Multilocus sequence types in Czech neonatal GBS strains from 2004 to 2008.

The first multilocus sequence typing results of Czech neonatal Streptococcus agalactiae isolates are presented in this paper. The aims of the study were to prove if the Czech isolates belong to potential invasive clonal complex CC17 and to further analyse serotype-sequence type relationships. Twelve sequence types detected among 77 isolates were assigned to 5 clonal complexes. Sequence type variability was found for most of our collection serotypes. As many as 83 % of the invasive isolates were covered by as few as 3 sequence types: S17, ST23 and ST19. ST17 was the only sequence type strictly tied to serotype III. "Hyperinvasive" ST17 was identified in 85 % of the cerebrospinal fluid isolates (6 out of 7), but in 32 % of the blood isolates only.

Arrangements of human telomere DNA quadruplex in physiologically relevant K+ solutions.

The arrangement of the human telomeric quadruplex in physiologically relevant conditions has not yet been unambiguously determined. Our spectroscopic results suggest that the core quadruplex sequence G(3)(TTAG(3))(3) forms an antiparallel quadruplex of the same basket type in solution containing either K(+) or Na(+) ions. Analogous sequences extended by flanking nucleotides form a mixture of the antiparallel and hybrid (3 + 1) quadruplexes in K(+)-containing solutions. We, however, show that long telomeric DNA behaves in the same way as the basic G(3)(TTAG(3))(3) motif. Both G(3)(TTAG(3))(3) and long telomeric DNA are also able to adopt the (3 + 1) quadruplex structure: Molecular crowding conditions, simulated here by ethanol, induced a slow transition of the K(+)-stabilized quadruplex into the hybrid quadruplex structure and then into a parallel quadruplex arrangement at increased temperatures. Most importantly, we demonstrate that the same transitions can be induced even in aqueous, K(+)-containing solution by increasing the DNA concentration. This is why distinct quadruplex structures were detected for AG(3)(TTAG(3))(3) by X-ray, nuclear magnetic resonance and circular dichrosim spectroscopy: Depending on DNA concentration, the human telomeric DNA can adopt the antiparallel quadruplex, the (3 + 1) structure, or the parallel quadruplex in physiologically relevant concentrations of K(+) ions.

Incidence of invasive pneumococcal disease in the Czech Republic and serotype coverage by vaccines, 1997-2006.

We studied the incidence of invasive pneumococcal disease (IPD) in the Czech Republic by analysing two sources of data. The incidence of pneumococcal meningitis based on routine notification data varied between 0.4 and 0.6/100 000 population between 1997 and 2006. The incidence of IPD based on laboratory surveillance varied between 2.3 and 4.3/100 000 population between 2000 and 2006. The annual IPD incidence remained stable during the study period. Estimates of absolute IPD case-load in the entire country varied from 235 to 437 per year. The age-specific incidence was highest in the <1 year age group, reaching 4.3/100 000 for pneumococcal meningitis in routine notification and 15.7/100 000 for IPD in laboratory-based surveillance data, respectively. A total of 1236 Streptococcus pneumoniae isolates from cerebrospinal fluid and sterile body sites were investigated. The most frequent serotypes causing IPD in all ages were 3, 4, 14, 8 and 19F, accounting for 41.5% of all isolates. The most frequent serotypes by age group were: <1 year (6B and 19F); 1-4 years (14, 6B and 23F); 40-64 years (3, 8 and 4), and > or = 65 years (3, 4, 9N and 14). The coverage of serotypes in all age groups by pneumococcal vaccines ranged from 41.5% for 7-valent conjugate vaccine to 67.9% for 13-valent conjugate vaccine. The coverage of serotypes causing IPD is significantly different between infants/children and adults/elderly. PCV-7 coverage by age group was: <1 year (66.0%), 1-4 years (65.1%), 40-64 years (34.4%) and > or = 65 years (39.3%). Similar age differences between infants/children and adults/elderly were found in coverage by PCV-9, PCV-11 and PCV-13. The distribution of serotypes in the total population and individual age groups was stable during the period 2000-2006.

[Invasive pneumococcal disease in the Czech Republic in 2000-2006]. / Invazivni pneumokokové onemocemní v Ceské republice v letech 2000-2006.

STUDY OBJECTIVE: To assess the incidence of invasive pneumococcal disease and coverage of the causative serotypes by pneumococcal vaccines in the Czech Republic. METHODS: The incidence and fatality rates of reported cases of pneumococcal meningitis (EPIDAT) in 1997-2006 are analyzed. In addition, the data of the National Reference Laboratory for Streptococci and Enterococci from 2000-2006 are presented. The incidence of invasive pneumococcal disease was calculated for the catchment population of the collaborating laboratories. Pneumococcal typing was performed in the National Reference Laboratory. The percentages of pneumococcal serotypes isolated from invasive disease in 2000-2006 and included in the pneumococcal vaccines were calculated. RESULTS: The incidence of invasive pneumococcal disease ranged from 2.30 to 4.28/100000 population. The age-specific incidence of invasive pneumococcal disease in 2000-2006 was the highest in the lowest age groups 0-11 months (15.75/100000) and 1-4 years (8.22/100000), followed by the age group of 65 years and older (7.3/100000). The total fatality rate of pneumococcal meningitis in 1997-2006 was 13.7%. The highest age-specific fatality rate was recorded in 65-year olds and over (24%). In the age group 0-11 months, the coverage of pneumococcal serotypes is 66% by the 7-valent conjugate vaccine, 76% by the 10-valent conjugate vaccine and 82% by thel3-valent conjugate vaccine. In the age group 1-4 years, the respective rates are 65.1%, 76.4% and 85.8%. The coverage of serotypes by conjugate vaccines is higher in the youngest age groups (0-11 months and 1-4 years) compared to adults (40-64 years and 65 years and older). CONCLUSION: Based on laboratory data, the incidence of invasive pneumococcal disease in the Czech Republic in 2000-2006 is comparable with the rates reported in other European countries. The highest incidence rates were observed in the youngest age groups. The distribution of serotypes causing invasive pneumococcal disease differs between children and adults. The coverage of serotypes by conjugate pneumococcal vaccines is higher in children (66-65%) compared to adults (34-65%). It is desirable to launch a nationwide programme of surveillance of invasive pneumococcal disease. Furthermore, it is recommended that a conjugate pneumococcal vaccine should be included in the children's immunization schedule.

Emergence of a novel macrolide-resistant Streptococcus pyogenes emm53 strain.

An unusual emm53, T-28/T-non-typeable, iMLS(B) phenotype clone represented a substantial proportion (28.6%) of invasive erythromycin-resistant group A streptococcus (GAS) isolates in the Czech Republic during 2003. Clonal analysis of emm53 isolates between 2001 and 2004 revealed four pulsed-field gel electrophoresis (PFGE) patterns and two emm subtypes. Isolates produced identical PFGE patterns regardless of their invasiveness and/or tetracycline resistance. Multilocus sequence typing classified all isolates as ST340. An ST5 (emm83) isolate, a potential ancestor of ST340, was isolated in the Czech Republic from an impetigo patient in 1988. The Czech emm53/ST340 isolates shared only three of seven alleles with the original Lancefield emm53/ST11 isolate.

Characteristics of Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and Staphylococcus aureus isolated from the nasopharynx of healthy children attending day-care centres in the Czech Republic.

Nasopharyngeal carriage of potential pathogens was studied in 425 healthy 3- to 6-year-old children attending 16 day-care centres (DCCs) in nine Czech cities during the winter 2004-2005. The overall carriage of pathogens was 62.8% (Streptococcus pneumoniae, 38.1%; Haemophilus influenzae, 24.9%; Moraxella catarrhalis, 22.1%; Staphylococcus aureus, 16%). An age-related downward trend was observed for colonization with respiratory pathogens in contrast to Staph. aureus whose carriage was significantly higher among older children. The following serotypes of colonizing S. pneumoniae were the most predominant: 23F (20.6%), 6A (15.1%), 6B (12.7%), 18C (7.8%), 15B and 19F (6% each). The majority (94.3%) of H. influenzae isolates were non-typable; among capsulated isolates, serotype b was not found. Decreased susceptibility to penicillin was determined in 3% of pneumococci; 4.6% of H. influenzae strains and 85.1% of M. catarrhalis strains produced beta-lactamase. As for non-beta-lactam antibiotics, pneumococci resistant to trimethoprim-sulphamethoxazole were the most common (15.7%) among the attendees.

[Disribution of Streptococcus pneumoniae serotypes and serogroups among patients with invasive pneumococcal diseases in the Czech Republic in 1996-2003: background data for vaccination strategy]. / Distribuce sérotypu a séroskupin Streptococcus pneumoniae u pacientu s invazivními pneumokokovými onemocneními v Ceské Republice v letech 1996-2003: podklady pro vakcinacní strategii.

STUDY OBJECTIVE: To map distribution of Streptococcus pneumoniae serotypes and serogroups in the Czech Republic in 1996-2003 in order to provide background data for effective strategy of vaccination with type specific pneumococcal conjugated vaccine. METHODS: Quellung typing with sera from the Statens Serum Institute (Denmark) was used to differentiate 1553 S. pneumoniae strains isolated from patients with invasive pneumococcal infections in the Czech Republic. RESULTS: Strains of serotypes 3, 19F, 9V, 23F, 1, 14 and 4 were most frequently isolated from clinical specimens (blood, cerebrospinal fluid, autopsy specimens, lung puncture aspirate, bronchoalveolar lavage fluid, sputum) of patients with invasive pneumococcal infections. Some serotypes were detected regardless of patient age (1, 3, 6A, 9V, 14, 19F and 23F), while serotype 6B was most frequent in the age group 0-2 years, serotypes 4, 6B, 7F and 18C were typically found in the age group >2-5 years and types 4, 7F, 8, 9N, 10A and 11A were common in patients aged >5 years. The 7-valent conjugated pneumococcal vaccine (PNCMR7) covers 63.1%, 62.3% and 34.3% of the serotypes identified in 152 patients aged 0-2 years, 168 patients aged >2-5 years and 1233 patients aged >5 years, respectively. It includes 38.3% of the serotypes identified among 1055 strains isolated from blood, cerebrospinal fluid and autopsy specimens regardless of patient age. Over the period moni- tored, the percentage of strains allocated to one of the serotypes included in the vaccine increased from 45.5% to 70.6%, from 54.3% to 38.7% and from 21.8% to 38.7% in patients aged 0-2 years, >2-5 years and >5 years, respectively, and from 30.0% to 39.4% for the isolates from blood, cerebrospinal fluid and autopsy specimens. A similar trend was found in the distribution of serogroups. These shifts can also be ascribed to the continuous increase in the percentages of strains of serotypes 4 (from 1.0% to 8.9%) and 9N (from 0.0% to 5.9%). CONCLUSION: The PNCRM7 vaccine is recommended for use in the Czech Republic in spite of the lower prevalence of vaccination serotypes among strains isolated from Czech patients with invasive pneumococcal infections which is a limitation to its efficacy. Nevertheless, results of the longterm monitoring of serotype distribution in the Czech Republic are suggestive of the need for inclusion of types 1 and 3 in the vaccine in the future.

[Surveillance of serious diseases caused by group A streptococci in the Czech Republic in 2003--the Strep-EURO project]. / Surveillance závazných onemocnení pusobených streptokoky skupiny A v Ceské republice v roce 2003--projekt Strep-EURO.

Results obtained in the first year of active surveillance of serious diseases caused by group A streptococci in the Czech Republic carried out within the Strep-EURO project are summarized. From January to December 2003, 28 Czech laboratories referred to the National Institute of Public Health 59 group A streptococcal strains isolated from invasive diseases meeting the Strep-EURO definition. At the same time, clinical-epidemiological data and information on the catchment area population were provided. For 2003, the total morbidity and case fatality rates were calculated to be 1.3/100,000 population and 13.5%, respectively. A high proportion (23.7%) of the 59 strains were of type emm 1, initially labeled M1. As many as 24.1%, 17.2% and 20.7% of the strains tested were resistant to erythromycin, clindamycin and tetracycline, respectively. The seemingly lower morbidity rate and higher case fatality rate from serious diseases caused by group A streptococci reported in the Czech Republic for 1994-1996, i.e. 0.4/100,000 and 46.5%, respectively, in comparison with the latest data can be explained by the use of softer international criteria for the Strep-EURO definition of invasive disease.

Vaginal & rectal carriage of Streptococcus agalactiae in the Czech Republic: incidence, serotypes distribution & susceptibility to antibiotics.

BACKGROUND & OBJECTIVES: Streptococcus agalactiae (group B streptococcus, GBS) is the predominant bacterial agent responsible for invasive perinatal infection. To obtain reliable data on vaginal and rectal carriage of S. agalactiae in pregnant women in Czech Republic, and to formulate a prevention programme of neonatal GBS disease for the Czech Republic, women at childbirth were screened for vaginal and anorectal carriage of GBS. The isolates were serotyped and tested for susceptibility to antimicrobials including those recommended for intrapartum prophylaxis. METHODS: A total of 586 women at childbirth were screened for GBS carriage in vaginal and anorectal regions using the non-enrichment and selective culture media. The isolates were serotyped by precipitation with antisera raised against various serotypes and antigenic extracts prepared according to Lancefield's modification. Mueller Hinton agar with 5 per cent defibrinated sheep blood was used for antimicrobial susceptibility testing. MIC values were evaluated according to the NCCLS criteria. RESULTS: Using selective media, GBS was detected in 172 (29.3%) of 586 women screened, vaginal and anorectal colonization was found in 21.7 and 24.4 per cent of them, respectively, concomitant vaginal and anorectal colonization was recorded in 16.5 per cent of the women studied. Serotypes III (33.2%), Ia (22.0%) and V (13.9%) prevailed among 172 isolates tested. All isolates were susceptible to penicillin, ampicillin and cefotaxime. The rates of GBS resistance to tetracycline, erythromycin and clindamycin were 83.9, 3.8 and 3.2 per cent, respectively. INTERPRETATION & CONCLUSION: GBS carriage in pregnant women in the Czech Republic is rather high as compared with that reported in literature. The most frequent serotypes III, Ia and V, identified in GBS-colonized pregnant women in the Czech Republic, were among those predominant in the USA and Western Europe. Our findings confirm uniform susceptibility of GBS isolates from pregnant women to penicillin and other beta-lactam antibiotics tested. Resistance to erythromycin remains low in the Czech Republic.

Comparison of Streptococcus pneumoniae serotypes causing acute otitis media & invasive disease in young children in the Czech Republic.

BACKGROUND & OBJECTIVES: The availability of a type-specific pneumococcal vaccine for children is a worldwide problem. It is necessary to study the serotypes prevalent in a country before introducing a type-specific vaccine. The objective of the present study was to analyse the prevalence of Streptococcus pneumoniae serotypes in children suffering from acute otitis media or invasive pneumococcal disease and to compare a coverage of serotypes by individual pneumococcal vaccines. METHODS: Children suffering from acute otitis media and invasive pneumococcal disease were analysed in the Czech Republic from October 1999 to November 2000. Serotyping was performed by the quellung technique using antisera from Statens Serum Institute (Denmark). RESULTS: The most frequent serotypes in patients with acute otitis media were 3, 19F, 23F, 14, 9V, 1, 6B, 11A and 28F. Vaccine coverage for the identified serotypes in acute otitis media patients was 52.1 per cent for the 7-valent vaccine, 57.8 per cent for the 9-valent vaccine and 75.7 per cent for the 11-valent form of the vaccine. In 108 patients with invasive pneumococcal disease, the most frequent serotypes were 6B, 9V, 14, 19F, 3 and 23F. Vaccine coverage for the identified serotypes in patients with invasive pneumococcal disease was 62 per cent for the 7-valent vaccine, 66.4 per cent for the 9-valent vaccine and 77.5 per cent for the 11-valent form of the vaccine. INTERPRETATION & CONCLUSION: Vaccine coverage for the identified serotypes for the 11-valent pneumococcal vaccine was better than the other two vaccines.

Active surveillance of early onset disease due to group B streptococci in newborns.

BACKGROUND & OBJECTIVES: Early onset disease (EOD) due to group B streptococci (GBS) poses a serious threat in many countries. In the Czech Republic neither summarized data on the EOD incidence are available nor a nationwide prevention program has been initiated. The present surveillance was initiated to establish the incidence of EOD due to GBS in newborns in the Czech Republic, distribution of GBS serotypes and GBS susceptibility to antimicrobials. METHODS: Both invasive and carrier GBS isolates from newborns and the data on the newborns' clinical status and maternal colonization and intrapartum prophylaxis were collected from 30 microbiological and clinical centres all over the Czech Republic within prospective active surveillance. HCl extracts of the GBS strains were precipitated with rabbit polysaccharide (I-VIII) and protein (c,R) antisera. RESULTS: Between January 2001 and September 2002, GBS isolates from 239 full-term and 46 preterm newborns were collected. Of the 285 GBS positive newborns, 105 had invasive EOD, 42 showed suspected EOD, and in 56 clinical diagnosis was not specified. Eighty two GBS isolates were obtained from healthy colonized infants. The isolates obtained from newborns with confirmed invasive EOD were mostly of serotype III (42%), followed by serotypes V a Ia (13% each). Types Ia (26%), III (22%) and II (20%) were most frequent among the isolates from colonized individuals. Protein antigens (c protein, R protein) either coupled with polysaccharide or alone were found in 70 per cent (30 and 40 %, respectively) of the study isolates. INTERPRETATION & CONCLUSION: The incidence of EOD due to GBS found in the Czech Republic 0.7-1.0 per 1000 live births was comparable with the rates reported in the countries where the prevention programme has been implemented nationwide. Serotypes III, V and Ia prevailing among the isolates from Czech newborns with EOD belonged to those most frequently identified in the USA and Western European countries.

[Antibiotic resistance in invasive pneumococci and their serotypes in the Czech Republic]. / Antibiotická rezistence invazivních pneumokoku a jejich sérotypy v Ceské republice.

BACKGROUND: Among strains of S. pneumoniae (pneumococci), isolated from blood or cerebrospinal fluid of 483 patients between January 2001 and October 2003 as many as 5.2% strains were penicillin-non-susceptible. METHODS AND RESULTS: Incidence of penicillin-non-susceptible S. pneumoniae (PNSP) strains was highest in children of the youngest age group (12.7%). Cefotaxim-non-susceptibility was detected in 2.7% of strains. Pneumococci tested were resistant to co-trimoxazol (9.9%), tetracycline (8.9%), chloramphenicol (4.6%), erythromycin (2.3%), clindamycin (1.4%), levofloxacin (0.6%) and rifampicin (0.2%); none of the strains was resistant to linezolid. Comparison of the results from 1996-1999 shows a permanent occurrence of invasive PNSP strains ranging about 5%. The PNSP strains were of 7 serotypes, with serotype 9V being the most frequent. The Czech collection of invasive pneumococcal strains is five times smaller than that of any other European country with comparable populations over the same period of time. CONCLUSIONS: Low incidence of invasive S. pneumoniae strains seems to be related to the underestimated significance of blood culturing. Valid data necessary for the safety of antibiotic therapy in terms of efficacy and prevention of resistance development cannot be obtained from some localities of the Czech Republic.

[Differentiation of serologic group A streptococci from group C and G using disks with various bacitracin levels]. / Odlisení streptokoku sérologické skupiny A od skupiny C a G pomocí disku s ruzným obsahem bacitracinu.

It was revealed that a diameter of the inhibition zone of 10 mm or more surrounding a disk with 0.04 U bacitracin differentiates reliably strains of the serological group A form strains of group C and G with the exception of 7.3% strains of group C and G with a high susceptibility for bacitracin. The disks with a higher bacitracin content gives less reliable results.

[Biological properties of Streptococcus group B: the interrelations of the virulence in mice, the adhesion to human epithelial cells and the production of type-specific antigen]. / Biologicheskie svoistva streptokokkov gruppy B: vzaimootnosheniia mezhdu virulentnost'iu dlia myshei, adgeziei k épitelial'nym kletkam cheloveka i produktsiei tipospetsificheskogo antigena.

The results presented in this work confirm the possibility of selecting the subpopulations of group B streptococci by the passage of these microorganisms through mice. This process was accompanied by the accumulation of cells with a high level of type-specific antigen (TSA). The passage of group B streptococci in the presence of type-specific antibodies led to the selection of avirulent microorganisms with low TSA production and high adhesiveness. These data may be considered to be the indirect evidence of the screening effect of TSA contained in the capsule of group B streptococci with respect to the ligand structures of these microbes. This suggestion is confirmed by the behavior of the variants of group B streptococci, obtained in the course of this investigation, on virus-infected tissue when TSA+ strains lost their ability to recognize viral polypeptides serving as receptors for TSA- variants of the streptococci.

Immuno-electronmicroscopic demonstration of capsules on group-B streptococci of new serotypes and type candidates.

The location of type polysaccharides on the cells of reference strains of group-B streptococci of serotypes IV and V and new type candidates NT6 and 7271 was investigated by electronmicroscopy of the bacteria after incubation with homologous type-specific antiserum. A distinct capsular layer was found on the surface of the cells of all these strains. Sialic acid, an integral part of all the conventional type polysaccharides of group-B streptococci, was also detected in all the strains examined.

Group B streptococci in the milk of lactating mothers.

A total of 1132 samples of maternal milk expressed from the disinfected breast on the first days after parturition were subjected to microbiological examination. Contamination by group B streptococci (Streptococcus agalactiae) was demonstrated in 40 samples (3.53%). Strains carrying the antigens Ia, Ia/c, Ib/c, II, III and R were represented among the isolates. Type Ia/c was the commonest; antigens II and III were always combined with the R antigen. The milk was either very strongly contaminated with almost pure culture demonstrable by direct cultivation of the milk sediment, or only isolated colonies were obtained in primary culture, often not until after enrichment. If the first samples gave dense growths, second-sample cultures were also positive. The strains isolated from samples of the individual mothers milk differed by their antigenic type structure; this gave evidence that the infection was not of nosocomial origin. Immunoglobulin levels in the whey of positive samples were not different from the whey of mothers not shedding group B streptococci. The health of both mothers and infants did not deviate from the general average for normal mothers and infants. No signs common to all the mothers shedding group B streptococci in their milk and no marked effect on the infants' health up to the age of 1-2 years were established.

A search for new group-B streptococcal serotypes.

New serotypes were sought among 165 clinical isolates of group-B streptococci that were untypable by antisera for the conventional types Ia, Ib, II and III. The strains were tested for sialic acid, an integral component of the group-B streptococcal type-polysaccharides; trypsin-treated bacteria were tested by slide-agglutination with the sialic-acid-specific lectin from the snail Cepaea hortensis. Sialic acid was detected in 96 of the strains; in 95 of these, new type antigens were identified serologically (type IV, 52; provisional type V, 34; candidate type NT6, seven; provisional type V and candidate type NT6, one; candidate type 7271, one); the remaining strain was found to possess a small amount of Ia antigen. Sialic acid was not detected in 69 strains, and none of these possessed a polysaccharide type-antigen. Chemical measurement of the sialic-acid content of cultures by Aminoff's method gave results in conformity with the lectin-agglutination test and the presence of polysaccharide type-antigens.