**4. Discussion**

As a consequence of the COVID-19 pandemic, the WHO predicts that the epidemiological situation of tuberculosis will deteriorate worldwide [13]. The pandemic caused significant changes in the functioning of health care systems, other important epidemiological problems were neglected, and the diagnosis of numerous infectious diseases, including tuberculosis, became less important. This may result in weaker national TB programmes [14] and increased TB incidence in the near future [15]. There has been a downward trend in TB incidence rates in Poland since 1957. In 2020, the incidence of TB was 8.8, significantly lower than in 2018 and 2019 (14.3 vs. 13.9) [2]. Unfortunately, the low rate in 2020 was a result of the COVID-19 pandemic. The ERLTB-Net-2 network of European reference mycobacterial laboratories published a report on the impact of the COVID-19 pandemic on TB laboratory services in Europe. They found that the most severe disruption of TB NRL services occurred at the beginning of the pandemic and coincided with a significant decrease in the number of samples received, by about 30% [16]. A similar analysis conducted by the National Reference Laboratory for Mycobacteria in Poland found that the number of TB tests decreased by as much as 45% during a single year of the

pandemic [2]. A study by Migliori et al. in 33 centres from 16 countries [17] assessed patient attendance at TB health care units by comparing data from 4 months of the COVID-19 pandemic (January–April 2020) within the same period in 2019. Most centres reported a decrease in the number of newly diagnosed TB cases and the total number of outpatient visits for active disease. In some centres, medical staff working with TB patients have been seconded to work with COVID-19 patients. In addition, the fewer clinic visits were due to patients' fear of COVID-19 exposure or difficulty accessing medical services [18].

As our comparative analysis has shown, the breakdown of the sex and age of TB patients in Poland has remained unchanged for years. The highest incidence of TB is among Poles over the age of 44. It is primarily men who ge<sup>t</sup> sick. Three times more men die from TB than women in Poland [6]. In 2020, men between the ages of 45 and 64 were also the largest group of patients (26%). Among women, most patients were over 65 years old (24%). Children under 14 years of age accounted for only 0.36%. The incidence of tuberculosis in the paediatric population mirrors the epidemiological situation of tuberculosis among adults. The new incidence of tuberculosis in children indicates that mycobacteria are being transmitted in the environment and that the disease is not completely controlled [19].

When comparing the results obtained in the three consecutive studies, secondary drug resistance was found to be statistically significantly more frequent than primary drug resistance. At the same time, the number of patients excreting mycobacteria with MDR resistance was more common among the previously treated patients than in the newly diagnosed ones. The proportion of patients excreting MDR-resistant mycobacteria ranged from 0.6% in 2012 to 5.3% in 2020 (Table 7).

**Table 7.** Comparison of results in newly diagnosed and previously treated patients in Poland 2012, 2016, and 2020.


In the three studies from 2012, 2016, and 2020, the highest proportion of newly diagnosed patients excreted mycobacteria that was resistant to a single drug (3.1%, 4%, and 3.9%, respectively). Patients with tuberculosis resistant to two drugs accounted for 0.8% of all newly diagnosed patients. The highest percentage of three-drug resistance was recorded in 2016 at 0.7%, while in 2012 and 2020, it was 0.3% and 0.4%, respectively. In the newly diagnosed patients in the 2012 and 2016 studies, four-drug resistance was found to be 0.2%, whereas this group constituted 1% in 2020 (Figure 2a).

Among the previously treated patients, as with the group of newly diagnosed patients, the greatest number of them excreted mycobacteria that was resistant to a single drug, about 6%. The number of strains resistant to two drugs decreased steadily from 2.6% in 2012 to 1.1% in 2020. The number of mycobacteria resistant to three drugs remained stable at 2.2%. The percentage of TB patients resistant to the four SIRE drugs also increased to 2.1% in 2020 (Figure 2b).

This may be one of the effects of the COVID-19 pandemic, resulting in fewer available records and less monitoring of TB treatment in Poland.

**Figure 2.** Resistance to one or more drugs among newly diagnosed (**a**) and previously treated (**b**) patients in Poland in 2012, 2016, and 2022.

Tracking the rise of mycobacterial resistance and implementing prevention methods is an important method for surveillance of the spread of tuberculosis. *Mycobacterium* drug resistance is the result of insufficient inhibition of mycobacteria growth by drugs' suboptimal concentrations caused by the administration of inappropriate drug combinations at inappropriate doses, for example, ref. [6]. Multi-drug resistant MTB strains are a growing health problem and a major challenge for TB control programmes. Knowledge of the prevalence of resistant strains in a population provides essential information about the epidemiology of the disease in a country. Most of the TB data obtained for 2020 in Poland are significantly lower than in previous years, demonstrating the limited availability of TB diagnosis and treatment during the COVID-19 pandemic. It is no coincidence that the areas of the world projected to be most affected by the social and economic consequences of COVID-19 are also the areas with the highest TB burden [20]. This is because TB is both a social and infectious disease. Poorer, malnourished people living in densely populated areas are more vulnerable to TB, and TB exacerbates poverty by increasing costs, reducing income and being associated with stigmatisation and discrimination [21–25].

Molecular analysis of the incidence of major SITs in Poland in 2020 revealed 35 different spoligotypes and six patterns not registered in the global SITVIT2 database among strains resistant to at least one antimycobacterial drug. It is noteworthy that in the group of patients excreting drug-resistant mycobacteria, in addition to the T family, which prevails in the European population (26.8%), the same percentage of strains was also registered for the Beijing family. The *Mycobacterium tuberculosis* genotype with the canonical spoligotype SIT1 was first described in 1995 and is now the predominant strain among TB patients in many Asian countries though it is increasingly being identified in all seven geographical areas of the world [26]. In Europe, Beijing strains have emerged as endemic and dominant genotypes in countries of the former Soviet Union, often in association with drug resistance [27–30]. Due to human migration and mobility, significant changes in the breakdown of MTB strains have been observed in other European countries, such as Ireland and Germany [31,32]. In Poland, non-Beijing genotypes were most common among drug-resistant strains until 2016, whereas since 2017, the Beijing genotype has prevailed. This is due to the fact that until recently, Beijing TB was identified in Poland mainly in foreigners from Eastern Europe and Asia, with a rise in cases identified among Poles since 2017. Considering all this, TB-control programmes should also use molecular epidemiology to track the transmission of high-risk strains and the diversity of TB in a given area. In European countries with a low incidence of tuberculosis, the intercontinental migration of people for recreation, work, or because of armed conflicts can dramatically change the socio-epidemiological situation. Among sensitive strains, there were 73 spoligotypes and 30 patterns that were not registered in the database. Unregistered patterns accounted for 11.9% of the 252 strains tested. Orphan spoligotypes represent patterns and were identified for the first time in a group of Polish patients in this study. These may indicate recent and/or sporadic TB

transmission in the study area [12,25]. The SITVIT2 database shows that this origin is more common among susceptible strains than drug-resistant strains in Poland. Among MTB strains sensitive to antimycobacterial drugs, SIT53 was the most common spoligotype in Poland. Large-scale migration from countries with high TB incidence rates can lead to unexpected changes in epidemiological indicators through the transmission of MTBC strains not previously recorded in the population. Therefore, it seems that the molecular identification of circulating clades is extremely important in controlling the epidemiological situation of tuberculosis worldwide [12].

**Author Contributions:** Conceptualization, D.B.-T., A.Z. and E.A.-K.; methodology, D.B.-T., A.Z., M.K. and E.A.-K.; writing—original draft preparation, D.B.-T.; writing—review and editing, E.A.-K.; supervision, E.A.-K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Science Centre, gran<sup>t</sup> number 2019/35/ B/NZ7/00942.

**Institutional Review Board Statement:** The study was approved by the Ethics Committee of the National Tuberculosis and Lung Diseases Research Institute (KB-55/2014) as a part of research on mycobacterial diseases in human.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data supporting reported results can be found in source data collected in National Tuberculosis and Lung Diseases Research Institute.

**Conflicts of Interest:** The authors declare no conflict of interest.
