**1. Introduction**

Tuberculosis (TB) is an infectious disease caused by mycobacteria of the *Mycobacterium tuberculosis* complex. Until the outbreak of the coronavirus pandemic (COVID-19), TB was the leading cause of death from a single infectious agent, ranking higher than HIV (AIDS). Unfortunately, the COVID-19 pandemic set back years of progress in the fight against tuberculosis, causing a global decline in the number of newly diagnosed and reported TB patients. The number of newly diagnosed TB patients has declined by approximately 20%, to the level recorded in 2012 (from 7.1 million in 2019 to 5.8 million in 2020) [1], representing a setback of at least 5 to 8 years in the fight against TB due to the COVID-19 pandemic. Limited access to diagnosis and antimycobacterial treatment has also resulted in an increase in deaths. In 2020, for the first time in more than a decade, an increase of more than 100,000 deaths was recorded, reaching 1.3 million [1]. The consequences of not having access to basic TB diagnostic and treatment services are expected to rise in future years. Additionally, the number of patients treated for drug-resistant TB decreased by 15%, from 177,000 in 2019 to 150,000 in 2020 [1]. Poland is one of the EU member states demonstrating low TB incidence rates (13.9 cases per 100,000 in 2019). In 2010, a decline in TB incidence to <20 per 100,000 population was first reported, and the downward trend has continued since then [2]. It is worth mentioning that the incidence rates of TB in post-war Poland were extremely high, at >290 per 100,000; thus, a substantial percentage of the population was infected with mycobacterium tuberculosis, and the immediate eradication of the disease was not possible. The radical decrease of epidemiological indicators in tuberculosis is certainly one of the greatest successes in Polish medicine [3]. In 2019, 5321 cases of

**Citation:** Borkowska-Tatar, D.; Zabost, A.; Kozi ´nska, M.; Augustynowicz-Kope´c, E. Tuberculosis in Poland: Epidemiological and Molecular Analysis during the COVID-19 Pandemic. *Diagnostics* **2022**, *12*, 1883. https://doi.org/10.3390/ diagnostics12081883

Academic Editor: Philippe A. Grenier

Received: 29 June 2022 Accepted: 2 August 2022 Published: 3 August 2022

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tuberculosis were registered in Poland, which is 166 cases of tuberculosis fewer than in the previous year and 2188 cases fewer than in 2010. The incidence of all forms of TB was 13.9 in 2019, down 2.8% from 2018 and down 29.4% from 2010, when it was 19.7 [4]. In 2020, in the midst of the COVID-19 pandemic, 3388 TB cases were registered, which was 1993 fewer than the previous year. The incidence of all forms of TB was 8.8 in 2020, a decrease of 36.7% compared to 2019 [2]. Despite significant improvement in the epidemiological situation, the prevalence of tuberculosis is slightly higher than the figures in other Western European countries: for example, 8.1 per 100,000 inhabitants in Germany, 7.7 in France, or 8.2 in Sweden [5]. Among the methods of diagnosing TB, microbiological methods are the gold standard and are crucial, as they allow for correct diagnosis and rapid initiation of treatment with the most effective regimen. Most clinical features of TB have low specificity, which can lead to misdiagnosis and unnecessary treatment [6].

The drug-resistant form of TB, in particular multidrug-resistant (MDR), pre-extensively drug-resistant, and extensively drug-resistant (XDR), constitutes a recent health concern and challenge for TB-control programmes worldwide. Monitoring the drug resistance of *Mycobacterium tuberculosis* strains to antituberculosis drugs is an important aspect of TB surveillance and is helpful in identifying the predominant MDR-TB strains and in indicating the quality of TB control in a country. Early detection and diagnosis of patients prevents transmission of drug-resistant strains in the environment [7]. The priority should be to restore access to essential TB services and increase spending on diagnostics, treatment, and prevention so that detection and treatment levels can return to at least those of 2019 [1].

The aim of this study was to investigate the effect of the COVID-19 pandemic on the diagnosis of tuberculosis in Poland and the patterns of resistance to basic antimycobacterial drugs shown by *M. tuberculosis* strains isolated in both newly diagnosed and previously treated patients. The epidemiological analysis of the 2020 strains was a cyclic study (conducted every 4 years) at the National Reference Laboratory for Mycobacteria at the Institute of Tuberculosis and Lung Diseases in Warsaw, according to the WHO protocol. The results were compared not only with data obtained in 2012 and 2016 but also with data reported to the National Tuberculosis Registry (NTR) [2,8,9]. Molecular analysis was performed on 20% of the strains susceptible to the four primary antimycobacterial drugs and all of the resistant strains in order to determine the frequency of specific molecular patterns of shared international type (SIT) spoligotype in the group of strains susceptible and resistant to antimycobacterial drugs.

#### **2. Materials and Methods**

The study was retrospective and prospective, based on the results of routinely performed microbiological tests at Mycobacterium Tuberculosis Laboratories in Poland. *Mycobacterium tuberculosis* complex strains cultured in regional laboratories were sent to the National Reference Laboratory for Mycobacteria at the Institute of Tuberculosis and Lung Diseases in Warsaw, together with a questionnaire containing information on the strain (the specimen from which it was cultured, basic identification tests, and drug resistance determined in the field laboratory) and data on the patient (sex, age, form of tuberculosis, and previous antimycobacterial treatment). The isolation was performed using Löwenstein– Jensen medium or BD Bactec MGIT system (Becton Dickinson Microbiology Systems, Cockeysville, MD, USA), with species identification based on niacin tests, the use of BD MGIT TBc identification test (TBc ID), and nucleic acid amplification test (NAAT). Drug susceptibility testing used the proportion method in Löwenstein–Jensen medium or using the BD Bactec MGIT 960 system.

The total number of *Mycobacterium tuberculosis* strains analysed in 2020 was 1383. The results were compared to studies conducted in 2012 and 2016. The programmes in these years followed the same WHO protocol and included approximately 9000 TB patients. Molecular analysis entailed spoligotyping for 20% (252) of the strains susceptible to 4 antimycobacterial drugs and for all (82) of the strains resistant to at least 1 drug from 2020. This is a pilot study. In the next stages, spoligotyping will be performed for the remaining 80% of strains sensitive to antituberculosis drugs.

#### *Method for Spacer Oligonucleotide Typing (Spoligotyping)*

Spoligotyping was performed by amplifying direct repeat regions in the genome of *M. tuberculosis* complex with the primers DRa and DRb and an available spoligotyping kit (Ocimum Biosolutions, Hyderabad, India) according to the protocol [10]. The amplified products were then hybridised to a membrane pre-coated with spacer oligonucleotides that characterise the spacer region of the identified sequence. After incubation with streptavidinperoxidase and enhanced chemiluminescence detection, the presence of spacers was visualised on X-ray films as black squares [11,12]. *M. tuberculosis* H37Rv was used as a positive control. The resulting spoligotypes were compared to the patterns registered in the SITVIT2 international database, available at http://www.pasteur-guadeloupe.fr:8081/SITVIT2 (accessed on 1 February 2022).

Among the 96 drug-resistant strains analysed, hybridisation patterns were obtained for 82. The strains were cultured from 64 (78%) Polish citizens and 18 (22%) foreigners living in Poland (from Ukraine, Georgia, Moldova, Vietnam, Nepal, and the Philippines). As with the resistant strains, genotyping was performed by spoligotyping. Hybridisation patterns were obtained for 252 out of 1287 sensitive strains. Strains were cultured from 201 (79.8%) Polish citizens and 51 (20.2%) foreigners living in Poland (from Ukraine, India, Nepal, Bangladesh, South Africa, and other countries).
