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Veterinary Microbiology 04/2012; 159(1-2):269-71. · 3.33 Impact Factor
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ABSTRACT: 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.
Two PCR-based genotyping methods: spoligotyping and mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTR) typing were used.
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.
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.
The Journal of infection 02/2012; 64(6):596-608. · 4.13 Impact Factor
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ABSTRACT: 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.
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.
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.
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.
Pneumonologia i alergologia polska: organ Polskiego Towarzystwa Ftyzjopneumonologicznego, Polskiego Towarzystwa Alergologicznego, i Instytutu Gruzlicy i Chorob Pluc 01/2012; 80(3):209-13.
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ABSTRACT: The diagnosis of latent tuberculosis infection (LTBI) is currently based on the century-old tuberculin skin test (TST). However a positive reaction can result from infection by Mycobacterium tuberculosis, BCG vaccination or cross-reaction with nontuberculous mycobacteria. T-SPOT.TB assay is a new test to diagnose tuberculosis infection by measuring in vitro T-cell interferon gamma release in response to two Mycobacterium tuberculosis-specific antigens: ESAT-6 and CFP 10.
T-SPOT.TB assay has been performed on whole blood samples (n = 137) from March to September 2010. A tuberculin skin test result was available for 96 of participants. A positive TST result was considered if the induration was 10 mm or more.
Of the 137 patients tested, T-SPOT.TB assay results were positive in 37 (27%), negative in 98 (71.5%) and indeterminate in only 2 (1.5%) persons. We analyzed T-SPOT.TB and TST results in the 96 patients for whom both test were available. Concordance between T-SPOT.TB and TST results (10 mm skin reaction interpreted as positive) was 79%. Fifteen (15.6%) patients had a positive TST result and a negative T-SPOT.TB and 5 (5.2%) patients had a negative TST result and a positive T-SPOT.TB. We observed good correlation between positive T-SPOT.TB results and the size of induration ≥ 15 mm in TST results.
T-SPOT.TB offers a more accurate approach than TST for identification tuberculosis infection. The study shows that the test T-SPOT.TB is a good diagnostic tool in identifying persons with tuberculosis infection. For full confirmation of this assessment, it is necessary to examine more cases.
Pneumonologia i alergologia polska: organ Polskiego Towarzystwa Ftyzjopneumonologicznego, Polskiego Towarzystwa Alergologicznego, i Instytutu Gruzlicy i Chorob Pluc 01/2011; 79(4):264-71.
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ABSTRACT: Tuberculosis (TB) is a curable disease and its spread can be prevented by using appropriate diagnostic methods and effective treatment. The obstacle to the rapid eradication of the disease from a population may be strains resistant to essential and most effective antibiotics. In many places in the world MDR, pre-XDR and XDR-TB was reported. These forms of TB do not respond to the standard six-month treatment with first-line anti-TB drugs and the therapy should be conducted two years or more with drugs that are less potent, more toxic and much more expensive.
This study included MDR-TB strains isolated from 297 patients in 2000-2009. To determine the XDR-TB population structure, the 19 isolates were genotyped by spoligotyping and MIRU-VNTR (mycobacterial interspersed repetitive units-variable number of tandem repeats) method.
Among 297 MDR-TB cases, 36 (12.1%) were pre-extensively drug-resistant (pre-XDR), 19 (6.4%) were XDR and 1 (0.3%) was pre-totally drug-resistant (pre-TDR). Four of the 19 XDR isolates exhibit a unique spoligopattern, while the rest 15 belonged to one of 5 clusters. The MIRU-VNTR analysis reduced the number of clustered isolates to 11.
The study documented the emergence of pre-extensively and extensively drug-resistant tuberculosis in Poland among patients with multidrug-resistant TB. Genotyping methods showed clonal similarity among XDR strains and may suggest the possible transmission among patients with newly diagnosed and with recurrent TB.
Pneumonologia i alergologia polska: organ Polskiego Towarzystwa Ftyzjopneumonologicznego, Polskiego Towarzystwa Alergologicznego, i Instytutu Gruzlicy i Chorob Pluc 01/2011; 79(4):278-87.
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Monika Kozińska,
Ewa Augustynowicz-Kopeć,
Zofia Zwolska,
Sylwia Brzezińska,
Anna Zabost,
Monika Anielak,
Magdalena Klatt,
Agnieszka Napiórkowska,
Maria Bełzowska,
Mirosława Dabrowska,
Robert Grzesica,
Maria Kowalska,
Dorota Krawiecka,
Lidia Maciak,
Bozena Piskuła,
Alicja Sankowska,
Krzysztof Słodowski,
Wioletta Szymkowicz,
Wojciech Zulikowski
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ABSTRACT: The aim of this study was estimation of usefulness of two molecular methods for Mycobacterium tuberculosis typing in the epidemiological research of tuberculosis (TB). We determined molecular patterns of M. tuberculosis strains isolated from 66 patients, members of 29 families living in 9 voivodeships of Poland. We also analysed drug susceptibility of the strains as well as some demographic characteristics of the patients.
The genotype analysis of the 66 clinical isolates was performed by using spoligotyping and IS6110-Mtb1/Mtb2 PCR.
Of the 29 families examined in this study, in 23 each family member was infected with the same M. tuberculosis strain. Three drug-resistant strains and two members of the Beijing family were identified.
We found that strains within each of the 23 families had the same genetic patterns, whereas those of the strains identified in the rest 6 families were different. Among those 6 families, in 3 differentiation of the strains was obtained with both spoligotyping and IS6110-Mtb1/Mtb2 PCR analysis, while in another 3 only with spoligotyping method.
Based on the results from this study, the two genotyping methods used were demonstrated as an efficient approach for investigating the epidemiological relatedness of TB cases.
Przegla̧d epidemiologiczny 02/2008; 62(1):55-62.
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ABSTRACT: Isoniazid (INH) is a drug widely used in the treatment of tuberculosis. INH is metabolized to acetylisoniazid by N-acetyltransferase (NAT) in the liver. The rate of INH acetylation is genetically determined. NAT isozymes are encoded at 2 loci; one encodes NAT1, formerly known as the monomorphic form of the enzyme, while the other encodes the polymorphic NAT2, which is responsible for individual differences in the ability to acetylate certain compounds. The objective of the present study was to apply the genotyping of the fast and slow acetylators for personalized therapeutic dose.
Plasma concentrations of INH were determined with biological method in the authors modification. This method warrants high accuracy and secured repeatable results. Genomic DNA was isolated from the blood samples. DNA extracted by Blood DNA Kit and amplified by PCR by Spurr [1] with two primers. PCR product was cut separately with 4 different restriction enzymes: Dde1, Kpn1, Tag1, and BamH1.
Four different NAT 2 alleles were detected in the study population. The presence of any 2 mutant alleles defines the slow-acetylator genotype, whereas rapid acetylators have 1 or 2 wild-type NAT2*4 alleles.
On the basis of our results we suggest the using of NAT 2 genotyping for discrimination of the fast and slow acetylators in monitoring of tuberculosis therapy.
Pneumonologia i alergologia polska: organ Polskiego Towarzystwa Ftyzjopneumonologicznego, Polskiego Towarzystwa Alergologicznego, i Instytutu Gruzlicy i Chorob Pluc 02/2007; 75(2):134-9.
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ABSTRACT: The control of tuberculosis (TB) requires methods for rapid detection and tracing sources of infection, so that further transmission can be arrested. Recent developments in molecular biology have resulted in techniques that allow prompt identification and tracking specific strains of M. tuberculosis as they spread through the population. Most of these techniques take advantage of M. tuberculosis DNA polymorphism and are based on various repetitive DNA elements as genetic markers. Each method yields strain-specific genetic profiles (fingerprints). Strains showing identical fingerprints are referred to as clustered and are usually associated with recent transmission, whereas strains whose fingerprints are unique are presumed to represent remote transmission, a reactivation of infection acquired in the distant past.
In recent years, spoligotyping has become one of the most widely used genotyping method for epidemiological studies of TB. Spoligotyping is a PCR-based method allowing to analyze strain-dependent polymorphisms observed in spacer sequences present within the direct repeat (DR) genomic region of M. tuberculosis complex strains. Spoligotyping provides some important advantages over other genotyping techniques. These are simplicity, rapidity, high reproducibility and stability of the results, with the latter being expressed in a simple digital pattern, readily named and databased, and the ability to perform spoligotyping directly on clinical samples, without the need for prior culture. However, spoligotyping has relatively low discriminatory capacity, which makes it necessary to use secondary fingerprinting methods to prove clonality between isolates.
The aim of this study was to evaluate the usefulness of spoligotyping in epidemiological investigations of TB by analyzing 16 isoniazid-resistant M. tuberculosis strains isolated from patients with pulmonary TB in the central region of Poland. A total of 11 distinct spoligopatterns were obtained. 9 isolates were represented by a unique pattern, whereas 7 were clustered in 2 groups of 5 and 2 isolates, respectively. When compared with an international spoligodatabase SpolDB4, 13 isolates shared already described spoligotypes, whereas 3 did not match any existing spoligopattern in database and were defined as orphans. Spoligotyping overestimated the number of clustered isolates in one of its two clusters when compared to IS6110 Mtb1/ /Mtb2 PCR. Strains clustered using the latter method were assumed to be closely epidemiologically related.
This report demonstrates the utility of spoligotyping as an initial screening technique, to be supplemented by another typing method of greater discriminatory power, such as the IS6110 Mtb1/Mtb2 PCR in order to better recognize the epidemiological links between TB patients.
Pneumonologia i alergologia polska: organ Polskiego Towarzystwa Ftyzjopneumonologicznego, Polskiego Towarzystwa Alergologicznego, i Instytutu Gruzlicy i Chorob Pluc 02/2007; 75(1):22-31.
