This study demonstrates that, among all myco-bacteriological culture-positive pulmonary disease cases, 6.4% were NTM, based on nationwide surveillance of drug-resistant tuberculosis. Pulmonary NTM infection was more frequent in southern China, particularly southern coastal areas with high humidity. The most prevalent SGM was the MAC, which comprised seven subspecies, among which, M.intracellulare was predominant and distributed widely across northern and southern China. The most prevalent RGM was the MABC, comprising three subspecies, with M.abscessus the predominant subspecies, and mainly distributed in southern China. The results of DST indicated that the drug-resistance spectrum varied greatly across different strains and subspecies. NTM showed relatively low resistance rates to macrolides and amikacin in vitro.
Distinguishing NTM from MTBC infection is of great clinical significance, as it can direct accurate and rapid clinical treatment [14-16]. The screening method used for NTM species is based on p-Nitrobenzoic acid, a time-consuming and difficult method, which most reports from China indicate can inhibit M. tuberculosis complex growth . The laboratory diagnosis methods used for identification of mycobacterial species have evolved over the decades . With the development of several extraction methods that enhance the amount of bacterial proteins available for MALDI-TOF MS identification, and the increasing amount of mycobacteria data available in commercial databases, MALDI-TOF MS technology has been implemented for NTM identification in many laboratories [8, 19]. Several studies have demonstrated that this method can achieve more than 95% agreement with results from DNA sequencing of variable genomic regions (including the 16S rRNA, hsp65, rpoB, and ITS genes) [5, 20]. In our research, we obtained a 98.4% NTM detection rate and achieved 93.4% agreement with 16S rRNA, hsp65, ITS, and rpoB gene sequencing. Although we could not identify NTM strains that were not contained in the Bruker MBT strains database, MALDI-TOF MS was able to identify most clinically relevant NTM in a rapid, reliable, and inexpensive manner.
The overall NTM pulmonary infection rate was approximately 6% in our study, similar to that reported in a systematic review and meta-analysis of NTM infections, which demonstrated that the prevalence of NTM infections among patients with suspected tuberculosis was 6.3% in mainland China . The geographic variability in both the prevalence of NTM infections and mycobacterial species composition was clearly demonstrated by our study. A previous investigation in southern-central China demonstrated that the NTM infection rate is 4.0%, with the two most prevalent species the M.avium-intracellulare and Mycobacterium chelonae-abscessus complexes , while a report from Shanghai found an overall rate of NTM isolation from mycobacterial culture-positive patients of 5.9%, with M.kansasii the most frequently identified species, with an increasing trend from 3.0% in 2008 to 8.5% in 2012 . In our study, the most frequent species in Shanghai province was also M.kansasii (7/11 isolates), with a further increase in the NTM prevalence rate to 11% in 2013. In another study in Guangdong and Shanghai provinces, M.intracellulare was the most commonly isolated NTM in Shanghai, while M.abscessus was the most frequently isolated species in Guangzhou . Some reports from eastern and northern regions of China have demonstrated NTM prevalence rates of around 2.0%–3.0%, with M.intracellulare the predominant species, followed by M. abscessus [5, 21-23]. In our study, NTM infection was more prevalent in southern than northern China and more frequent in eastern than western China. The most epidemic NTM species were MAC, which was widely distributed, and MABC which is mainly distributed in southeastern China. In addition, we isolated 16 M.marseillense strains of the MAC from sputum samples. Pulmonary disease caused by M. marseillense warrants increased attention, as it is infrequently reported [24, 25].
In addition to Mycobacteria spp., we also identified some acid-fast-staining-positive non-mycobacteria. As shown in Figure 1, we randomly selected 60 non-mycobacteria from 286 contaminated or other species for species identification, including: 15 Gordonia, 2 Nocardia, 2 Streptococcus, and 1 Tsukamurella (data not shown). The 15 Gordonia (comprising 8 G. sputi, 4 G. bronchialis, and 3 G. rubripertincta) isolates were distributed across nine provinces. Interestingly, the presence of Gordonia is consistent with a previous report from China . Two Nocardia species, which often cause chronic lung disease, were isolated, as previously reported in China . In addition, a case of Tsukamurella has previously been reported in Jiangxi province, Southern-central China . In addition to NTM and MTBC infection, Gordonia and Nocardia species should also be tested for when using acid-fast staining to diagnosis pulmonary infection.
We evaluated the susceptibility of RGM and SGM from China to antimicrobials by measuring MIC values using the RAPIDMYCOI and SLOWMYCOI Sensititre™ panels, according to CLSI protocol M24-A2. No such simple commercial kits for MIC measurement are available in China, despite the increase in patients with NTM infections, and information on drug susceptibility of NTM isolates is lacking. We mainly analyzed the susceptibility of MABC isolates, which were the most common clinical RGM isolates. Inducible macrolide resistance leads to differences in treatment outcome between patients with M. abscessus and M.massiliense infections. Consistent with previous reports [26-28], we found that M.abscessus had a higher inducible resistance (65.67% vs 2.22%, p < 0.01) and acquired resistance (17.91% vs 8.89%, p = 0.2841) rates for clarithromycin than M.massiliense. These results further emphasize the importance of M.abscessus and M.massiliense subspecies identification, to inform appropriate clinical treatment using different strategies. Amikacin was the most active antimicrobial agent against MABC species, showing a 94.74% overall susceptibility rate, similar to the overall susceptible rate observed in previous studies from China and Australia [29, 30]; however, higher resistance rates, from 28.2% to 76.0%, have been observed in Japan and South Korea [15, 31]. After amikacin, cefoxitin was the second most effective antimicrobial agent against MABC, with a 16.67% resistance rate, unlike results from South Korea , where the second most effective antimicrobial agent was linezolid, but consistent with findings from Japan . The resistance rate to cefoxitin was higher in M.abscessus (19.40%) than M.massiliense (11.11%). Linezolid, with a resistance rate of 33.33%, could be used as an alternative therapy choice against RGM isolates. Given the high resistance rates to the other drugs tested in our study, they may not be appropriate for treatment of MABC infections; however, studies of clinical therapeutic effects are required.
For SGM, we mainly analyzed the susceptibility of the MAC and M.kansasii, the two most frequent SGM species. Consistent with previous studies , macrolides and amikacin, as the first-line therapeutic agent for lung diseases caused by MAC infection, showed excellent in vitro activity against MAC isolates, with 90% susceptibility. Patients with MAC pulmonary diseases are frequently administered a combination of clarithromycin, ethambutol, and rifampicin; however, one study suggested that treatment with clarithromycin and ethambutol is not inferior to treatment with clarithromycin, ethambutol, and rifampicin for MAC lung disease . Our data support the two treatment regimen, since the resistance rates to ethambutol and rifampicin in vitro were 58.33% and 91.67%, respectively. Some researchers have reported differential drug susceptibility patterns of M.chimaera and other members of the MAC . In our study, we only obtained one M.chimaera strain. We compared the drug susceptibility patterns of the two most frequent species of MAC, and found no significant difference between M. intracellulare and M.marseillense. As M.marseillense infections are rare in humans [24, 25, 35], our drug susceptibility data add to knowledge of this species.
M. kansasii, which was the second most frequent SGM infection species identified in this study, showed a higher susceptibility rate to the majority of first- and second-line antibiotics recommended by CLSI (M24, 3rd Edition). These drug susceptibility patterns were markedly different from those reported by a previous study, which included DST of a total of 78 M. kansasii strains from 13 provinces of China . Except for ethambutol (83.87% vs 20.5%), the resistance rates in our study were lower than those reported by the previous study , as follows: clarithromycin (0 vs 20.5%), amikacin (0 vs 5.1%), rifampicin (6.45% vs 56.4%), rifabutin (3.23%% vs 34.6%), moxifloxacin (0 vs 16.7%), and linezolid (3.23%% vs 32.1%). Our results are similar to those of a study using 85 M. kansasii isolates from eight countries in Europe and Asia . In this study, all 85 M. kansasii isolates were susceptible to rifampicin, amikacin, rifabutin, moxifloxacin, and linezolid. Although all 31 M. kansasii isolates included in our study were from 13 provinces in China, more isolates should be tested to evaluate drug susceptibility patterns of M. kansasii, given the relatively small number of strains and regional disparities.
No NTM were identified from some provinces in the northwestern region, likely due to the small sample size. Also, there were a limited number of strains of each species. We have prepared to collect more samples from these regions to complete our analysis of NTM infection and drug resistance status in China, and plan to evaluate NTM infection using the isolates collected during nationwide surveillance of tuberculosis.