Non-tuberculous mycobacteria (NTM) have emerged as clinically significant opportunistic pathogens responsible for a broad spectrum of pulmonary and extrapulmonary diseases. Their importance is particularly evident in tuberculosis (TB)-endemic settings, where substantial overlap in clinical presentation, radiological findings, and microbiological characteristics with Mycobacterium tuberculosis complicates accurate diagnosis, often leading to misclassification and suboptimal management [1]-[9].

The increasing global burden of NTM disease has been attributed to multiple converging factors, including an ageing population, rising prevalence of chronic lung diseases, expanded use of immunosuppressive therapies, and improvements in diagnostic methodologies [2][4]. Despite these advances, the clinical relevance of NTM isolation remains incompletely defined, and adherence to standardized diagnostic and therapeutic guidelines continues to be variable across settings. NTM are ubiquitous environmental organisms, widely distributed in soil, water, and biofilms, with the capacity to colonize or infect susceptible hosts. Among the various clinical manifestations, non-tuberculous mycobacterial pulmonary disease (NTM-PD) represents the most common presentation and is frequently associated with underlying structural lung abnormalities, including bronchiectasis, cystic fibrosis, and chronic obstructive pulmonary disease (COPD) [1]-[3][5][7]-[14][15].

NTM are broadly classified into rapid- and slow-growing groups based on culture characteristics. Slow-growing mycobacteria (SGM), requiring more than 7 days for visible growth, comprise a diverse group of clinically relevant species. To date, over 200 NTM species have been identified, many of which are implicated in human disease [2][7][11][13][14][16]. Among these, the Mycobacterium avium complex (MAC), comprising M.avium, M.intracellulare, and M.chimaera, is most frequently associated with pulmonary disease globally. Other important species include Mycobacterium kansasii, Mycobacterium simiae, and Mycobacterium gordonae[3][7][10][11].

Data from North India remain limited, although available reports suggest an NTM isolation rate of approximately 12.1% in Delhi [17]. Globally, NTM-associated mortality has shown an increasing trend, particularly among elderly populations. Furthermore, significant geographic variability in species distribution has been well documented, with MAC predominating worldwide [12].

In this context, the present study was undertaken to comprehensively characterize the species distribution and antimicrobial susceptibility patterns of slow-growing mycobacteria isolated from pulmonary and extrapulmonary clinical specimens in Delhi, North India, over an eight-year period (2018-2025). Traditionally, NTM has been classified according to the Runyon classification system, which categorizes species into four groups based on growth rate and pigment production. Accordingly, Groups I-III consist of slow-growing mycobacteria, further classified as photochromogens, scotochromogen, and non-chromogens, while Group IV includes rapidly growing species that typically exhibit visible growth within seven days (Table 1).

Table 1. Runyon classification of slow-growing mycobacteria species identified in the study (2018-2025).

This retrospective study was conducted at Dr Lal Path Labs, National Reference Laboratory (NRL), New Delhi, India, over an eight-year period from January 2018 to December 2025. All patients with culture-confirmed non-tuberculous mycobacteria (NTM) isolates from pulmonary and extrapulmonary clinical specimens were included. Application of the American Thoracic Society (ATS) diagnostic criteria for NTM pulmonary disease was not feasible due to the unavailability of detailed clinical and radiological data; accordingly, the analysis was restricted to microbiological and laboratory-based characterization of NTM isolates. Species identification was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with the MALDI Bio typer Compass/IVD reference library (2023). Antimicrobial susceptibility testing was conducted by broth microdilution using the Sensititre system and interpreted in accordance with Clinical and Laboratory Standards Institute (CLSI) M24-A2 guidelines [19].

Among 197,943 clinical specimens processed during the study period, 34,776 (17.5%) yielded acid-fast bacilli in culture. Of these, 30,343 (87.3%) were identified as members of the Mycobacterium tuberculosis complex, whereas 4433 (12.7%) were classified as non-tuberculous mycobacteria (NTM). Among the NTM isolates, 2588 (58.4%) were rapidly growing mycobacteria and 1845 (41.6%) were slow-growing species. Pulmonary specimens accounted for 63.2% of the slow-growing mycobacteria isolates, while 36.8% recovered from extrapulmonary samples. Within the slow-growing group, Mycobacterium avium complex is the most frequently isolated slow NTM species. Detailed APC (Annual Percent Change) and segmented trend analysis are provided in Table 2.

Table 2.Temporal trends in NTM isolation rates among tuberculosis-suspected patients, 2018-2025.

A total of 34,776 acid-fast bacilli (AFB) culture samples were analyzed between 2018 and 2025, of which 4433 were identified as non-tuberculous mycobacteria (NTM), yielding an overall prevalence of 12.7%. The annual proportion of NTM isolation demonstrated variability over time, ranging from 7.4% in 2020 to 17.3% in 2025. An initial increase was observed from 11.5% in 2018 to 13.6% in 2019, followed by a transient decline in 2020, and a subsequent gradual rise through 2025, suggesting apparent increase in recent years; however, this was not statistically significant.

Despite these fluctuations, trend analysis using the Cochran-Armitage Chi-square test did not demonstrate a statistically significant temporal change (p > 0.05), indicating the absence of a consistent directional trend. This was further supported by Joinpoint regression analysis, which showed a minimal and non-significant increase in NTM proportions over time (Annual Percent Change [APC]: +0.15% per year; p = 0.963). Overall, these findings suggest that, despite year-to-year variability and a modest rise in recent years, the proportional burden of NTM remained stable without statistically significant temporal variation across the study period.

The distribution of slow-growing mycobacteria species isolated from various clinical specimens during 2018-2025 demonstrated that pulmonary specimens accounted for many isolates (1166/1845; 63.2%), with sputum (37.1%) and bronchoalveolar lavage (BAL) (26%) representing the predominant sources. In contrast, extrapulmonary specimens constituted 36.8% of the isolates.

Among the non-respiratory specimens, 171 isolates (9.2%) were categorized under other specimen types, followed by pus samples (145; 7.9%), tissue specimens (126; 6.8%), and body fluids (120; 6.5%). A smaller proportion of isolates were recovered from endometrial samples (57; 3.1%), urine (31; 1.7%), and menstrual blood (29; 1.6%) (Figure 1).

Figure 1. Distribution of slow growing mycobacteria isolates across clinical specimen types.

Slow-growing mycobacteria isolates were categorized according to the Ernest H. Runyon classification system based on pigment production. Most isolates belonged to the non-chromogenic group, accounting for approximately 67.8% (n = 1251) of all isolates. This group was predominantly represented by species such as Mycobacterium chimaera,Mycobacterium avium,Mycobacterium intracellulare,M.colombiense, and several other related species.

Photochromogens accounted for 26.7% (n = 493) of isolates, including Mycobacterium kansasii, M.simiae, M.marinum, and M.asiaticum, while scotochromogens comprised 5.5% (n = 101), represented by M.gordonae, M.scrofulaceum, M.szulgai, M.lentiflavum, and M.paragordonae. Overall, non-chromogenic slow-growing NTM predominated, followed by photochromogens and scotochromogens. Most isolates across all Runyon groups were derived from pulmonary specimens, with fewer from extrapulmonary sites; however, non-chromogenic species also showed notable extrapulmonary representation (Table 3).

Table 3. Pulmonary vs. extrapulmonary distribution of slow-growing mycobacteria isolates (2018-2025).

A total of 1845 slow-growing mycobacteria (SGM) isolates were recovered from various clinical specimens during the study period. Most isolates originated from pulmonary samples, accounting for 1166 (63.2%) of all isolates. The remaining 36.8% (679/1845) were obtained from extrapulmonary specimens, Among the identified species, members of the Mycobacterium avium complex (MAC) accounted for 55.7% of the slow-growing mycobacteria isolates. Within this group, Mycobacterium chimaera was the most frequently isolated species, representing 954 isolates (51.7%), followed by Mycobacterium avium (39 isolates) and isolates belonging to the Mycobacterium intracellulare (35 isolates). These closely related species were collectively analyzed as MAC due to their phylogenetic relatedness and similar clinical relevance. Most MAC isolates originated from respiratory samples, underscoring the predominance of pulmonary infection.

The next most frequently identified species were Mycobacterium simiae (240 isolates; 13.0%) and Mycobacterium kansasii (235 isolates; 12.7%), both predominantly recovered from pulmonary specimens. Less frequently encountered species included Mycobacterium parascrofulaceum, Mycobacterium gordonae, and Mycobacterium colombiense, along with several rare isolates such as Mycobacterium europaeum, Mycobacterium heckeshornense, Mycobacterium xenopi, and Mycobacterium triplex. It is important to note that some of these species may reflect environmental contamination or colonization rather than true clinical infection.

Notably, while most species were predominantly isolated from pulmonary samples, several organisms—including Mycobacterium farcinogenes,Mycobacterium marinum,and Mycobacterium arupense—were more frequently associated with extrapulmonary specimens, particularly pus and tissue, suggesting their involvement in soft-tissue or disseminated infections.

Overall, the data demonstrate that respiratory specimens represent the principal source of slow-growing mycobacteria isolates, with Mycobacterium chimaera emerging as the dominant species, followed by Mycobacterium simiae and Mycobacterium kansasii. The identification of multiple SGM species from both pulmonary and extrapulmonary specimens highlights the diversity and increasing clinical relevance of non-tuberculous mycobacteria in clinical settings (Table 4).

Table 4. Distribution of slow-growing mycobacteria species in different pulmonary and extrapulmonary samples.

The age- and sex-wise distribution of SGM isolates is presented in Figure 2. Among the 1845 culture-positive cases, the largest proportion was observed in individuals aged ≥65 years (n = 516; 27.9%), followed by those aged 36 - 50 years (n = 496; 26.9%), 51 - 65 years (n = 452; 24.5%), and 13 - 35 years (n = 381; 20.7%). No cases were recorded in children aged 0 - 12 years (n = 0). The ≥65-year age group constituted the largest cohort and showed male predominance. Overall, males predominated in most age groups, except in the 36 - 50-year group, where females were slightly more prevalent.

Figure 2. Distribution of slow growing mycobacteria (SGM) group of organisms by age and gender of patient.

AST was intentionally performed on a subset of 204 slow-growing mycobacteria isolates, in accordance with clinician-directed requests and real-world diagnostic practices. Given that routine susceptibility testing for all isolates is neither universally recommended nor always clinically indicated, particularly for certain NTM species, this targeted approach ensured clinically relevant and guideline-aligned testing.

Overall, most isolates showed high susceptibility to several important antimicrobial agents. Amikacin demonstrated the greatest activity, with 99.5% of isolates classified as susceptible and only 0.5% as resistant, reflecting excellent in vitro efficacy against slow-growing mycobacterial species. Clarithromycin also showed strong activity, with 97.5% susceptibility and 2.5% resistance, reinforcing its well-established role as a key drug in the treatment of many NTM infections.

High susceptibility rates were also observed for Linezolid (95.2% susceptible; 1.9% resistant) and Moxifloxacin (94.6% susceptible; 4.4% resistant), suggesting that these agents may serve as effective alternative or adjunct therapeutic options in selected cases. Moderately high susceptibility was observed for Cotrimoxazole (93.2% susceptible; 6.8% resistant). In contrast, Ciprofloxacin and Minocycline demonstrated 88.7% susceptibility, with resistance rates of 11.3% and 10.8%, respectively. Similarly, Doxycycline showed 86.2% susceptibility, with 2.5% intermediate susceptibility and 11.3% resistance.

Overall, the AST profile indicates that Amikacin and Clarithromycin exhibited the highest in vitro activity against slow-growing mycobacteria isolates, followed by Linezolid and Moxifloxacin. Conversely, comparatively higher resistance rates were observed with Doxycycline, Ciprofloxacin, and Minocycline. These findings underscore the importance of species-level identification and antimicrobial susceptibility testing to guide appropriate therapeutic management of NTM infections (Figure 3).

Figure 3. Cumulative interpretation of antimicrobial susceptibility (%) among slow growing mycobacteria (SGM) group of organisms in diverse samples.

Antimicrobial susceptibility testing was performed for 126 isolates of Mycobacterium chimaera and 78 isolates of other species of slow-growing mycobacteria (SGM).

Overall, both groups demonstrated high susceptibility to several key antimicrobials, although minor differences were observed between M.chimaera and other SGM species. Among M.chimaera isolates, Amikacin exhibited the highest activity, with 99.2% susceptibility and only 0.8% resistance, while other SGM isolates showed complete susceptibility (100%). Similarly, Clarithromycin demonstrated excellent activity, with 96.8% susceptibility in M.chimaera and 98.7% susceptibility among other SGM isolates, indicating its strong efficacy and clinical importance in NTM treatment regimens.

High susceptibility rates were also observed for Linezolid (95.3% in M.chimaera and 94.8% in other SGM) and Moxifloxacin (96.0% and 92.3%, respectively). These findings suggest that these agents may represent useful therapeutic options in the management of slow-growing mycobacteria infections.

Moderate susceptibility patterns were observed with Cotrimoxazole, showing 93.7% susceptibility in M. chimaera and 92.3% in other SGM isolates. In contrast, relatively higher resistance rates were noted for Ciprofloxacin, with 9.5% resistance among M.chimaera and 14.1% among other SGM isolates. Likewise, Doxycycline exhibited 84.9% susceptibility in M.chimaera and 88.5% in other SGM, with resistance rates exceeding 11% in both groups.

Among tetracycline derivatives, Minocycline demonstrated 86.5% susceptibility in M.chimaera compared with 92.3% in other SGM isolates, with resistance observed in 12.7% and 7.7% of isolates, respectively.

Overall, the findings indicate that Amikacin and Clarithromycin exhibited the highest in-vitro activity against both M.chimaera and other slow-growing mycobacteria species, followed by Linezolid and Moxifloxacin. In contrast, comparatively higher resistance rates were observed with Ciprofloxacin, Doxycycline, and Minocycline, highlighting the importance of species-level identification and antimicrobial susceptibility testing to guide appropriate therapy for NTM infections (Table 5).

Table 5. Comparative antimicrobial susceptibility profile of M.chimaera and other slow-growing mycobacteria.

This study presents a large-scale, laboratory-based characterization of slow-growing mycobacteria (SGM) isolated from tuberculosis (TB)-suspected patients in a high-burden setting in North India over an eight-year period. NTM accounted for 12.7% of culture-positive mycobacterial isolates, reflecting a consistent presence in routine diagnostic practice. Although global studies have reported a rising prevalence of NTM, the present analysis demonstrated a stable proportional burden without a statistically significant temporal increase [2]-[6][8][9][11][12][15][17].

This trend is further supported by global mortality data, which show an increase in NTM-associated deaths from 0.36 per million in 2000 to 0.77 per million in 2022, particularly among older populations, underscoring the growing clinical importance of NTM infections [12]. The transient decline observed in 2020 is attributable to disruptions in healthcare access and diagnostic services during the COVID-19 pandemic [17]. The predominance of pulmonary specimens (63.2%) aligns with global data indicating that the respiratory tract remains the principal site of NTM recovery. However, the inclusion of a TB-suspected cohort inherently enriches for pulmonary samples and may overestimate the relative contribution of respiratory isolates [1][2][4][8][10][11][13]. The substantial proportion of extrapulmonary isolates further reinforces the broad ecological and pathogenic spectrum of NTM, encompassing soft tissue, body fluid, and genitourinary specimens. Notably, certain species such as Mycobacterium marinum, M.farcinogenes, and M.arupense demonstrated a predilection for extrapulmonary sites, consistent with their known clinical associations [16].

A key finding of this study is the predominance of the Mycobacterium avium complex (MAC), which accounted for more than half of all slow-growing mycobacteria isolates. Within this group, M.chimaera was the most frequently identified species. While MAC dominance is consistent with global trends, the high proportion of M.chimaera should be interpreted cautiously. Species-level differentiation within MAC using MALDI-TOF mass spectrometry remains challenging due to spectral overlaps, and misclassification with closely related species, such as M.intracellulare cannot be excluded in the absence of molecular confirmation.

In contrast, several international studies have reported M.avium and M.intracellulare as the predominant MAC species [3][6]. Nevertheless, the overall predominance of MAC observed in the present study remains aligned with global trends [3][4][8][11][13][18]. Conversely, studies from other regions have identified Mycobacterium kansasii and Mycobacterium simiae as the leading species, highlighting substantial geographic variability in NTM species distribution [1-2] [15][16]. These findings are in broad agreement with global data, where species such as Mycobacterium abscessus, Mycobacterium fortuitum, MAC, and Mycobacterium kansasii are among the most frequently encountered NTM [4][7][10][11][13][17][18].

The identification of thirty-seven distinct slow-growing mycobacteria species in this study highlights the expanding spectrum of mycobacteria recognized in clinical microbiology, driven by advances in diagnostic technologies. However, the clinical significance of many of these species remains uncertain. Notably, several organisms detected in this cohort, including Mycobacterium gordonae and other scotochromogen species, are well-recognized environmental contaminants. In the absence of correlated clinical, radiological, and repeat microbiological evidence, differentiation between true infection, colonization, and transient contamination remains challenging. Therefore, the present findings should be interpreted as reflecting patterns of laboratory isolation rather than definitive disease burden.

The Mycobacterium tuberculosis complex (MTBC) remained the predominant pathogen, consistent with TB-endemic settings [1][2][13]. In contrast, NTM predominates in low-TB-incidence regions, reflecting an epidemiological transition driven by declining TB burden and improved diagnostics [18]. The demographic distribution observed in this study, with a higher prevalence among older individuals and a slight male predominance, is broadly consistent with known epidemiological patterns of NTM isolation [5][12][16]. However, other studies have reported a female predominance in clinically significant NTM infections, indicating variability across populations [2]. Nonchromogenic species predominated, consistent with MAC dominance. Followed by photochromogens species such as Mycobacterium simiae,M.kansasii were identified nonchromogenic, whereas scotochromogen represent a smaller proportion, in line with international reports [4][7][10].

Antimicrobial susceptibility testing revealed high in vitro activity of amikacin and clarithromycin against slow-growing NTM isolates, consistent with their established role as key agents in the treatment of NTM infections [4][6][7][9][10]. Linezolid and moxifloxacin also demonstrated favorable susceptibility profiles, indicating their potential as adjunctive therapeutic options in selected clinical settings. However, application of the American Thoracic Society (ATS) diagnostic criteria for NTM pulmonary disease was not feasible in this study due to the absence of detailed clinical and radiological data. Therefore, the findings are restricted to microbiological characterization and should not be interpreted as evidence of definitive clinical disease.

Importantly, for most NTM species, in vitro susceptibility does not consistently correlate with clinical outcomes, with the notable exception of macrolide susceptibility in Mycobacterium avium complex (MAC) infections. Although the clinical use of certain agents, such as linezolid, may be constrained by toxicity, they remain valuable components of treatment regimens when first-line options are unsuitable or ineffective [7][13]. The variable susceptibility observed for fluoroquinolones and tetracyclines further underscores the intrinsic heterogeneity of NTM and the limitations of extrapolating in vitro findings to clinical decision-making. Collectively, these results reinforce current recommendations emphasizing the importance of accurate species-level identification and individualized, susceptibility-guided therapy rather than reliance on empirical treatment strategies.

This study has several notable strengths, including its large sample size, extended duration, and standardized laboratory workflow incorporating both liquid and solid culture systems, as well as MALDI-TOF-based species identification. These features provide a comprehensive overview of NTM isolation patterns in a high-volume reference laboratory.

Nevertheless, important limitations must be acknowledged. Foremost, the retrospective design and lack of clinical and radiological correlation precluded application of established diagnostic criteria for NTM disease. As a result, differentiation between true infection, colonization, and environmental contamination was not feasible. Second, species identification relied solely on MALDI-TOF mass spectrometry without molecular confirmation, which may have affected accuracy, particularly within closely related complexes such as MAC. Third, antimicrobial susceptibility testing was performed on a limited subset of isolates, potentially introducing selection bias. Finally, the study population comprised TB-suspected patients, which may limit the generalizability of findings to the broader community.

This study highlights the persistent and diverse presence of slow-growing NTM in a TB-endemic setting, with MAC predominance and substantial representation across both pulmonary and extrapulmonary specimens. The findings underscore the diagnostic challenges posed by NTM in routine practice and the critical importance of accurate species identification. However, the absence of clinical correlation necessitates cautious interpretation, and future prospective studies integrating microbiological, clinical, and radiological data are essential to delineate the true burden and clinical significance of NTM disease in this region.

Formal analysis: P.S., S.M.; Investigation: P.S.; Writing—original draft preparation: P.S.; Writing—review and editing: P.S., S.M.; Visualization: P.S., S.M., V.L. All authors have read and agreed to the published version of the manuscript.

We express our sincere gratitude to Dr. Reena Nakra, Principal Laboratory Director (Operations), National Reference Laboratory, Dr Lal Path Labs, Delhi, for her invaluable operational support. We also thank the Microbiology Team, with special acknowledgment to Ramesh Chandra Masiwal, for their technical assistance and support in data retrieval for this study.

The study was based on anonymized laboratory data without patient identifiers; therefore, ethics committee approval was waived.