The knowledge of the prevalence of drug resistant tuberculosis in the population is important information about the epidemiology of the disease in the country. In Poland, the incidences of drug-resistant tuberculosis have been monitored since the early years of chemotherapy. In the period from 1997 to 2012 were four prospective studies covering the whole country. The aim of this study was to determine the prevalence primary and acquired drug resistance of M. tuberculosis strains isolated from patients in Poland in 2012, compared with the results obtained in previous studies (1997-2008). Material and methods: The material for the work was taken from 4781 tuberculosis patients excreting susceptible and drug resistant bacilli during the 12-months from 1st January to 31st December 2012. Data about patients were collected based on the results of routine diagnostic tests carried out in tuberculosis laboratories. Division by age group, verification and analysis of data was performed according to WHO recommendations. Drug susceptibility testing was performed on solid medium (L-J) by proportion method and liquid medium using the Bactec MGIT 960 system. Results: In this study we were included 4136 patients with tuberculosis (86.9% new and 13.1% treated cases). Among 3596 studied untreated patients 157 (4.4%) excreting drug resistant bacilli. MDR-TB was found in 12 (0.6%) patients and bacilli resistant to 4 drugs in 7 patients (0.2%).Among previously treated patients 63 (11.7%) excreting drug resistant bacilli. MDR-TB was found in 24 patients (4.4%), bacilli resistant to 3 drugs SM+INH+RMP (11 patients – 2.0%) and 4 drugs SM+INH+RMP+EMB (6 patients – 1.1%). Conclusions: Among untreated patients 157 (4.4%) excreting drug resistant bacilli, in previously treated patients 63 (11.7%). Excreting drug resistant bacilli MDR-TB was more often in previously treated (4.4%) than untreated patients (0.6%).

Objective: The most important measures in the control of the spread of drug-resistant tuberculosis (DR-TB) are the early detection of smear-positive patients, implementation of appropriate treatment, and tracing the chain of transmission of infection. Research on TB transmission in the environment of patients indicates that the risk of infection between close contacts, especially family members, is very high. The purpose of this study was to investigate the transmission of DR-TB within 6 family-households identified during the period of 2006-2016 in Poland. Methods: Two PCR-based genotyping methods were used: spoligotyping, and mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTR) typing. Results: According to spoligotyping and MIRU-VNTR results, in all households, patients had identical Mycobacterium tuberculosis isolates, implying intrafamilial transmission. Isolates from 2 families with Beijing-TB represented the pre-XDR and XDR phenotypes. Conclusions: This study demonstrates the household setting as an important pathway of drug-resistant Mycobacterium tuberculosis transmission, and thereby reinforces the need for routine extensive screening of the housemates of TB patients.

Major issues are currently associated with Tuberculosis (TB) treatment, particularly in patients infected by Multi-drug Resistant Tuberculosis/Extensively Drug Resistant Tuberculosis (mdr-TB/XDR-TB) resistant mycobacteria. A new threat recently reported in various Asian countries is totally Drug Resistant Tuberculosis (TDR). The presence of such Mycobacterium tuberculosis strains is disturbing also for the reasons they spread beyond the continent of Asia. The currently recommended tuberculosis treatment regimen is not well received by patients due to its minimum six-month, complexity, and common adverse events. The prevalence of MDR-TB and XDR-TB are inversely correlated with the quality of TB control and the proper use of second-line anti-TB drugs. Moreover, cost is extraordinary high. Since the mid-1960s only two new anti-TB drugs, bedaquiline and delamandine, have come to market; however, these drugs are not available in many regions and are limited to severely resistant cases. Currently, new derivatives such as spectinoamide are of interest in tuberculosis treatment. In vitro results and animal studies are used to aid in drug development. There is an urgent need for treatment improvement through enhancement of existing agents. Namely, individual differences in absorption and excretion of the primary anti-TB drugs, isoniazid and rifampin, require consideration. Recently, several studies attempted to evaluate the effect of anti-TB drug concentrations on treatment outcomes. Authors showed that 50-76% of the tested patients had low concentrations of INH (Isoniazid) and RMP (Rifampin). Because Therapeutic Drug Monitoring (TMD) was performed in small numbers of selected patients with comorbidities or slow treatment responses, the studies did not clearly demonstrate the effect of low drug levels on treatment outcomes. Future coordinated research is required. New molecular tests allow for research using supervised, individualized treatment of tuberculosis. In addition, effective tuberculosis outcomes require coordinated action multiple parameters for patient detection through implementation of rapid microbiological and clinical tests as well as reliable drug resistant tests of Mycobacterium tuberculosis. This leads to a break in the chain of transmission, and prevents the spread of disease in community. Education plays in important role for patients and families concerning the causes of disease and prevention methods. Additionally, medical staff should also themselves improve the level of diseases knowledge. Behaviour changes in tuberculosis infection control among medical personnel is also required. Keep in mind that one of the reasons for the relapse of tuberculosis is its disregard.

Leprosy or Hansen disease is caused by an infection of Mycobacterium leprae. The large number of undetected cases (2000-2012 years 4 mln people) remains a threat to the elimination of leprosy. Leprosy is an unheard in Poland and generally is considered a condition so "exotic" that it is not worth to spend more attention to it. Forgotten disease in developed countries still thrives in an environment of poor and uneducated. Regardless of the conclusion that in the 21st century none infectious disease should not be treated as a disease on the designated regions of the world, other than our own, it should be recalled that the M. leprae was discovered in Europe, where for many years there were leprosaria and still infectious hospitals in Great Brittan, France or Spain get patients suspected of leprosy. The mobility of the inhabitants of the globe caused by wars, ethnic conflicts or a simple tourism causes that any infectious disease can not be treated as solely limited to distant us regions. The best proof of this were the viral diseases, formerly found in only in Asia or Africa, and currently transmitted to Europe [1]. At any moment, we can stand up against the problem of diagnostics of humans toward leprosy. Many medical reports indicate that leprosy as a disease with many symptoms encountered difficulties in its diagnosis. Only the experience of medical professionals and good microbiological diagnosis may speed up the diagnosis of leprosy

Rapid identification of drug-resistance in Mycobacterium tuberculosis infections is crucial for early treatment and control of transmission. The MTBDRplus assay is potentially a practical and rapid alternative to the slower phenotypic drug susceptibility testing (DST) for detection of drug resistant tuberculosis (TB). The purpose of this study was to determine the diagnostic accuracy of the MTBDRplus assay. Sixty strains of Mycobacterium tuberculosis isolated from TB patients diagnosed in different parts of Poland in the two year period 2015-2016 were used. All strains were analyzed for drug susceptibility using both a conventional indirect proportion method and the MTBDRplus assay. The MTBDRplus assay identified 72% (43) of isolates as multidrug-resistant TB (MDR-TB), 7% (4) as INH-monoresistant and 5% (3) as RIF-monoresistant. The sensitivity and specificity of the MTBDRplus assay were 96% and 100% with respect to both RIFand INH-resistance, while sensitivity of the assay was lower (91%) for MDR-TB. In our study, codon 531 of rpoB gene and codon 315 of katG gene were found to have highest mutational frequency for RIF resistance (74% – 34 strains) and INH resistance (89% – 42 strains) respectively. The use of MTBDRplus assay as a supplement to the gold standard DST in obtaining a rapid preliminary DST result may contribute to early optimization of treatment.

BACKGROUND:Mutations in several genetic loci have been implicated in the development of resistance to second-line anti-tuberculosis (TB) drugs (SLDs). The purpose of this study was to investigate the prevalence of resistance to SLDs and its association with specific mutations in multidrug-resistant (MDR) Mycobacterium tuberculosis clinical isolates.MATERIALS AND METHODS:The study included 46 MDR-TB isolates. Mutation profiling was performed by amplifying and sequencing the following six genes: gyrA/gyrB, rrs, tlyA, and ethA/ethR, in which mutations are implicated in resistance of tubercle bacilli to ofloxacin (OFX), amikacin (AMK), capreomycin, and ethionamide (ETH), respectively.RESULTS:Of the strains analyzed, 14 (30.4%) showed resistance to at least one of the four SLDs tested. Mutations in the gyrA gene occurred in 34 (73.9%) strains, with the most common amino acid change being Ser95Thr. The Asp94Asn and Ala90Val substitutions in the gyrA were present exclusively in OFX-resistant strains, yet represented only 40% of all OFX-resistant strains. The only mutation in the gyrB gene was substitution Ser447Phe, detected in one OFX-resistant isolate. None of the AMK-resistant strains carried a mutation in the rrs gene. Mutations in the ethA/ethR loci were found in one ETH-resistant and 11 ETH-susceptible strains.CONCLUSIONS:The results of this study challenge the usefulness of sequence analyses of tested genes (except gyrA) for the prediction of SLD resistance patterns and highlight the need for searching other genetic loci for detection of mutations conferring resistance to SLDs in M. tuberculosis.

INTRODUCTION In 2007, Poland, the Czech Republic, and Slovakia joined the Schengen Agreement, abolishing restrictions on people crossing the borders. Currently, these areas are places of population movements for economic, family, and touristic reasons. This favors the transmission of infectious diseases, including tuberculosis, and requires enhanced control over the spread of the source of infection in the population of patients living in the border areas. OBJECTIVES The aim of this study was to investigate the genetic relatedness among Mycobacterium tuberculosis complex strains isolated from patients living in 3 border areas: Poland, the Czech Republic, and Slovakia. PATIENTS AND METHODS The study group consisted of 209 patients with tuberculosis diagnosed and treated between 2007 and 2011 in health care facilities in the Silesia Province in Poland (121 patients [58%]), Žilina in Slovakia (57 [27%]), and the Moravian-Silesian Region in the Czech Republic (31 [15%]). Genotyping of strains was performed using spoligotyping and IS6110-Mtb1-Mtb2 polymerase chain reaction. RESULTS Among 209 strains, 23 molecular families (clusters) were identified. Seventeen clusters were identified as national. Six international clusters consisted of 30 strains isolated from patients of various nationalities. CONCLUSIONS We identified 6 potential outbreaks of tuberculosis transmission between patients of different nationalities. The circumstances favorable to potential contacts of patients included mainly travelling to the neighboring countries, hospital stays, and addictions. However, there was no evidence of an epidemiological link between these patients, so it may be assumed that if they had come in contact with one another, it was accidental. We observed that the greater incidence of tuberculosis on the Polish territory did not affect the incidence in the Czech Republic or Slovakia over the analysis period.

The diagnosis of cutaneous tuberculosis poses a serious challenge due to many skin diseases of different etiology resembling the lesions caused by the TB (tuberculosis) bacillus, and difficulties in confirming the disease. The presented case concerns skin lesions in a hobby aquarist stung in the finger of the left hand by a fish. The resulting inflammatory infiltration was to be cutaneous tuberculosis or mycobacteriosis caused by MOTT (Mycobacterium other than tuberculosis). Laboratory, pathomorphologic, genetic and microbiologic tests of samples obtained from the patient, fish and water in the aquarium gave ambiguous results. A multidisciplinary discussion is presented on the difficulties in the differential diagnosis, problems with a clear interpretation of the results of various conducted tests, and possible ways of transmission of the infection, relevant to the described example.

OBJECTIVES:Progress in the detection of drug-resistant TB has been underpinned by the development and implementation of new, reliable and rapid diagnostic tools. These rely mostly on the detection of specific mutations conferring resistance to anti-TB drugs. The aim of this study was to search for mutations associated with isoniazid resistance among Mycobacterium tuberculosis clinical isolates.METHODS:A collection of 150 M. tuberculosis strains, including 50 MDR, 50 isoniazid-monoresistant and 50 pan-susceptible strains, was used. For all the strains, seven structural genes (katG, inhA, ahpC, kasA, ndh, nat and mshA) and two regulatory regions (mabA-inhA promoter and oxyR-ahpC intergenic region) were PCR amplified and sequenced in their entirety.RESULTS:Sixty-six distinct mutations were detected at all nine loci investigated, accounting for 109 (72.7%) of the strains tested. The number of strains with any mutation among the MDR, isoniazid-monoresistant and pan-susceptible groups was 49 (98%), 37 (74%) and 23 (46%), respectively. Mutations in the katG gene predominated, with 29 different types distributed among 46 (92%) MDR, 31 (62%) isoniazid-monoresistant and 2 (4%) pan-susceptible strains. Twenty-nine and 19 mutations were found exclusively in MDR and isoniazid-monoresistant strains, respectively.CONCLUSIONS:This study revealed 17 mutations, previously unreported, that might be of potential use as new surrogate markers of isoniazid resistance. Their diagnostic accuracy needs to be confirmed on larger strain samples and from different geographical settings. For isoniazid resistance detection, molecular approaches should still be a complement to rather than a replacement for conventional drug susceptibility testing. This is supported by the lack of mutations in any of the nine genetic loci investigated in 18 isoniazid-resistant strains from this study.

Introduction. The most detrimental impact on the epidemiology of tuberculosis (TB) in Poland may have the spread of tubercle bacilli from outside the country. This is because Poland is geographically situated in close proximity to the former Soviet Union countries, such as Latvia, Estonia, Ukraine, and Russia, where the incidence of TB, including drugresistant (DR) TB is exceptionally high. Aim. To describe the genetic diversity of DR Mycobacterium tuberculosis isolates, circulating in four provinces (Lublin, Podkarpacie, Podlasie, and Warmia-Masuria) constituting the so-called “Eastern Wall” of Poland. Material and methods. A total of 44 DR M. tuberculosis isolates were spoligotyped. The results were compared with the international spoligotype database (SpolDB4). SpolDB4 and the web-based program SpotClust were used to classify isolates into phylogenetic clades. Results. Spoligotyping resulted in 25 distinct patterns, of which five were not recorded in the SpolDB4. Of the remaining 20 types, 16 (80%) had already been reported in Poland. Most of the spoligotypes (56%) identified were found to be present in Poland’s neighbors, such as Germany, the Czech Republic, Latvia, and Russia. At the phylogenetic level, the T and Haarlem families accommodated 80% of the isolates. Conclusions. The M. tuberculosis population in Eastern Poland displayed features characteristic of a European country. Given the low frequency of imported genotypes and the absence of foreign-born patients, DR-TB in Eastern Poland appears to be due to the local transmission of M. tuberculosis strains that have been active in Poland for many years, rather than to the importation of strains from neighboring countries.

Summary Introduction. The most detrimental impact on the epidemiology of tuberculosis (TB) in Poland may have the spread of tubercle bacilli from outside the country. This is because Poland is geographically situated in close proximity to the former Soviet Union countries, such as Latvia, Estonia, Ukraine, and Russia, where the incidence of TB, including drug- resistant (DR) TB is exceptionally high. Aim. To describe the genetic diversity of DR Mycobacterium tuberculosis isolates, cir- culating in four provinces (Lublin, Podkarpacie, Podlasie, and Warmia-Masuria) constitut- ing the so-called Wall of Poland. Material and methods. A total of 44 DR M. tuberculosis isolates were spoligotyped. The results were compared with the international spoligotype database (SpolDB4). Spol- DB4 and the web-based program SpotClust were used to classify isolates into phyloge- netic clades. Results. Spoligotyping resulted in 25 distinct patterns, of which five were not recorded in the SpolDB4. Of the remaining 20 types, 16 (80%) had already been reported in Poland. Most of the spoligotypes (56%) identified were found to be present in Poland's neighbors, such as Germany, the Czech Republic, Latvia, and Russia. At the phylogenetic level, the T and Haarlem families accommodated 80% of the isolates. Conclusions. The M. tuberculosis population in Eastern Poland displayed features characteristic of a European country. Given the low frequency of imported genotypes and the absence of foreign-born patients, DR-TB in Eastern Poland appears to be due to the lo- cal transmission of M. tuberculosis strains that have been active in Poland for many years, rather than to the importation of strains from neighboring countries.

Objectives: To determine the prevalence of isoniazid resistance-conferring mutations among multidrug-resistant (MDR) isolates of Mycobacterium tuberculosis from Poland.Methods: Nine genetic loci, including structural genes (katG, inhA, ahpC, kasA, ndh, nat and mshA) and regulatory regions (i.e. the mabA-inhA promoter and oxyR-ahpC intergenic region) of 50 MDR M. tuberculosis isolates collected throughout Poland were PCR-amplified in their entirety and screened for mutations by direct sequencing methodology.Results: Forty-six (92%) MDR M. tuberculosis isolates had mutations in the katG gene, and the katG Ser315Thr substitution predominated (72%). Eight (16%) isolates (six with a mutated katG allele) had mutations in the inhA promoter region and two such isolates also had single inhA structural gene mutations. Mutations in the oxyR-ahpC locus were found in five (10%) isolates, of which all but one had at least one additional mutation in katG. Mutations in the remaining genetic loci (kasA, ndh, nat and mshA) were detected in 12 (24%), 4 (8%), 5 (10%) and 17 (34%) MDR isolates, respectively. All non-synonymous mutants for these genes harboured mutations in katG. One isolate had no mutations in any of the analysed loci.Conclusions: This study accentuates the usefulness of katG and inhA promoter mutations as predictive markers of isoniazid resistance. Testing only for katG 315 and inhA -15 mutations would detect isoniazid resistance in 84% of the MDR M. tuberculosis sample. This percentage would increase to 96% if the sequence analysis was extended to the entire katG gene. Analysis of the remaining genetic loci did not contribute greatly to the identification of isoniazid resistance.

Multidrug-resistant tuberculosis (MDR-TB) poses a serious challenge to the global control of the disease. The purpose of this study was to characterize MDR-TB patients from Poland and to determine the extent of MDR-TB disease attributable to recent transmission. The study included all 46 patients diagnosed with MDR-TB in Poland in 2004 and followed up for 6 years (until 2011). For each patient, sociodemographic and clinical characteristics, treatment outcomes, and bacteriological data were collected by the review of medical and laboratory records. Mycobacterium tuberculosis isolates from all patients were characterized using spoligotyping, mycobacterial interspersed repetitive unit–variable number tandem repeat (MIRU-VNTR) typing, IS6110 restriction fragment length polymorphism (RFLP) analysis, and sequencing analysis of drug resistance-associated loci (katG, mabA-inhA, rpoβ, rpsL, and embB). The majority of patients were male (86.9 %), 40–64 years of age (60.8 %), with a history of TB treatment (84.8 %), and producing smear-positive sputa (86.9 %). Twenty-two (47.8 %) patients suffered from concomitant diseases and 28 (60.8 %) were alcohol abusers. Treatment outcome assessment revealed that 8 (17.4 %) patients were cured or completed therapy, while 15 (32.6 %) died of TB, 11 (23.9 %) defaulted, 8 (17.4 %) failed, and 1 (2.2 %) was transferred and lost to follow-up. Upon genotyping, 10 (21.7 %) isolates were allocated in four clusters. These were further subdivided by mutational profiling. Overall, in 6 (13 %) patients, MDR-TB was a result of recent transmission. For 4 (8.7 %) of these patients, a direct epidemiological link was established. The study shows that the transmission of MDR-TB occurs at a low rate in Poland. Of urgent need is the implementation of a policy of enforced treatment of MDR-TB patients in Poland.

Until recently, the basic test to identify latent tuberculosis infection (LTBI) was the tuberculin skin test, despite its limitations in the form of low sensitivity and specificity. Currently, Interferon Gamma Release Assays from peripheral blood are used for a rapid diagnosis of LTBI and measurement of the interferon gamma (IFN-g) levels secreted by specific T cells stimulated with Mycobacterium tuberculosis antigens. Detection of LTBI is important in the control of people potentially at risk of TB disease, such as people remaining in close contact with BK (+) tb patient and for patients evaluated for biological treatment. The paper presents the value of IGRA in three selected clinical situations: in two cases of latent tuberculosis infection and in one case of active tuberculosis.

Currently, mutations in three genes, namely rrs, rpsL, and gidB, encoding 16S rRNA, ribosomal protein S12, and 16S rRNA-specific methyltransferase, respectively, are considered to be involved in conferring resistance to streptomycin (STR) in Mycobacterium tuberculosis. The aim of this study was to investigate the spectrum and frequency of these mutations in M. tuberculosis clinical isolates, both resistant and susceptible to STR. Sixty-four M. tuberculosis isolates recovered from as many TB patients from Poland in 2004 were included in the study. Within the sample were 50 multidrug-resistant (32 STR-resistant and 18 STR-susceptible) and 14 pan-susceptible isolates. Preliminary testing for STR resistance was performed with the 1% proportion method. The MICs of STR were determined by the Etest method. Mutation profiling was carried out by amplifying and sequencing the entire rrs, rpsL, and gidB genes. Non-synonymous mutations in either rrs or rpsL gene were detected in 23 (71.9%) of the STR-resistant and none of the STR-susceptible isolates. Mutations in the gidB gene were distributed among 12 (37.5%) STR-resistant and 13 (40.6%) STR-susceptible isolates. Four (12.5%) STR-resistant isolates were wild-type at all three loci examined. None of the rrs, rpsL or gidB mutations could be linked to low, intermediate or high level of STR resistance. In accordance with previous findings, the gidB 47T→G (L16R) mutation was associated with the Latin American-Mediterranean genotype family, whereas 276A→C (E92D) and 615A→G (A205A) mutations of the gidB gene were associated with the Beijing lineage. The study underlines the usefulness of rrs and rpsL mutations as molecular markers for STR resistance yet not indicative of its level. The gidB polymorphisms can serve as phylogenetic markers.

In the fourth part of an ongoing study dealing with the search for new rifamycin antibiotics, the reaction of 3-formylrifamycin SV (1) with secondary amines (a.o. pyrrolidine) and ketones of general structure R1–CH2–CO–R2 (R1=H or alkyl and R2=alkyl or aryl) has been investigated. A new synthetic method for the preparation of stable zwitterionic rifamycin derivatives with α,β-unsaturated pyrrolidinium substituents at C3 of the naphthol chromophore has been developed. The structures of the isolated microcrystalline rifamycins and a general mechanism have been proposed on the basis of mass spectrometry results, FTIR, 1D and 2D NMR as well as TLC methods. Rifamycins displaying an aromatic substituent within the pyrrolidinium fragment at C3 displayed a good stability against hydrolysis and proved, in preliminary tests, to be active against TB and MOTT, the activity being for some particular strains (Myc. tbc. Bovis and Myc. scrofulaceum) higher than that observed with the reference drugs (RMP, RBT).