**3. Results**

#### *3.1. Sex and Age of Tuberculosis Patients in 2020*

Among the 1383 patients from whom mycobacteria belonging to *Mycobacterium tuberculosis* complex were cultured, the most numerous group included men aged 55–64 years (26.03%). Among women, most patients were over 65 years old (24.32%). The incidence of TB among men was higher than among women. There were 1087 cases registered in men and 296 cases in women. There were five children (0.36%) in the study group: two boys and three girls. They belonged exclusively to the group of newly diagnosed patients. Three of them were under 5 years of age (Table 1).

**Table 1.** Tuberculosis incidence in Poland in 2020, by age and sex.


Comparing the sex of TB patients in our three original studies—from 2012, 2016, and 2020—with data from the NTR in Poland, we found that the percentages of female and male patients in all groups were similar. Men were more likely to contract the disease in all three studies. In the 2012 and 2016 patient groups, the female-to-male ratio averaged 1:2.5, while it reached 1:3.7 in the 2020 patient group. Similarly, data from the NTR show that in 2020, the proportion of male patients increased by about 5 p.p. over previous years, reaching 76.3% (Table 2).



Data on patients with incomplete clinical information on the course of treatment, preventing patients from being classified as newly diagnosed or previously treated, were excluded from the analysis. The percentages of newly diagnosed and previously treated cases did not change significantly among the three studies by the authors, amounting to 87% and 13%, respectively. In contrast, in absolute numbers, there was a significant decrease in patients reported in 2020 compared to previous years. The number of strains submitted dropped threefold, from 4136 in 2012 to 1383 in 2020. The NTR shows that reported TB cases halved, from 5070 in 2012 to 2655 in 2020 (Table 3).

**Table 3.** Comparison of data on new cases and relapse tuberculosis cases between the authors' studies and data from the National Tuberculosis Registry (NTR) in Poland in 2012, 2016, and 2020.


#### *3.2. Analysis of Primary and Acquired Drug Resistance in Authors' 2020 Study*

Newly diagnosed patients (1174) accounted for 84.9% of the cases, with 1102 of them (93.9%) isolated strains that were sensitive to all tested drugs. Resistant mycobacteria were isolated from 72 patients (6.13%). There were 46 (3.9%) mono-resistant strains, most commonly resistant to isoniazid (I) and streptomycin (S), at 25 (2.13%) and 19 (1.62%), respectively. Nine patients showed resistance to two drugs, and five patients to three drugs. Resistance to all four primary drugs was observed in 12 patients. Multiple-drug resistance, i.e., resistance to at least isoniazid (I) and rifampicin (R), was found in 19 patients (1.6%). Among MDR strains, resistance to the following four antimycobacterial drugs prevailed (SIRE): streptomycin, isoniazid, rifampicin (R), and ethambutol. Three drug-resistant (SIR) and two drug-resistant (IR) strains were isolated from five patients (Table 4).

Among the 187 patients (13.5%) previously treated with antimycobacterial drugs, 164 (87.7%) isolated strains were sensitive to the primary drugs, and 23 (12.3%) isolated strains were resistant to at least 1 drug. Twelve patients (6.42%) were infected with mycobacteria resistant to a single drug, and four patients each had a strain with mono-resistance to streptomycin, isoniazid, or rifampicin. MDR-TB drug resistance was found in 10 cases (5.3%). Among the MDR strains, four-drug resistance (SIRE) was the most common, manifesting in four patients. Resistance to three drugs was found in four patients—three SIR and one IRE—while two patients showed resistance to two drugs (IR). Only one strain showed resistance with a different phenotype, that of the SIE type (Table 4).

Information on history of TB disease and treatment was not obtained for 21 of the patients (1.5%).


**Table 4.** Resistance patterns of *Mycobacterium tuberculosis* strains isolated from newly diagnosed patients (primary drug resistance) and from previously treated patients (acquired drug resistance) in Poland in 2020.

The analysis of total drug resistance (the proportion of individual drugs in all resistance patterns) shows that isoniazid resistance prevailed in the groups of newly diagnosed and previously treated patients, with 51 (70.8%) and 15 (65.2%), respectively; ethambutol resistance was the least common, with 12 (16.7%) and 6 (26.1%), respectively. There were 43 (59.7%) and 12 (52.2%) cases, respectively, of streptomycin resistance. Resistance to rifampicin was found in 21 (29.2%) and 14 (60.9%) cases, respectively (Figure 1).

#### *3.3. Molecular Analysis of Mycobacterium Tuberculosis Strains Resistant to at Least 1 Drug (Compared to 20% of Sensitive Strains), Poland 2020*

Among the 82 hybridisation patterns (drug-resistant strains), the SITVIT2 international database identified spoligotypes most commonly belonging to the Beijing 22 (26.8%) and T 22 (26.8%) families, followed by Haarlem 18 (21.9%), URAL 6 (7.3%), LAM 5 (6.1%), and EAI 2 (2.4%) families. Twenty-four individual (unique) spoligotypes were identified. The following spoligotypes were the most abundant among resistant strains cultured in 2020: SIT1 14 (17%), SIT53 8 (9.7%), SIT265 8 (9.7%), and SIT139 6 (7.3%). Seven patterns had no counterparts in the database, with a 15-digit octagonal number only. Two isolates had the same pattern: 777737607420771 (Table 5).

**Figure 1.** Total resistance to I, S, R, and E in *M. tuberculosis* strains isolated from new (primary resistance) and previously treated patients (acquired resistance), 2020.

In order to determine or exclude differences in the molecular patterns of drug-sensitive and drug-resistant strains, 20% of the strains were randomly selected among the sensitive strains isolated from patients in 2020. The SITVIT2 international database identified spoligotypes most commonly belonging to the T 77 (30.5%), Haarlem 64 (25.4%), URAL 17 (6.7%), and Beijing 15 (5.9%) families, followed by CAS 10 (4%), LAM 9 (3.6%), EAI 3 (1.2%), and X 3 (1.2%) and S 2 (1.2%). Forty-eight unique spoligotypes were identified. Spoligotypes were the most abundant among the susceptible strains grown in 2020: SIT53 35 (13.9%), SIT50 21 (8.3%), SIT47 17 (6.7%), and SIT1 14 (5.5%). Another 52 patterns were not found in the database. The most common among them was the pattern 770000777660731, which represented 10 strains of *Mycobacterium tuberculosis* (Table 6).

**Table 5.** Prevalence of the most common spoligotypes of resistant strains of *Mycobacterium tuberculosis* in Poland in 2020 (n = 82).



**Table 6.** Prevalence of the most common spoligotypes of sensitive strains of *Mycobacterium tuberculosis* in Poland in 2020 (n = 252).
