Epidemiology of non-tuberculous mycobacterial diseases in Slovakia during the years 2016–2021

Nontuberculous mycobacteria (NTM) are opportunistic human pathogens found worldwide, primarily in the environment. They predominantly affect the lungs, especially in individuals with compromised immune systems. Recent studies suggest an increasing incidence of NTM disease; however, their actual clinical impact in Slovakia remains uncertain. In this study, we conducted a retrospective analysis using a representative collection of NTM cases in the country. We searched the national database for patients with positive NTM cultures between January 2016 and December 2021. A total of 1355 NTM-positive cultures were identified in Slovakia, with no significant increase observed during the study period. Among these, 358 cases (26.4%) were confirmed as NTM disease. The incidence of the disease was notably higher in individuals over 55 years old (p<0.0001). Moreover, women diagnosed with NTM disease exhibited a significantly higher average age than men (p=0.0005). The majority of NTM disease cases were attributed to Mycobacterium (M.) intracellulare (39.9%) and M. avium (38.5%). Geographically, the highest incidence of NTM disease was observed in the Bratislava region (10.69 per 100,000 population).


Introduction
Non-tuberculous mycobacteria (NTM) are a group of environmental bacteria that are commonly found in water and soil (Ratnatunga et al., 2020). Advances in molecular techniques, such as whole genome sequencing (WGS), have led to the identification of more than 180 NTM species, out of which 60 have been confirmed as pathogens in humans (Dohál et al., 2021;Mortaz et al., 2018). The most commonly observed NTM species causing infections in humans are the Mycobacterium (M.) avium complex (MAC), which includes M. intracellulare, M. avium, and M. chimerae, as well as M. kansasii and M. abscessus complex (Zulu et al., 2021). Although NTM colonization is often asymptomatic, it poses a significant risk of morbidity and 5-year mortality, particularly among high-risk populations such as individuals with bronchiectasis, cystic fibrosis, or asthma (Cassidy et al., 2009). NTM-induced diseases can manifest with both pulmonary and extrapulmonary symptoms, resembling infections caused by M. tuberculosis or other respiratory disorders. This highlights the importance of accurate diagnosis, particularly in countries where tuberculosis (TB) is highly prevalent (Chindam et al., 2021). Treatment for NTM infections typically involves a combination of antibiotics, which can be expensive and may have notable side effects (van Ingen et al., 2018).
In recent years, the prevalence of NTM-induced diseases has experienced a significant increase. However, compared to other pulmonary infections, the epidemiology of these diseases is complex and not as well understood (Hermansen et al., 2017;Lee et al., 2021). For instance, the number of NTM lung infections among individuals with cystic fibrosis has seen a dramatic rise in the United States and other developed countries (Martiniano et al., 2017). The upward trends in NTM infections can be attributed to various factors, including improved diagnostic methods, heightened physician awareness, greater exposure to contaminated water sources, a growing elderly population with increased comorbidities and immunosuppression (Brode et al., 2015;Larsson et al., 2017;Park et al., 2019). Additionally, a lower cross-immunity to M. tuberculosis has been associated with a higher proportion of mycobacterial diseases induced by NTM, possibly due to a decreased incidence of TB in many countries (Brode et al., 2014).
One of the limitations in evaluating the clinical impact and epidemiological characteristics of NTM infections is the inadequate differentiation between disease and colonization, coupled with a scarcity of epidemiological data. In numerous countries, these diseases are not reported to public health authorities (Ahmed et al., 2020;Stout et al., 2016). Moreover, many studies of NTM pulmonary diseases (PD) are retrospective or restricted to specific regions within countries, lacking multi-center collaboration (Chalmers et al., 2020).
In Slovakia, there has been only one published study on NTM infections, resulting in limited knowledge regarding the incidence and prevalence of the disease (Porvaznik et al., 2017). The diagnosis of NTM within the country is solely performed by the National Reference Mycobacteriology Laboratory at the National Institute for Tuberculosis, Lung Diseases, and Thoracic Surgery in Vyšné Hágy. This centralized setup facilitates access to patient information and all recorded NTM cases in recent years. In the present study, we have gathered data on all confirmed NTM cases in Slovakia between 2016 and 2021, aiming to enhance our understanding of NTM epidemiology and identify effective strategies for the prevention and treatment of these infections.

Data source and study design
This retrospective nationwide cohort study included all patients with positive NTM culture from January 2016 to December 2021 retrieved at the National reference laboratory for mycobacteria (NRL) in Slovakia (the only one in Slovakia). Sputum was primarily collected for the clinical assessment of NTM disease. However, in cases where sputum could not be obtained, the physician had the discretion to opt for either a bronchoscopic wash/lavage or a lung biopsy. NTM cultures were considered positive if the presence of at least one NTM species has been confirmed. Patients who received their initial diagnosis prior to the 2016 period were excluded from the study. NTM species identification was carried out using GenoType Mycobacterium CM/AS® (Hain Lifescience, Nehren, Germany) according to the manufacturer´s instructions. NTM were classified into the following groups: M. avium, M. intracellulare, M. gordonae, M. chelonae, M. xenopi, M. mucogenicum, M. abscessus, M. fortuitum, M. kansasii, M. peregrinum, M. species, M. lentiflavum, M. interjectum, M. scrofulaceum. For definitive confirmation of NTM disease, at least one of the following microbiological criteria had to be met: (1) at least two positive cultures from sputum, (2) one positive culture in the case of bronchoscopic wash or lavage, or (3) a transbronchial or other lung biopsy with a positive culture for NTM or compatible histopathological features such as granulomatous inflammation or stainable acid-fast bacilli (AFB), and one positive sputum or bronchial wash culture for NTM regardless of the mycobacterial strain (Griffith et al., 2007). Other NTM-positive cultures were considered as NTM colonization, which is not clinically relevant.
For all patients with confirmed NTM disease, we retrieved sociodemographic data (including age, gender, and region of residence). Slovakia is divided in 4 different regions: Bratislava, Western, Central and Eastern Slovakia. All four regions are classified based on territoriallegal division developed by the European Commission (Eurostat) ("Portrait of the regions -Volume 7: Slovakia (PDF) -Products Statistical Books -Eurostat," 2001). Population density data were obtained from Statistical Yearbook of the Slovak Republic 2022 published by Statistical office of the Slovak Republic.

Statistics
All statistical analyses were performed using the GraphPad Prism v. 8.0.1. All statistical analyses were performed using the GraphPad Prism v. 8.0.1. To evaluate differences in population proportions we used Chisquare test (with Yates correction when only two groups were compared). We performed Shapiro-Wilk normality test to evaluate data distribution and accordingly we performed t-test to evaluate mean age in men and women. ANOVA was applied to analyze mean age of patients in different Slovak regions. P values < 0.05 were considered statistically significant.

Ethics approval
This study was approved by the Ethics committee of Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Slovakia (EK 72/2018).

Results
In the period 2016-2021, 1355 cultures positive for NTM were identified in the NRL. Of these, NTM disease was confirmed in 358 cases (26.4%), while NTM colonization was suspected in 997 cases (73.6%). The incidence rate of NTM disease ranged from 0.88 to 1.43 per 100,000 (Table 1). However, comparing also previous data, since 2010 the incidence has significantly increased (p < 0.0001, 95% CI = 0.05982 -0.3071). The major increase in incidence was observed between the years 2015 and 2016 (p < 0.0001, 95% CI = 0.1332 -0.4741). The data visualization can be found in Fig. 1B.
The median patient age was 67 years old. We observed an significant increase in incidence of NTM disease with age. In the 0-14, 15-24 and 25-54 age group, the incidence was 0.85, 1.28 and 2.10 per 100.000, while in the 55-64 and > 64 age group, the incidence was 8.39 (p = 0.0352) and 19.19 (p = 0.004) per 100.000, respectively (Fig. 3, Table 2). The findings further indicated associations between age, sex, and the occurrence of NTM disease. Specifically, women diagnosed with NTM disease exhibited a significantly higher average age than men (p = 0.0005). The mean age difference between women (average age 64.5) and men (average age 58.0) was 6.5 years.
The geographical distribution of NTM strains and the incidence of NTM disease differed in the 4 main regions of Slovakia (Fig. 4, Table 3). Most cases occurred in the Western (n = 112) and Eastern (n = 115) Slovakia region, but the highest incidence of NTM disease was confirmed in the Bratislava region (the capital). Slow-growing species prevailed in all regions (most of them belonging to the MAC). There was no significant difference in the mean age in different Slovak regions (p = 0.4910). However, we have found significantly different male patients proportion according to the regions (p = 0.0124). Only 32.5% of patients on average from Central region were men while in both Eastern and Western regions men represented a slight majority (52%).

Discussion
Several studies have shown an alarmingly increasing incidence of NTM disease in the last decade (Hermansen et al., 2017;Lee et al., 2021). It is known that NTM can cause progressive lung disease or can reside in the lungs and cause respiratory failure in some cases. Until now, the situation regarding mycobacteriosis in Slovakia was reported from years 2010 -2015, and a total of 77 cases were registered (Porvaznik et al., 2017). To characterize the incidence of NTM disease and circulation of specific NTM species in Slovakia, we conducted a nationalwide epidemiological study including all NTM identified at the NRL during years 2016-2021. During the studied period, 1355 NTM-positive cultures were isolated, of which NTM disease was confirmed in 358 cases (26.4%; incidence range from 3.4 to 5.4 per 100.000). Compared to other European countries (including neighboring countries), Slovakia is among the countries with the highest prevalence of NTM disease (Kwiatkowska et al., 2018;Modrá et al., 2019). The previous study demonstrated a steady patient count for NTM disease diagnoses from 2010 to 2015. However, our study revealed a substantial and significant increase (p < 0.0001) in the number of patients diagnosed with NTM disease beginning in 2016. We assume that the upward trend in the number of NTM isolates and incidence of mycobacteriosis is associated with increased testing and the introduction of novel molecular-genetic methods as part of routine diagnostics. However, the reasons behind this NTM increase need to be further elucidated.
The first study describing the species distribution of NTM patient isolates in European countries (including Slovakia) was published in 2013 and revealed geographic differences (Hoefsloot et al., 2013). The results showed the predominance of M. kansasii in Slovakia and Poland. In our study, M. kansasii was identified only in 8 cases, indicating significant changes in the circulation of individual NTM strains in recent years. Currently, M. gordonae is the prevalent species; however, it is more appropriate to consider it as contamination from tap water, as none of our cases met the criteria for confirmation as a causative agent of the disease. Among disease-causing isolates MAC is the most prevalent NTM found in respiratory samples. Consistent with our findings, research conducted in various European and non-European countries has demonstrated that M. intracellulare and M. avium are the most frequently isolated NTM species, accounting for up to 85% of all NTM disease cases (Dolezalova et al., 2021;Thangavelu et al., 2021;Prevots and Marras, 2015;van Ingen et al., 2021).
Considering the gender difference, NTM disease was confirmed only slightly more often in women (50.5%) than in men (49.5%). However, considering the factor of age, there was a significant difference in the average age between women and men diagnosed with NTM disease (p = 0.0005). Women had a considerably higher average age of 64.5 years, while men had an average age of 58.0 years. Similar    epidemiological results were reported in the previous studies showing a higher risk of developing NTM disease in women over 50 years of age (Furuuchi et al., 2019;Kartalija et al., 2013;Morimoto et al., 2017;Park et al., 2021;Prevots et al., 2010). A possible explanation for the susceptibility of women to NTM disease could be attributed to aberrant expression of adipokines, sex hormones, and/or transforming growth factor beta (TGF-β) (Chan and Iseman, 2010). The correlation between age and the rising incidence of the disease is further substantiated by additional studies, which confirm that NTM disease is more commonly observed in elderly patients with potential comorbidities and increased life expectancy (Kim et al., 2022;Santos et al., 2022). The number and incidence of NTM disease among pediatric patients (<15 years) was low (Fig. 3, Table 2). These outcomes are consistent with results from other studies conducted within the last decade (Lee et al., 2019;Schoenfeld et al., 2016;Zimmermann et al., 2017). Geographic data revealed distinct variations in the incidence and distribution of NTM based on the specific region. Most cases were reported in Eastern Slovakia, but the incidence was highest in the Bratislava region. The Bratislava region is the part of Slovakia with the lowest number of TB patients, therefore we assume that one of the explanations for the high number of NTM cases is low cross-immunity to M. tuberculosis (May et al., 2009). Due to the variability in the dataset, further studies are needed to identify possible environmental reservoirs of NTM and human risk factors, such as the number of patients with cystic fibrosis, HIV/AIDS, and other immunosuppressive diseases/therapies in individual regions.
We are aware that our study has some limitations. Disease confirmation was based on microbiological criteria adapted from the American Thoracic Society and the Infectious Diseases Society of America. In several cases, we recorded only one positive culture, which ruled out disease. Since we have no further data on some patients, we assume that the incidence of NTM disease may be underestimated in our study. Moreover, we did not have access to data on underlying diseases as our analysis was restricted to microbiological results. The majority of patients were classified in the medical health record system as having non-specific pneumonia or other respiratory conditions without detailed documentation of specific comorbidities. Furthermore, the inability to accurately classify the species of certain mycobacteria represents another limitation of this study. To address this limitation in future research, we intend to utilize WGS to enhance species identification in such cases. Also, we had no access to information on treatment effectiveness, which would help us to evaluate the overall severity of NTM diseases.
In summary, our study contributed to a better understanding of the dynamics of NTM diseases in Slovakia in the last decade. The results confirmed the increasing trend in the incidence of NTM diseases since 2016 and the prevalence of NTM belonging to MAC. This study highlights the need to adopt an appropriate uniform approach to the diagnosis of NTM and more studies to answer the real threat of NTM.