6% vs. 19.4%, P=0.002) than patients with M. abscessus infection, but both groups had negative TB-RNA and GeneXpert results, with no other characteristics significant differences. The results of DST showed that M. intracellulare had high susceptibility rate to moxifloxacin (95.9%), amikacin (90.1%), clarithromycin (91.7%), and rifabutin (90.1%). M. abscessus had the highest susceptibility rate to amikacin (71.0%) and clarithromycin (71.0%). The clinical features of M. intracellulare pneumopathy and M. abscessus pneumopathy are highly similar. It may be easily misdiagnosed, and therefore, early strain identification is necessary. M. intracellulare has a high susceptibility rate to moxifloxacin, amikacin, clarithromycin, and rifabutin, while M. abscessus has the highest susceptibility rate to amikacin and clarithromycin. This study provides an important clinical basis for improving the management of NTMPD.


Introduction
e incidence of nontuberculous mycobacteria (NTM) diseases is increasing every year, and it has become a public health concern due to the difficulty of diagnosis, the long course and high cost of treatment, and resistance to most antimicrobial drugs [1][2][3][4]. Over 90 species of NTM have been identified in humans [5]. NTM can be found in drinking water systems [6], domestic plumbing, showerheads, and potting soil [7][8][9]. e estimated NTM prevalence increased from 2.4 per 100,000 persons in 1980 to 15.2 per 100,000 persons in 2013 in the United States of America [10]. Similar figures are observed in Canada [11], the United Kingdom [12], Denmark [13], and Germany [14]. e results of three consecutive epidemiological sampling surveys in China in 1990China in , 2000China in , and 2010 showed that the isolation rate of NTM was gradually increasing with 4.9%, 11.1%, and 22.9%, respectively [15]. NTM disease is clinically similar to tuberculosis and therefore can be misdiagnosed as such in the absence of microbiological identification [3]. Besides, most NTM are resistant to antimycobacterial drugs, resulting in poor efficacy of such drugs [16][17][18][19][20].
Among all types of NTM disease, nontuberculous mycobacterial pulmonary disease (NTMPD) is the most common one [21,22]. In Western countries, the prevalence of NTMPD is higher than the prevalence of tuberculosis [23]. In the United States of America, Mycobacterium kasassii is the second most common cause of pulmonary infections, and those bacteria are also responsible for infections in the United Kingdom [2,24]. Mycobacterium intracellulare is most isolated in China, with an isolation rate up to 40%-60% in Northern China [25,26]. In Southern China, Mycobacterium abscessus is also highly isolated in addition to Mycobacterium intracellulare [25,26]. Still, a comprehensive analysis of the clinical characteristics, computed tomography (CT) findings, and drug susceptibility testing (DST) results of M. intracellulare and M. abscessus has been rarely reported in pulmonary disease. erefore, this study aimed to investigate the clinical characteristics, CT findings, and DST results of NTMPD patients diagnosed with M. intracellulare or M. abscessus infection at their first visit. e results of this study are expected to enrich the epidemiological data of NTM diseases and facilitate the early diagnosis and treatment of NTMPD.

Study Design and Patients.
is retrospective study included patients with NTMPD who were first diagnosed with M. intracellulare infection or M. abscessus infection at Anhui Chest Hospital between January 2019 and December 2021. e study was approved by the ethics committee of Anhui Chest Hospital (approval number: K2021-007). Informed consent was obtained from the patients.
NTM strains were isolated from 772 inpatients during the three years; then, a total of 633 patients with M. intracellulare or M. abscessus isolated were initially included in the study for further research, and eventually 152 patients were analysed, of whom 121 had M. intracellulare pulmonary disease, and 31 had M. abscessus pulmonary disease.
All included patients had complete clinical data, appropriate laboratory findings, and imaging findings. Exclusion criteria were (1) NTM infection but no NTM disease, including tuberculosis [16], tumors, COPD, and chronic lung diseases such as bronchial and other diseases; (2) loss of follow-up after no definite diagnosis during hospitalization; . Strains with positive results by the colloidal gold method in the PNB identification medium (Zhuhai Baso Biotechnology Co., Ltd.) were initially identified using the MPB64 antigen detection kit (Hangzhou Genesis Biodetection & Biocontrol Co., Ltd.). If the bacteria grew in the PNB medium or the MPB64 antigen test showed negative, the strain was tentatively identified as NTM (Figure 3). e strain preliminarily identified as NTM was then identified to species using the MeltPro Myco assay, which targets the intergenic transcribed spacer (ITS) region between the 16S rRNA and 23S rRNA genes of mycobacteria using a panmycobacterial primer set (Zeesan Biotech, Xiamen, China) [28] and the Bruker MALDI-TOF MS identifier [29,30]. Six technicians with 6 to 15 years of experience, including two deputy chief technicians and four chief technicians, performed the NTM culture and species identification in this study.

Data Collection.
All data were collected from the electronic medical record system (Winning Health CIS 5.5.0.10    antituberculosis treatment, clinical symptoms, laboratory findings, imaging findings (lesion sites and morphological features), and DST results.

Statistical
Analysis. SPSS 25.0 (IBM, Armonk, NY, USA) was used for statistical analysis. Continuous data that conformed to a normal distribution (according to the Shapiro-Wilk test) are expressed using means ± standard deviations and were analysed using Student's t-test. Data that did not conform to a normal distribution were expressed as "medians (ranges)" and were analysed using the Mann-Whitney U-test. Categorical data were expressed as "n (%)" and were analysed using the chi-square test or Fisher's exact test. A two-sided P value <0.05 was considered statistically significant. ere were no significant differences between the two groups in terms of age, sex, comorbidities, clinical symptoms, previous antituberculosis treatment, mean duration of antituberculosis treatment, and laboratory findings (all P > 0.05) ( Table 1).

Pulmonary Imaging Features.
e comparison of the imaging findings between the two groups of patients is shown in Table 2. Patients with M. intracellulare lung disease had a higher rate of cavitation than patients with M. abscessus lung disease according to pulmonary imaging findings (49.6% vs. 19.4%, P � 0.002). ere were no significant differences between the two groups in terms of lesion location, imaging findings of the lesions, mediastinal lymph node enlargement or calcification, emphysema, alveoli, lung injury, and bronchodilation (all P > 0.05).

Discussion
e incidence of NTM disease is increasing year by year, but analysis of the clinical features, CT findings, and DST results of Chinese patients has rarely been reported. e results of this study indicate that the clinical features of M. intracellulare pneumopathy and M. abscessus pneumopathy are highly similar, necessitating the identification of molecular biology strains. DST results showed that M. intracellulare was highly sensitive to moxifloxacin, amikacin, clarithromycin, and rifabutin, while M. abscessus had the highest sensitivity to amikacin and clarithromycin.
Studies indicated that it takes a long time to diagnose NTMPD (7 to 8 years on average), and most patients were first misdiagnosed with pulmonary tuberculosis (92.8%) [36,37]. A study in Iran showed that among 714 positive   [38]. Furthermore, many NTMPD patients were infected by a wide range of atypical mycobacteria [39]. erefore, it is of great importance to differentiate NTM from Mycobacterium tuberculosis and to make reliable identification in clinical practice to reduce misdiagnosis and improve treatment efficiency. Before diagnosis, most patients had received repeated antituberculous drug treatment. Our research indicated that most patients were misdiagnosed before they were diagnosed with NTMPD as nearly 70% of the patients in both groups had a history of antituberculosis treatment, which lasted for 4 to 5 months on an average and 3 years on the longest, which was consistent with the previous study. e high rate of misdiagnosis of NTMPD, the difficulty in confirming the diagnosis, and the high cost of treatment when patients are misdiagnosed make it important to find diagnostic markers for NTMPD.
In the present study, out of 633 patients infected with M. intracellulare or M. abscessus, only 152 were diagnosed with NTM disease at the first visit, 38 were diagnosed with NTMPD at the second visit, 20 were diagnosed with mixed infection with Mycobacterium tuberculosis and NTM, 31 were not diagnosed and lost to contact, and the remaining 423 were excluded due to the absence of NTMPD. Such results suggest that NTM strains isolated in the laboratory only prove the possibility of NTM infection. Clinicians should still identify NTMPD based on diagnostic criteria.
Patients of both groups had a high positive rate of acidfast smears. ose with M. intracellulare pulmonary disease having a positive rate of 66.1%, and those with M. abscessus pulmonary disease showing 45.2%, which is in agreement with the findings of Riello et al. [40]. However, TB-RNA and GeneXpert results were negative in both groups, suggesting that if a patient has a positive acid-fast smear and a negative TB-RNA and GeneXpert result, then he/she may have NTM disease.
At imaging, lesions of NTMPD are various and diffuse, and double lung lesions are more common in NTMPD than in pulmonary tuberculosis [16,26,41,42]. In this study, patients with M. abscessus pulmonary disease had a higher rate of cavitation than patients with M. intracellulare pulmonary disease based on lung imaging findings (49.6% vs. 19.4%, P � 0.002).
ere were no significant differences between the two groups in lesion location, lesion severity, the characteristics of pulmonary shadow, pleural thickening or pleural effusion, and bronchiectasis, which was in line with the results of a previous study [26].
Most NTM are resistant to traditional antituberculosis drugs, and their resistance patterns vary greatly with  [43][44][45]. . e consensus of experts on diagnosis and treatment of nontuberculous mycobacterium disease [16] clearly points out that drug sensitivity test before treatment is still very important, and the consensus believes that the correlation between drug sensitivity test results and clinical effect is still difficult to determine, but it is still suggested to base the drug sensitivity results and medication history as far as possible when making chemotherapy regimen for NTM disease. M. intracellulare belongs to slowly growing mycobacteria (SGM) of nontuberculous mycobacteria. Expert consensus [16] and An Official ATS/ERS/ESCMID/IDSA Clinical Practice Guideline [27]  ). Except azithromycin, the drug sensitivity results in this study were in good agreement with literature reports. In this study, the sensitivity rate of azithromycin (18.2%) was low, which was significantly different from that reported in the literature. e results for clarithromycin predict those for azithromycin, for which testing is problematic as a result of poor solubility at the high concentrations of drug that must be used [32]. e susceptibility rate of clinical Mycobacterium intracellulare isolates to moxifloxacin was 95.9%. Wang et al. [46] reported that fluoroquinolone moxifloxacin also had good antibacterial activity against Mycobacterium intracellulare in vitro. is study was consistent with literature reports, but moxifloxacin was not among the consensus-recommended drugs. M. abscessus belongs to rapidly growing mycobacteria (RGM) of nontuberculous mycobacteria. M. abscessus occupies the largest proportion of RGM that is naturally resistant to and possess acquired resistance to most commonly used antibiotics, commonly treated with antituberculous drugs; also, M. abscessus is known as a nightmare bacterium. Expert consensus [16] and An Official ATS/ERS/ESCMID/ IDSA Clinical Practice Guideline [27] suggested that the treatment of M. cheloniae abscess includes clarithromycin, azithromycin, amikacin, and cefoxitin. e drug sensitivity test results of this study showed that the drug sensitivity rate of M. abscessus was clarithromycin (71.0%),azithromycin (32.3%), amikacin (71.0%), linezolid (51.6%), and cefoxitin (67.7%).
is study has limitations. First, it studied only two NTM and had a small sample size so that the results might be biased. Second, due to limited testing competence and methodology, the study did not investigate the resistancerelated genetics.

Conclusions
In conclusion, there are still many difficulties in diagnosing and treating NTMPD, and NTM disease can be easily misdiagnosed. Elderly people are more likely to have NTMPD, and delayed diagnosis of NTMPD and improper antituberculosis treatment are common in clinical practice due to highly similar clinical symptoms and signs, laboratory examinations, and imaging. M. intracellulare has a high susceptibility rate to moxifloxacin, amikacin, clarithromycin, and rifabutin, while M. abscessus has the highest susceptibility rate to amikacin and clarithromycin.

Data Availability
All the data generated or analysed during this study are included in this published article.

Conflicts of Interest
e authors declare no conflicts of interest regarding the publication of this paper.

Authors' Contributions
Dongping Wang contributed to the design and drafted the manuscript. Wenhong Lin contributed to the conception and critically revised the manuscript. Hongyan Cheng contributed to acquisition of the data and analysis of the data. Xundi Bao contributed to analysis of the data. Dongfang Xu and Suo Liang contributed to interpretation of the data. Yue Jiang and Chao Wang contributed to acquisition of the data. All the authors have read and approved the final manuscript.