Epidemiological analysis of pneumococcal serotype 19A in healthy children following PCV7 vaccination.

After the introduction of conjugate vaccines, a strong rearrangement of pneumococcal serotypes was observed globally. Probably most concerning was the emergence of serotype 19A, which has not only high invasive disease potential, but also high antibiotic resistance. In the current study we focused on the increased prevalence of serotype 19A after the PCV vaccination rate became widely used in Hungary. A total of 2262 children aged 3-6 years were screened for pneumococcus carriage using nasal swabs. Children were divided into two groups according to the vaccination rates, low level (group 1) vs. high level (group 2). While the carriage rate did not change over time (average 32·9%), the serotype distribution differed greatly in the two groups. The prevalence of serotype 19A increased >eightfold. Almost all 19A isolates had high-level macrolide resistance and elevated penicillin minimum inhibitory concentrations. Genotyping methods revealed that these new 19A isolates are different from the previously frequent Hungary19A-6 PMEN clone. Both the carriage rate and the overall penicillin and macrolide resistance remained stable over time, but while several serotypes were represented in group 1, serotype 19A alone was clearly dominant in group 2.

Varying fitness cost associated with resistance to fluoroquinolones governs clonal dynamic of methicillin-resistant Staphylococcus aureus.

The purpose of this study was to investigate the impact of fluoroquinolone resistance on the existence and dynamic of MRSA clones. Resistance to ciprofloxacin was induced in strains of community-acquired (CA) MRSA from various sequence types and the fitness cost suffered by mutant derivatives measured in a propagation assay. In addition, the fitness of fluoroquinolone resistant health care-associated (HA) MRSA isolates from major clones prevalent in Hungary were compared with each other and with those of the CA-MRSA derivatives. The genetic background of fluoroquinolone resistance and fitness cost in CA-MRSA was investigated. The fitness cost observed in the CA-MRSA derivatives proved diverse; the derivatives of the ST30-MRSA-IV strain suffered significantly greater fitness cost than those of the ST8-MRSA-IV and ST80-MRSA-IV isolates. Strains from the New York-Japan (ST5-MRSA-II), South German (ST228-MRSA-I) and EMRSA-15 (ST22-MRSA-IV) HA-MRSA clones proved more viable than CA-MRSA derivatives with similar MIC values to ciprofloxacin and HA-MRSA strains from the Hungarian/Brazilian clone (ST239-MRSA-III). Our strains from the New York-Japan, South-German and EMRSA-15 clones seem to have a competitive edge over the tested CA-MRSA isolates in the health care setting. The greater fitness observed in our New York-Japan and South-German strains could account for the replacement by them of the Hungarian/Brazilian clone in Hungary about ten years ago. Alterations in relevant genes were detected. The Ser80 → Phe mutation in the grlA gene may have seriously compromised viability. Surprisingly silent nucleotide substitutions in the grlB gene seemed to impact fitness in derivatives of the ST30-MRSA-IV isolate.

Phenotypic and genetic characterisation of methicillin-resistant Staphylococcus aureus strains isolated from the university hospitals of Debrecen.

The purpose of this study was to characterise methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in 2005 at the university hospitals of Debrecen, Hungary. Three hundred and thirty-nine MRSA strains were isolated from 102 patients at 18 different clinics. Their sensitivity to oxacillin and ten other antibiotics was determined. For genotypic analysis, phage typing and pulsed-field gel electrophoresis (PFGE) was performed. The rate of MRSA strains increased to 7.2% in 2005, especially at the clinics of surgery, pulmonology and paediatrics. No vancomycin- or teicoplanin-resistant strains were found. The resistance to erythromycin, clindamycin and ciprofloxacin was nearly 100% and multi-resistance was very frequent. Fifty-eight percent of the isolates belonged to mixed phage types and 8% was non-typable. One PFGE clone contained 58.2% of all strains and two further major clones were found at a separately located clinical block, indicating intra-hospital spread. We can conclude that MRSA exhibits an increasing nosocomial problem also in Hungary.

Molecular characterisation of bovine faecal Escherichia coli shows persistence of defined ampicillin resistant strains and the presence of class 1 integrons on an organic beef farm.

Antimicrobial use is heavily restricted on organic farms; however, few studies have been conducted to investigate the impact this has on the epidemiology of resistance in pathogenic and commensal bacteria. We investigated the persistence of antimicrobial resistant Escherichia coli within an organic beef herd over a period of 28 months. Faecal samples collected monthly from three calf cohorts and annually from adult cattle and environmental samples, were screened for the presence of ampicillin, apramycin and nalidixic acid resistant E. coli. The prevalence of ampicillin resistance ranged from 27.3 to 40.7% in the annual herd and environmental samplings (n=22-55) and was greater in the calf cohorts, with a peak cohort prevalence of >47% in all 3 years (n=16-18). Apramycin and nalidixic acid resistant E. coli were rare. Pulsed-field gel electrophoresis (PFGE) identified 10 main genotype groups within the herd, with evidence of strain transmission between different livestock groups, animal species and years. Multiple resistance was found in >44% of isolates tested, with ampicillin, neomycin, sulphamethoxazole and tetracycline carriage the commonest phenotype identified. PCR detected the presence of class 1 integrons in <5% of resistant isolates, 6/7 of which were of cattle origin. These data demonstrate that ampicillin resistant E. coli was common on the farm despite restricted antimicrobial use, although strain diversity was low. Persistence of defined genotype groups was observed between years, together with the transmission of resistant strains between different animal species on the farm.

The first steps towards fluoroquinolone resistance in Hungarian pneumococci.

The incidence of fluoroquinolone resistance among Hungarian routine laboratory Streptococcus pneumoniae isolates, collected in 2000-2002, in common with other European countries, was very low; only 5/304 strains (1.64%) were resistant to ciprofloxacin (MIC = 4 microg/ml), and the other fluoroquinolones showed full efficacy. However, we could identify the Lys-137-Asp amino acid change, caused by a point mutation in the QRDR of the parC gene, in five strains. Additionally, we observed a definite shift in the minimum inhibitory concentrations (MICs) of all fluoroquinolones towards higher values throughout the study period. These two findings, coupled with the increasing consumption figures of fluoroquinolones, suggest that pneumococcal resistance looks poised to develop in Hungary.

The relationship between serotypes and PFGE genotypes in isolates of Streptococcus pneumoniae from Hungary.

The relatedness of 112 penicillin-non-susceptible isolates of Streptococcus pneumoniae from Hungary was determined by pulsed-field gel electrophoresis (PFGE), serotyping and antibiotic susceptibility tests. The differences in PFGE patterns closely mirrored the changes in resistance. Some genotypes comprised multiple serotypes, and the genetic diversity among certain serotypes was considerable. Generally, serotyping alone was insufficient for epidemiological mapping of pneumococcal isolates. There was considerable serotype diversity, but the five most frequent international serotypes (6, 9, 14, 23, 19) were the most prevalent. In addition, the presence of some well-defined resistant international pneumococcal clones in the Hungarian population was identified.

Taguchi optimisation of ELISA procedures.

We propose a new method in the field of ELISA optimization using an experimental design called the Taguchi method. This can be used to compare the net effects of different conditions which can be both qualitative and quantitative in nature. The method reduces the effects of the interactions of the optimized variables making it possible to access the optimum conditions even in cases where there are large interactions between the variables of the assay. Furthermore, the proposed special assignment of factors makes it possible to calculate the biochemical parameters of the ELISA procedure carried out under optimum conditions. Thus, the calibration curve, the sensitivity of the optimum assay, the intra-assay and inter-assay variability can be estimated. The method is fast, accessing the results in one step, compared to the traditional, time-consuming 'one-step-at-a-time' method. We exemplify the procedure with a method to optimize the detection of ScFv (single chain fragment of variable) phages by ELISA. All the necessary calculations can be carried out by a spreadsheet program without any special statistical knowledge.

Constitutive heterochromatin in Ehrlich ascites carcinoma cells.

The study of the distribution of constitutive heterochromatin in the chromosomes of Ehrlich ascites carcinoma cells has revealed changes in both the amount and the spatial distribution of this class of DNA. The marker chromosomes were identified and the probable mechanism of their formation was discussed.

Fast method to remove UV absorbing agarose gel contamination from DNA samples.

Agarose gel electrophoresis and subsequent purification of DNA bands from the agarose gel is a widely used molecular biological method. There are different methods to achieve this goal, however they have different advantages and disadvantages. One major problem is the presence of different contaminants in the final sample. We developed a method which is effective in removal of the agarose contaminants.

Missing X chromosome and ring chromosome 21 in a case of acute myelomonocytic leukemia.

The karyotypic picture of a female patient with acute myelomonocytic leukemia (A.M.M.L.) consisted in the loss of a sex chromosome and a ring chromosome 21. It is suggested that in A.M.M.L. the loss of sex chromosome may represent an early event, the monosomic cells being the object of further chromosome rearrangements, which involve more frequently a chromosome 21.

Different cell clones in bone marrow and spleen of a patient with chronic myelocytic leukemia (CML) in blastic phase.

The chromosome study of a patient with chronic myelocytic leukemia in blastic phase revealed a 46,XY,Ph1/47,XY,Ph1,+8 cytogenetic constitution in bone marrow cells and a 46,XY,Ph1/48,XY,2Ph1,+19 cytogenetic constitution in spleen cells. As the cell clones exhibiting chromosome abnormalities in addition to the Ph1 chromosome evolved apparently independently, it is suggested that the acute transformation had a bifocal, myeloid and splenic origin.

Serological evidence of adenovirus infection in cats.

The widespread presence of adenoviruses in various species makes it probable that infection and the carrier state also exist in cats. On the basis of these considerations, investigations were carried out to find antibodies against adenovirus in sera from different cat populations kept under different conditions. For the antibody detection, purified adenovirus was used in an indirect ELISA. To produce positive serum, SPF cats were immunized with a purified hexon preparation. Altogether 632 field sera of different origin were tested. Among field samples, adenovirus seropositivity varied between 10-26%.

Characterization of intertype specific epitopes on adenovirus hexons.

The specification and differentiation of eighteen intertype specific (IT) epitopes of adenovirus hexons defined by monoclonal antibodies are given by two groups of hexon types: on which they are and on which they are not present. The close specificity relationship among some groups of epitopes determined by pairwise analysis according to their presence on the hexon types indicate that they could be continuous (sequential), overlapping or discontinuous (topographic) epitopes. Based on the identification of the hydrophilic regions and the localization of beta-turns sixteen IT epitope sites were predicted on human adenovirus (HAdV) type 2 and nineteen on HAdV-41 hexon's amino acid (aa) sequences beside the type and genus specific ones. The 16 predicted IT epitopes on HAdV-2 hexon show good coincidency with the 14 IT epitopes demonstrated with monoclonal antibodies (MAbs) on this hexon type. The predicted number of common epitopes between HAdV types 2 and 41 also corresponds well with the eight IT epitopes determined by MAbs, but the 17 predicted non-common epitopes indicate the possibility of the existence of more epitopes on these hexon types. The location of the predicted epitopes of HAdV-2 were determined by alignment of their sequence numbers on the three dimensional ribbon representation of the hexon subunit. Most of the predicted IT epitopes were found between the type and genus specific epitopes i.e. in the "upper" regions of pedestal domains P1 and P2 orientated toward the virion surface and in the "lower" part of loop 1 region orientated inside the virion. Two peptides representing potential IT epitopes were synthesized corresponding to residues 309-320 from the "lower" part of loop 1 and 399-408 from the "upper" part of P1 region of HAdV-2 hexon. Antibodies raised against the peptide-carrier conjugates recognized different purified native hexon types in ELISA.