Leprosy - one of the many forgotten tropical diseases.

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.

'TB or not TB?' Problems of differential diagnosis of cutaneous mycobacteriosis and tuberculosis--A Case Study and interdisciplinary discussion.

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.

Transmission of tuberculosis among people living in the border areas of Poland, the Czech Republic, and Slovakia.

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%]), Zilina 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.

Second-line anti-tuberculosis drug resistance and its genetic determinants in multidrug-resistant Mycobacterium tuberculosis clinical isolates.

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.

Mutation profiling for detection of isoniazid resistance in Mycobacterium tuberculosis clinical isolates.

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.

Screening for streptomycin resistance-conferring mutations in Mycobacterium tuberculosis clinical isolates from Poland.

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.

Detection of mutations associated with isoniazid resistance in multidrug-resistant Mycobacterium tuberculosis clinical isolates.

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.

[Interferon-Gamma Release Assays in the diagnosis of latent tuberculosis infection in clinical situations]. / Testy IGRA w diagnostyce zakazenia pratkami gruzlicy w wybranych sytuacjach klinicznych.

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.

Identification and analysis of mutations in the katG gene in multidrug-resistant Mycobacterium tuberculosis clinical isolates.

INTRODUCTION: A major role in the development of resistance of Mycobacterium tuberculosis to isoniazid (INH) is attributed to mutations in the katG gene coding for the catalase/peroxidase, an enzyme required for obtaining a pharmacologically active form of the drug. Analysis of mutations in the katG gene in M. tuberculosis strains may contribute to the development of reliable and rapid tests for detection of INH resistance. The aim of the study was to identify and characterize mutations in the katG gene in multidrug-resistant M. tuberculosis clinical isolates. MATERIAL AND METHODS: The study included 46 strains of M. tuberculosis, recovered from MDR-TB patients in Poland in 2004. Mutations in the katG gene were detected by comparing DNA sequences with the corresponding sequence of a wild-type reference laboratory strain (M. tuberculosis H37Rv). The obtained results were interpreted in the context of MIC values of INH and catalase activity of the strains tested. RESULTS: A total of 43 (93%) strains contained mutations in the katG gene. The most frequently observed were mutations at codon 315, found in 34 (74%) strains. Mutations at other codons were rare: 4 strains contained mutations at codon 463, 2 at codon 131 and another 2 at codon 234. Mutations at codons 68, 91, 101, 126, 128 and 194 were found in single strains only. Two strains, for which no mutations at codon 315 of the katG gene were identified, had a unique translation termination mutation, which would invariably result in polypeptide truncation leading to the generation of dysfunctional catalase polypeptides. Both these strains presented the highest MIC values for INH (80 and 100 µg/mL) and showed a complete loss of catalase activity. For the remaining 41 strains with katG mutations, the MICs of INH were within the range 0.2-10 µg/mL. Thirty-six (88%) of those strains retained their catalase activity. CONCLUSIONS: Mutations at codon 315 within the katG gene, depending on their type might be useful for the prediction of INH resistance. Whereas the missense mutations do not affect the catalase activity or the level of INH resistance, the nonsense mutations result in high-level resistance to INH and a total loss of catalase activity.

[The biological role of prokaryotic and eukaryotic N-acetyltransferase]. / Rola biologiczna prokariotycznych i eukariotycznych N-acetylotransferaz.

The N-acetyltransferases (NAT; E.C.2.3.1.5) are involved in the metabolism of drugs and environmental toxins. They catalyse the acetyl transfer from acetyl coenzyme A to an aromatic amine, heterocyclic amine, or hydrazine compound. NAT homologues are present in numerous species from bacteria to human. Sequence variations in the human NAT1 and NAT2 result in the production of NAT proteins with variable enzyme activity or stability, leading to slow or rapid acetylation. Therefore, genetic polymorphisms in NAT1 and NAT2 influence drug metabolism and drug-related toxicity. Epidemiological studies suggest that the NAT1 and NAT2 acetylation polymorphisms modify the risk of developing cancers of the urinary bladder, colorectal, breast, head and neck, and lung.

Correlation of N-acetyltransferase 2 genotype with isoniazid acetylation in Polish tuberculosis patients.

UNLABELLED: Isoniazid (INH), a key agent in the treatment of tuberculosis (TB), is metabolized primarily by the genetically polymorphic N-acetyltransferase 2 (NAT2) enzyme. Patients treated with INH can be classified as fast, intermediate, and slow acetylators. The objective of this study was to explore the relationship between NAT2 genotypes and the serum concentrations of INH. Blood samples from 130 patients were taken for the analysis, and plasma INH concentrations were determined by using the high-performance liquid chromatography (HPLC) technology. Acetylation genotype was determined on genomic DNA by using an allele-specific polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. Once the NAT2 genotypes were established, patients were classified into three categories: fast, intermediate, and slow acetylators. Of the 130 patients studied, 84 (64.6%) were slow, 39 (30%) were intermediate, and 7 (5.4%) were fast acetylators. Analysis of INH concentrations in the blood of patients receiving the approximate doses of the drug revealed that, at the time intervals examined, the average concentration of INH was 2- to 7-fold higher among slow acetylators compared to fast and intermediate acetylators. CONCLUSION: Determining mutations in the NAT2 gene enabled the identification of the INH acetylation type in patients and the genotyping results were consistent with the phenotype determined by methods of measurement of drug bioavailability.

Mutations in the embB gene and their association with ethambutol resistance in multidrug-resistant Mycobacterium tuberculosis clinical isolates from Poland.

Ethambutol (EMB) continues to be used as part of a standard drug regimen for the treatment of tuberculosis (TB). Mutations in the embB gene and those within its conserved EMB resistance determining region (ERDR) in particular have repeatedly been associated with resistance to EMB in Mycobacterium tuberculosis. The aim of this study was to examine the mutational "hot spots" in the embB gene, including the ERDR, among multidrug-resistant (MDR) M. tuberculosis clinical isolates and to find a possible association between embB mutations and resistance to EMB. An 863-bp fragment of the embB gene was sequenced in 17 EMB-resistant and 33 EMB-susceptible MDR-TB isolates. In total, eight embB mutation types were detected in 6 distinct codons of 27 (54%) M. tuberculosis isolates. Mutations in codon 306 were most common, found in both EMB-resistant (9) and EMB-susceptible (11) isolates. Only mutations in codons 406 and 507 were found exclusively in four and one EMB-resistant isolates, respectively. Sequence analysis of the ERDR in the embB gene is not sufficient for rapid detection of EMB resistance, and the codon 306 mutations are not good predictive markers of resistance to EMB.

[Polymorphism in the N-acetyltransferase 2 gene in patients with lung cancer. Short communication]. / Polimorfizm w genie N-acetylotransferazy 2 u chorych na raka pluca. Doniesienie wstepne.

INTRODUCTION: Individual's risk of developing lung cancer depends not only on exposure to tobacco smoke, but also on the activity of enzymes involved in the activation or deactivation of carcinogens. Arylamine N-acetyltransferase (EC 2.3.1.5) is an enzyme involved in biotransformation of xenobiotics, mainly aromatic and heterocyclic amines and hydrazines. The different acetylation phenotypes within a population are derived from mutations in the NAT 2 gene. These mutations influence the activity (specifically resulting in high or low activity) of the NAT enzyme. Some authors have demonstrated lung cancer predisposing role of slow acetylator phenotype, whereas other reported increased lung cancer risk for fast acetylators or neutral effect of the NAT2 polymorphism. The aim of this preliminary report was to determine the NAT2 gene polymorphism in patients with lung cancer. MATERIAL AND METHODS: 39 patients with inoperable lung cancer (29 - NSCLC and 10 - SCLC), median age 59 years (42- -72) entered the study. Acetylation genotype was determined in the genomic DNA using an allele-specific polymerase chain reaction. We investigated four genetic mutations, C481T, G590A, A803G i G857A, of the gene NAT2. RESULTS: There were 10 different NAT2 genotypes among the 39 patients. Fourteen patients with a NAT2*2 4/4, *4/5, *4/6 and *4/7 were classified as fast acetylators; and 25 patients with a NAT2*5/5, *5/6, *5/7, *6/6, *6/7 or *7/7 genotype were classified as slow acetylators. Among the 10 patients with SCLC - 4 were fast acetylators, and among 29 patients with NSCLC dominated slow acetylation type found in 19 patients (genotypes NAT2 *5/5 and NAT2 *5/6). CONCLUSIONS: Among patients with small cell lung cancer, there was no predominance of genotype of acetylation, whereas among patients with non-small cell lung cancer predominated NAT2*5/5 and NAT2*5/6 genotypes (slow acetylators).

[Molecular analysis of strains from tuberculosis patients in Polish prisons in 2004-2008. Initial analysis of the project]. / Molekularne dochodzenia epidemiologiczne wsród polskich wiezniów chorych na gruzlice w latach 2004­2008. Badania wstepne.

INTRODUCTION: Correctional facilities are recognised breeding ground for infectious diseases. As The World Health Organization reported the incidence of infectious diseases in prison's population is 10-100 times higher than in general population. The incidence of tuberculosis among correctional inmates in Poland in 2008 was 270/100000, that is around 10 times higher than among non-prisoners. MATERIALS AND METHODS: The study included 57 M. tuberculosis isolates from patients in Polish prisons in 2004-2008 (5% of all diagnosed TB patient in Polish prisons 2004-2008). Primary isolation was performed with Löwenstein-Jensen (L-J) medium, species identification was done with the niacin test and gene probes test. Bacterial DNA was extracted from the L-J medium slants with the cetyltrimethylammonium bromide (CTAB) method. Mycobacterium tuberculosis strains were analyzed with two methods: screening for epidemiological discrimination of M. tuberculosis - spoligotyping and high-throughput - MIRU/VNTR. RESULTS: Isolates that are grouped in clusters (33 isolates) were analyzed by means of MIRU/VNTRs. In MIRU/VNTRs all strains showed different genetic patterns. Most isolates of the prisoners were grouped into two clusters: T1 53 and H3 50. CONCLUSIONS: 1. MIRU/VNTR is a high-throughput method. 2. MIRU/VNTR is a promising method to diagnose TB transmission in Polish jails. 3. To identify the probable source of transmission, molecular analysis of strains from patients of the general population is needed.

Transmission of tuberculosis within family-households.

OBJECTIVE: The introduction of molecular typing methods in the 1990s to study the epidemiology of tuberculosis (TB) has significantly improved the possibilities of quantifying transmission of Mycobacterium tuberculosis in different human settings. The purpose of this study was to investigate transmission of TB in 35 family-households in Poland. METHODS: Two PCR-based genotyping methods: spoligotyping and mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTR) typing were used. RESULTS: Of 78 patients, 49 (63%), could be assigned to intra-household transmission on the basis of identical DNA fingerprints upon a combined typing approach. However, if a single spoligotype spacer or a single MIRU-VNTR locus variation was tolerated in the cluster definition, the intra-household transmission raised to 85% of all patients. For 12 patients in 6 households, the M. tuberculosis isolates were clearly distinct in either spoligotyping or VNTR typing or in both genotyping methods, suggesting that these patients were infected by the sources in the community. CONCLUSIONS: This study is the first to provide the results of a molecular epidemiological investigation performed within family-households in Poland. It shows the household setting as an important reservoir of M. tuberculosis transmission, and thus argues in favor of routine and extensive screening of the family contacts of TB patients.

Synthesis, structure and biological evaluation of novel bicyclic nitroimidazole derivatives.

A new series of 3-hydroxy-8-nitroimidazo[5,1-b]-1,4,5,6-tetrahydropyrimidine systems, being potential tuberculostatic agents, were synthesized. These products are close structural analogs of the basic structure of the known antitubercular bicyclic nitroimidazooxazine PA-824. The structures of the products obtained were confirmed by X-ray methods on the example of 3-hydroxy-8-nitro-1-phenylaminoimidazo[5,1-b]-1,4,5,6-tetrahydropyrimidine. Evaluation of these products for their anti-tuberculosis effects revealed interesting structure-activity relationships.

The capacity of Mycobacterium tuberculosis complex species and M. bovis BCG substrains specific identification - implications for optimized PCR-based diagnostics in adverse events following vaccination suspected cases.

The capacities of differentiation of Mycobacterium bovis BCG from other members of M. tuberculosis complex species using PCR-RFLP, multiplex PCR, and PCR-based genomic deletion analysis approaches were compared. In the study, mycobacteria isolated from patients suspected of adverse events following vaccination with BCG, primarily classified according presence of RD1 marker as virulent and avirulent mycobacteria, were used. The PCR-based genomic deletion analysis was found the best option for mycobacteria diagnostics improvement, as it was capable precisely differentiate virulent and avirulent mycobacteria or virulent species of M. tuberculosis complex. The routine confirmation of mycobacteria species in the cases of adverse events following BCG vaccination is highly expected, especially in clinical practice of patients with primary immunodeficiency.