Non-tuberculous mycobacteria (NTM) do not cause tuberculosis or leprosy, but can cause pulmonary disease resembling tuberculosis, lymphadenitis, skin disease, or disseminated disease. The pulmonary infection caused by NTM is difficult to clinically differentiate from that caused by MTB. However, most of the NTM strains are not susceptible to anti-tuberculosis drugs. The treatment plan is related to the species, infection site and the severity of infection (13). Therefore, rapid and accurate identification of Mycobacterium to species level is very important for managing these infections. PCR-fluorescence probe method (Tuberculosis and Non-Tuberculous Mycobacteria Real-time PCR Detection Kit, CapitalBio Technology Inc., Beijing, China) can be used to detect both MTB or NTM in the specimen directly in three hours, and there is no significant difference in the detection performance of this method compared to traditional methods (culture and microscopy) (10). In addition, the DNA microarray chip method (Mycobacterial Species Identification Array Kit, CapitalBio Technology Inc., Beijing, China) can detect and identify 17 species or groups of clinically common Mycobacteria, including M. tuberculosis complex, M. intracellular, M. avium, M. gordonae, M. kansasii, M. fortuitum, M. scrofulaceum, M. gilvum, M. terrae, M. chelonae /M. abscessus, M. phlei, M. nonchromogenicum, M. marinum /M. ulcerans, M. aureus, M. senegalense / M. malmoense, M. xenopi and M. smegmati. Using this method, results are available within 6 hours, which is helpful for early diagnosis and treatment of tuberculosis and NTM disease.
An analysis of the current study in relation to previous findings showed that the absolute numbers of both NTM and MTB positive samples increased yearly from 2013 to 2018. Notably, the proportion of NTM positive samples among the total positive samples for MTB and/or NTM as per the PCR-fluorescence probe method (Tuberculosis and Non-Tuberculous Mycobacteria Real-time PCR Detection Kit, CapitalBio Technology Inc., Beijing, China), increased from 15.6% in 2013 to 46.1% in 2018. This is in agreement with the results of the national tuberculosis epidemiological sampling surveys carried out in China in 1990 (4.9%), 2000 (11.1%) and 2010 (22.9%) (14, 15).This finding is consistent with those from multiple studies in diverse countries, demonstrating an increasing proportion of NTM infections in recent years (16, 17). Many factors may contribute to the observed rise in NTM detection, including improved clinical awareness of NTM infections, use of better and more sensitive NTM detection techniques, increased number of immunocompromised patients, aging of the population, the threshold to test a sample, and alteration of sample types for NTM detection (18, 19). As shown in Figure 1, as the number of samples submitted for MTB or NTM identification increased, so did the positivity rate for NTM species whilst that of MTB decreased. Besides, as shown in Table 4, the number of samples tested for NTM from cerebrospinal fluid (CSF) and lymph node has increased several times over the last three years. In addition, the NTM positive rate in CSF or lymph node sample is just lower than in sputum samples but higher than in any other samples. The increase in the numbers of CSF and lymph node samples tested may explain the observed increase in the number of NTM positive samples. Among the most common NTM pathogens, the incidence of MAC grew faster than that of M. chelonae /M. abscessus, from 2013 to 2018 (Table 2), suggesting a more prominent role for MAC in NTM infections. Furthermore, the fast increase in the proportion of M. gordona and M. fortuitum in NTM infections should be noted.
The major clinical manifestation of NTM is pulmonary disease, with MAC the most common species involved in infection (20, 21). In this retrospective study, M. intracellulare (65/193, 33.7%) was the most commonly detected species in respiratory tract samples, followed by M. chelonae / M. abscessus (37/193, 19.2%), M. avium (27/193, 14.0%), M. gordonae (24/193, 12.4%). So in this study, MAC (92/193, 47.7%) was still the most common NTM pathogen in respiratory tract, which is consistent with previous findings in Europe, US, Canada, Australia, Japan, Korea, Southern China and so on (18-23). Although MAC is the most common pathogen in NTM pulmonary disease, its relative frequency varies widely among different geographical regions. Many factors such as climate type and population density can affect the distribution of NTM species (2).
In the present study, NTM infection was much more common in women (55.2%) than men (44.8%). Furthermore, the age range of infected people was relatively wide, being most common in the 45-65 year age group (49.6%), probably due to some issues related to the function of the immune system (24). This finding is in agreement with those of multiple studies in US (25), Japan (26, 27) and South Korea (23), which all indicated that older women were more susceptible to NTM infection. According to a previous study, abnormal expression of adipokines, sex hormones, and/or TGF-β may predispose slender, older women to NTM infection (28). However, contrasting findings have been reported in Europe, where patients with NTM pulmonary disease were more likely to be male, possibly owing to smoking history and increased incidence of chronic obstructive pulmonary disease (COPD) (16, 27).
DNA microarray chip method (Mycobacterial Species Identification Array Kit, CapitalBio Technology Inc., Beijing, China) can accurately distinguish between M. avium and M. intracellulae, which have quite similar phenotypes. We found out that the prevalence of M. intracellulae (31.8%) was always higher than M. avium (13.5%) from 2013 to 2018. It is important to distinguish between M. avium and M. intracellulae because they show different pathogenic characteristics. M. intracellulae is more virulent compared to M. avium, indicating the need for a more intensive therapeutic strategy (29). However, the DNA microarray chip method (Mycobacterial Species Identification Array Kit, CapitalBio Technology Inc., Beijing, China) could not distinguish between M. chelonae and M. abscessus, and between M. marinum and M. ulcerans. This is because M. chelonae and M. abscessus have the same 16S rRNA gene sequences, and so are M. marinum and M. ulcerans. However, M. chelonae tends to cause disseminated infections (13), and M. ulcerans produces a cytotoxin (mycolactone) with immune-modulating properties that causes necrosis (30). Therefore, accurate identification of these strains to species level still has an important clinical significance. Microbiology laboratories could further identify these strains by 16S-23S Internal Transcribed Spacer Region sequencing and Sequencer-Based Capillary Gel Electrophoresis (31). Moreover, Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR) markers can be used for typing M. intracellulae clinical isolates for molecular epidemiological studies (32). Other than pulmonary infectious diseases, NTM can also cause lymph node and skin and soft tissue infections. In this study, lymph node and pus were the second most common specimen types, only less than respiratory tract infections.
This study has several limitations. First, most NTM strains are widely distributed in the environment, being found in the soil, and water, including even treated water. Therefore, caution must be exercised in interpreting positive results from specimens as this doesn’t necessarily mean that the patient is infected by the bacteria. Positive results may be due to bacterial colonization, or transient infection which is quickly cleared by the immune system, or contamination in sample collection and transportation (21). Second, the resolution power of our NTM detection method is limited. The Mycobacterial Species Identification Array Kit (CapitalBio Technology Inc., Beijing, China) cannot differentiate the following organisms into species level: M. chelonae and M. abscessus, M. marinum and M. ulcerans, M. szulgai and M. malmoense (11). In addition, the kit cannot discriminate NTM strains into sub-species level. Over recent years, researchers have modified the sub-species taxonomies of many NTM species (33, 34). The sub-species of M. abscessus, for instance, contains M. massiliense and M. bolletii, which are distinct in susceptibility to macrolides based on differences in the erm gene, thus leading to different therapeutic strategies if identified properly (34). Third, among all the NTM-positive samples collected (565), only 40% (223) were further subjected to species identification testing. This was because some patients were lost to follow up, and some patients could not afford to pay for further NTM species identification. As a result, our data may not contain the overall NTM species information.
Finally, among the common NTM species, MAC, M. abscessus and M. kansasii are highly pathogenic, and can cause lung diseases and lymph node, skin and soft tissue infections. However, M. gordonae and M. fortuitum rarely cause any infections, and only cause diseases when the patient’s general condition is particularly poor. Thus during the clinical diagnosis and treatment, the clinical significance of the strain should be considered by combining with the patient's symptoms, signs and imaging findings.