Biomedical subjects

Ewa Augustynowicz

Publications and source records attributed to Ewa Augustynowicz.

18 recordsLinked to original sources

PubMed2005

Amplified fragment length polymorphism (AFLP) versus randomly amplified polymorphic DNA (RAPD) as new tools for inter- and intra-species differentiation within Bordetella.

Automated amplified fragment length polymorphism (AFLP) and randomly amplified polymorphic DNA (RAPD) techniques with fluorescently labelled primers were used to track differences among isolates of the eight known species of the Bordetella genus. Eighty-one representative strains of these species from international and Polish bacterial collections were genotyped according to RAPD protocols using primer 1254 or 1247, and AFLP involving EcoRI/MseI or newly designed SpeI/ApaI restriction/ligation/amplification procedures. By comparing AFLP and RAPD data, it was concluded that the discriminatory power of AFLP is higher in comparison with RAPD for both intra- and inter-species differentiation of isolates of the Bordetella genus. The most precise level of inter-species discrimination and the highest level of intra-species discrimination of the Bordetella isolates of the eight species were observed in the AFLP EcoRI/MseI and SpeI/ApaI sets, respectively. Both techniques might provide alternative tools for the identification of Bordetella at the genomic species and strain levels, and thus may be valuable in human and veterinary diagnostics as well as in epidemiology. By applying the AFLP technique presented in this article, more precise data on the emergence of newly acquired and/or on expanded clones and transmission routes of isolates of the Bordetella genus in the human and animal environments might be obtained.

Animals↗

PubMed2005

[Diagnostic difficulties in identification of Haemophilus influenzae colonising the nasopharynx of children].

The frequency of capsulated or non-capsulated Haemophilus influenzae strains colonisation among children attending day-care centres or orphanages has been studied. Detection of capsulated or non-capsulated H. influenzae strains has been compared for agglutination test and PCR. Misdiagnosing of H. influenzae serotype with agglutination found in the study suggest that the frequency of Hib strains colonizing the nasopharynx might be lower that previously evaluated. Due to perspectives of the wider use of Hib Immunisation in the future, more efficient diagnosis scheme for identification/differentiation of capsulated and non-capsulated H. influenzae strain should be undertaken.

Carrier State↗

PubMed2005

[Transmission of Haemophilus influenzae type b strains among children attending day-care centers and orphanages].

Goal of the work was to evaluate the differences level among H. influenzae strains of b serotype isolated from children attending day-care centres and orphanages and among strains isolated from invasive infections. In the work PFGE in Hib strains transmission examination and for epidemiological studies among three sources of invasive infection was applied. Among 35 Hib strains tested and control strain 8 different pulsotyped were found. Among 21 strains colonising the nasopharynx of healthy children, and among 13 Hib strains isolated from cerebrospinal fluid, 6 and 1 pulsotypes were found, respectively. Results obtained show that healthy children might be colonizing with genotypes characteristic for Hib strains isolated from invasive infections. In this view wider Hib vaccination seems be expected, as Hib circulation is common.

Carrier State↗

PubMed2004

Sequence variation in pertussis S1 subunit toxin and pertussis genes in Bordetella pertussis strains used for the whole-cell pertussis vaccine produced in Poland since 1960: efficiency of the DTwP vaccine-induced immunity against currently circulating B. pertussis isolates.

The present study indicates that the appearance of the B. pertussis harbouring prn2 gene allele variant (not found among clinical isolates before 1990s) may have been induced by long-term vaccination in Poland with DTP-composed vaccine strains presenting exclusively prn1. However, ptxS1A allele of pertussis toxin subunit S1 encoding gene, predominant in the currently isolated B. pertussis strains, has been found in vaccine strains used for whole-cell pertussis component (wP) production of DTP vaccine in 1960-1978. This outrules the possibility that the appearance of ptxSIA allele might be related to vaccine pressure driven by non-ptxS1A vaccine strains used for long-term immunization with wP. Intranasal challenge animal model testing the efficiency of the clearance of B. pertussis strains harbouring different ptxS1/prn allele gene combinations revealed that currently produced DTwP vaccine may not contain adequate B. pertussis vaccine strains, since isolates with gene variants different from those observed in vaccine strains were eliminated from the lungs of the immunized animals with lower efficiency.

Animals↗

PubMed2004

Potency of pertussis component in the DTP vaccine--an overview of three decade study in Poland.

In Poland, similar to many highly immunized Western countries, a recent increase in cases of pertussis has been observed. This study aims to evaluate the level of potency fluctuations of the pertussis component of Polish-produced DTP vaccine due to the changes having occurred in production and potency testing procedures. We compared the potency of the pertussis component of DTP vaccine lots produced and evaluated in similar periods and with similar production and testing procedures. Records of Kendrick test results performed over a 30-year period were available for analysis. This study confirms the role of different manufacturers, changes in vaccine strain compositions, in-house reference preparations used as reference vaccines in the Kendrick tests, and in mice of single strain sources in the potency values obtained. In addition, the comparisons performed revealed a downtrend in potency levels since 1992. Potency decrease in vaccine lots produced during 1992-1997 has been positively correlated to the lowering of the number of IOU/dose. Strain compositions of the DTP vaccine pertussis component and in-house references have been found to be associated with the fluctuation in potency estimations, and confirmed their crucial role in ensuring vaccine efficacy. Our study reveals that relative efficacy of the DTP vaccine produced in 1992-1997 might be lower than that of vaccines produced in other periods. This might in turn explain the increase in pertussis cases among children aged 5-15 years which is presently being observed in Poland.

Adolescent↗

PubMed2004

Pertussis in Poland.

BACKGROUND: Since 1997, an unexpected 2-5-fold increase in the incidence of pertussis has been reported in Poland in comparison with the previous 10 years, although the introduction of the diphtheria-tetanus-pertussis (DTP) vaccination in 1960 reduced the incidence of pertussis approximately 100-fold in the 1980s. The aim of the study was to analyse all available data on pertussis in Poland to identify the risks associated with its re-emergence. METHODS: Available data on notification, incidence, mortality, hospitalization, geographical distribution, incidence according to age, and diagnosis of pertussis were collected from national surveillance monographs and statistically evaluated. RESULTS: Analyses performed in the study found two periods of rising and falling trends: in the incidence before and after 1989, respectively. Moreover, after 1989, the age-specific incidence among children aged 0-4 years decreased, and among 5-9, 10-14, and 15-19 year olds increased in comparison to the previous decade. The incidence rate of pertussis among infants was similar in both decades analysed. Clustering of pertussis incidence increase in provinces along a line from North East to South West was observed. CONCLUSIONS: As vaccination coverage did not decrease and diagnostics have not been improved since the 1980s, it is possible that waning immunity and the appearance of Bordetella pertussis vaccine escape mutants are involved in the changing pertussis epidemiological parameters. Further monitoring studies, together with improving diagnostics, might allow more precise epidemiological data to be obtained. An additional booster dose of acellular pertussis vaccine at age 6 years has been included in the current vaccination schedule.

Adolescent↗

PubMed2004

[Increase in the incidence of pertussis and quality of whole-cell pertussis component of the DTP vaccine produced in Poland. Part I. Potency of DTP vaccine].

This study aimed to identify and to evaluate the level of potency fluctuations of the pertussis component of Polish-produced DTP vaccine lots produced within 1972-2001 due to the changes having occurred in production and potency testing procedures. The study confirms that higher potency values were obtained for vaccine lots produced since 80-ties, e.g. after changes of: references lots (1975), vaccine strains (1978) and source of animals used in Kendrick tests (1979). Additionally, the comparisons performed revealed a down trend in potency levels within 1992-1999 correlating to the lowering of the number of IOU/dose.

Animals↗

PubMed2004

[Increase in the incidence of pertussis and quality of whole-cell pertussis component of the DTP vaccine produced in Poland. Part II. Consistency of control and production].

Animals↗

PubMed2003

The possible effect of a sanitization program on intraspecies differentiation of Listeria monocytogenes strains isolated from a fish processing plant.

Forty-seven Listeria monocytogenes strains isolated during a year in a selected Polish fish-processing plant as well as 7 L. monocytogenes strains of different origins (including a reference strain) were analyzed in our studies. Strains were isolated from raw fish fillets (flounder), frozen coated flounder fillets, coating ingredients, and the processing environment. Isolation of strains covered the period of a sanitization program introduced in the plant. L. monocytogenes was identified using conventional microbiological methods and the PCR technique. RAPD (random amplified polymorphic DNA) technique for fingerprinting was applied to analyze the intraspecies diversity. Six RAPD types (A-F) and seven unique strains were revealed as a result of fingerprinting with one persistent type isolated from July 1999 to February 2000. It was detected for the first time after one month of sanitization. Its occurrence could have been promoted by clone selection either due to ineffective disinfection or to resistance against the disinfectant. As L. monocytogenes mostly occurred on frozen products, this indicates that contamination could start during product freezing, cold storage, or handling. The results revealed that there is a crucial need for preparing sanitization schemes precisely targeted at L. monocytogenes to avoid its recurrence as persistent 'in-house' strains. The possibility of incorrect interpretations of classical microbiological test results as well as the necessity to introduce assays based on nucleic acid analysis into epidemiological investigations were emphasized.

Animals↗

PubMed2003

Comparison of usefulness of randomly amplified polymorphic DNA and amplified-fragment length polymorphism techniques in epidemiological studies on nasopharyngeal carriage of non-typable Haemophilus influenzae.

Randomly amplified polymorphic DNA (RAPD) and automated amplified-fragment length polymorphism (AFLP) techniques with fluorescently labelled primers were used to type non-serotypable Haemophilus influenzae (NTHI) isolates. Eighty-seven isolates from healthy children attending day-care centres or living at orphanages in southern Poland were investigated. Through comparison of the AFLP data with RAPD analysis, it has been concluded that the discriminatory power of AFLP for NTHI typing is higher than RAPD. Generally, the NTHI isolates analysed were highly heterogeneous, as detected with a HindIII/TaqI AFLP genotyping scheme on intra/inter similarity levels of 94 and 96 % using Pearson's correlation coefficient. The range of similarity values found for isolates from children permanently residing at a particular day-care centre was much wider than that for isolates from orphanages. AFLP can efficiently access NTHI strain diversity and can monitor their turn-over for comparative typing in local and inter-local epidemiological investigations.

Bacterial Typing Techniques↗

PubMed2003

[Genetic variability of Bordetella pertussis strains. Part I. Current knowledge].

Variability of B. pertussis genome concerning S1 subunit of pertussis toxin (ptxS1) and pertactin (prn) genes has recently been recognised. Studies clearly showed differences in the DNA patterns and ptxS1 and prn alleles present in B. pertussis strains used for production of pertussis component in the DTP vaccine, in comparison to strains presently isolated from clinical materials. This trend has been observed in highly immunized populations with a long history of pertussis vaccination (The Netherlands, Finland, Poland, USA). Different ptxS1 and prn allele distribution for different countries needs further careful monitoring.

Alleles↗

PubMed2003

[Genetic variability of Bordetella pertussis strains. Part II. Perspectives].

In some highly immunized countries with long history of pertussis vaccination (The Netherlands, Finland, Poland, USA, France, Great Britain) differences in the DNA patterns and ptxS1 and prn alleles present in B. pertussis strains used for production of pertussis component in the DTP vaccine, in comparison to strains presently isolated from clinical materials have been found. Studies on efficacy of whole-cell and acellular pertussis vaccines in elimination of B. pertussis strains harbouring different gene variants in a particular country might indicate a need for a change of B. pertussis strains used for production of the vaccine against pertussis.

Alleles↗

PubMed2003

[Polymorphism analysis of Haemophilus influenzae strains isolated from healthy children with respect to amplification reaction].

The random-amplified polymorphic DNA (RAPD) assay was used to generate DNA fingerprints for 44 isolates H. influenzae obtained from healthy children. Problems with reproducibility and discriminatory power, frequently cited in the literature, were overcome by optimization procedure allowing to achieve reliable conditions for H. influenzae analysed. Particular parameters of RAPD fingerprinting were evaluated with respect to selection of best working primer, DNA polymerase and DNA concentration for amplification pattern. This study proved high sensitivity and efficiency of optimized RAPD profiling applicable for searching the epidemiology traces.

Child, Preschool↗

PubMed2003

[Phenotype and genotype of Clostridium sp. strains producing botulism neurotoxin].

Neurotoxins produced by strains of Clostridium sp. are belonging to the most toxic biological substances. In the study phenotypes and genotypes of C. botulinum strains in animal studies in vivo and on the DNA level were evaluated, respectively. Additionally, the presence of genes encoding BoNT toxins of A, B, and E types among strains of Clostridium sp. were identified. In case of C. botulinum DNA was isolated from vegetative bacterial cells and from spores. Two different genes encoding two different neurotoxins harboured by three strains of Ae biotype/ae genotype, and by two strains of B biotype/be genotype were detected. Additionally, above E type C. botulinum strains, the presence of gene encoding E type neurotoxin, was found in genome of two C. baratii, two C. butyricum, and C. bifidobacterium, and C. oedematicum strains. C. bifidobacterium and C. oedematicum strains positive for presence of gene encoding E type neurotoxin, were found negative for E neurotoxin production in vivo in TN test. The study indicates that genes encoding BoNT/E neurotoxins are very common among Clostridium species. Phenotype and genotype analysis indicated co-presence of B phenotype together with be genotype and A phenotype together with ae genotype among C. botulinum strains.

Animals↗

PubMed2002

[Enterotoxic strains of Clostridium perfringens and sporulation].

Clostridium perfringens strains of A type capable of enterotoxin (CPE) synthesis may be a potential source of food-poisoning. Suitability of methods for CPE detection on the protein level is limited by difficulties in inducing sporulation in vitro. A number of unknown facts concerning coregulation the sporulation processes and CPE synthesis are recognised. The goal of the work was to determine the level of correlation between CPE synthesis and spores formation. Enterotoxin and cpe gen were detected by RPLA after sporulation induction test and by methods based on amplification on the DNA and mRNA levels. Sixty-four C. perfringens strains of A type isolated from patients with food poisoning symptoms and from food samples were analysed. Collection of isolates was differentiated as not enterotoxic, enterotoxic, and potentially enterotoxic strains based on appropriate strain profile: plc(+), cpe(-), CPE(-); plc(+), cpe(+), CPE(+); and plc(+), cpe(-), CPE(-), respectively. No significant difference between expression of cpe mRNA in vegetative and sporulation phase was found. The obtained results indicate that sporulation is not an essential factor for cpe gene expression.

Base Sequence↗

PubMed2002

Development of broad-range polymerase chain reaction (PCR) bacterial identification in diagnosis of infective endocarditis.

BACKGROUND AND AIMS OF THE STUDY: The study aim was to identify the value of broad-range bacterial PCR in infective endocarditis (IE) of bacterial etiology, and to determine its specificity and sensitivity. METHODS: Thirty blood samples were taken for analysis from patients with IE (diagnosed according to Duke criteria) and acquired valvular heart disease. Two control groups of patients with (n = 10) or without (n = 15) urinary tract infection were defined. DNA was isolated, and three different primer pairs for the region of the gene coding for 16S rRNA were tested, to determine the most specific pair. Amplification products were analyzed with gel electrophoresis, stained with ethidium bromide, and located under UV light. RESULTS: Positive blood cultures were found in 25 patients with IE. A typical echocardiography picture with bacterial vegetations was found in all patients with sterile blood cultures, and in 20 patients with positive blood cultures. The highest specificity was found for forward/reverse (F/R) primers, as the relevant amplified PCR product was present in all blood samples with IE, and in four of 10 patients with urinary tract infection. CONCLUSION: Broad-range PCR in bacterial endocarditis is a rapid, sensitive and inexpensive technique for the detection of bacteria, but is far more prone to contamination than species-specific PCR. However, under controlled conditions, broad-range PCR may be valuable for the identification of non-specific infection, permitting a more rapid clinical diagnosis of endocarditis.

Aortic Valve↗

PubMed2002

[Amplification reaction to bacterial DNA for diagnosing infective endocarditis].

The aim of this study was to evaluate the usefulness of broad-range bacterial PCR in infective endocarditis of bacterial etiology, and to determine its specificity and sensitivity. Twenty five blood samples were taken for analysis from patients with infective endocarditis and acquired valvular heart disease. Infective endocarditis was diagnosed according to Duke criteria. There were two control groups consisting of patients with acquired valvular heart disease: 10 patients with urinary tract infection and 15 patients without. Three different primer pairs for the region of the gene coding for 16S rRNA were tested, to find the most specific one. The highest specificity was found for F/R primers, as the relevant amplified PCR product was present in every blood sample with infective endocarditis, and also in 4 out of 10 patients with urinary tract infection. Broad-range PCR in bacterial endocarditis is a fast, sensitive and inexpensive tool for the detection of bacteria, but it is far more prone to contamination than species specific-PCR. However, in controlled conditions it may be valuable in the identification of non-specific infection allowing for a more rapid clinical diagnosis of endocarditis.

Bacteria↗

PubMed2002

[Broad-range fungal PCR in diagnosis of complicated infective endocarditis].

UNLABELLED: Infective endocarditis (IE) frequently causes problems in diagnosis, especially where blood cultures are negative and when IE has fungal etiology. The aim of this study was to evaluate the usefulness of broad-range fungal PCR in diagnosis of fungal super-infection of bacterial IE. Twenty five blood samples were taken for analysis from patients with infective endocarditis. IE was diagnosed according to Duke criteria including positive blood cultures. Suspicion of fungal superinfection was established in 5 patients on serological investigation and confirmed by blood culture in 2 patients. Control group consisted of 15 patients without infection. DNA was isolated using the commercially available S.N.A.P. kit. Amplification products were analyzed by gel electrophoresis. Fungal DNA was found in 2 patients with fungal super-infection of bacterial IE confirmed by culture. In the remaining patients with IE and controls no fungal DNA was found. CONCLUSION: Broad-range fungal PCR is a fast and inexpensive tool for the detection of fungal DNA. The method may be valuable in the diagnosis of fungal super-infection of bacterial IE.

Aspergillus↗