Sequence-based identification, genotyping and EUCAST antifungal susceptibilities of Trichosporon clinical isolates from Greece.

Trichosporon yeasts constitute emerging pathogens, implicated in organ-specific and systemic infections. In this first, comprehensive study of Trichosporon clinical isolates in Greece, 42 isolates were identified by sequencing the hypervariable D1/D2 domain of the Large Subunit (LSU) rDNA gene, while Trichosporon asahii were genotyped by sequencing the Intergenic Spacer 1 region, and antifungal susceptibilities were determined by the EDef 7.2 (EUCAST) method, in parallel with the CLSI standard. Trichosporon asahii was the primary species (37 isolates) followed by Trichosporon coremiiforme, Trichosporon dermatis, Trichosporon loubieri and Trichosporon mycotoxinivorans. One strain remained unidentified. Seven T. asahii genotypes were recorded. The major genotypes were: genotypes 4 (29%) and 3 (26%) followed by 1, 5 and 7 (9.5% each). Two novel genotypes were identified designated as 10 and 11. EUCAST MIC ≥2 mg/L was recorded in 58% of the isolates (amphotericin B), 41% (itraconazole), 41% (posaconazole) and 38% (voriconazole). Fluconazole MICs of ≥32 mg/L were recorded in 23.8% of the isolates. Analysis of variance performed on absolute values showed that the amphotericin B, itraconazole, posaconazole and voriconazole MICs of T. asahii were equivalent. Typically higher MIC values were displayed by fluconazole. Antifungal susceptibilities of the seven different genotypes were homogeneous. Agreements between EUCAST and CLSI ranged from 88.1 to 97.62%. Overall, the high MICs recorded among the Trichosporon isolates for all tested drugs justify routine susceptibility testing of clinical isolates.

Differences in the epidemiology between paediatric and adult invasive Streptococcus pyogenes infections.

In order to investigate for possible differences between paediatric and adult invasive Streptococcus pyogenes (iGAS) infections, a total of 142 cases were identified in 17 Greek hospitals during 2003-2007, of which 96 were children and 46 adults. Bacteraemia, soft tissue infections, streptococcal toxic shock syndrome (STSS), and necrotizing fasciitis were the main clinical presentations (67·6%, 45·1%, 13·4%, and 12·0% of cases, respectively). Bacteraemia and lymphadenitis were significantly more frequent in children (P=0·019 and 0·021, respectively), whereas STSS was more frequent in adults (P=0·017). The main predisposing factors in children were varicella and streptococcal pharyngotonsillitis (25% and 19·8%, respectively), as opposed to malignancy, intravenous drug abuse and diabetes mellitus in adults (19·6%, 15·2% and 10·9%, respectively). Of the two dominant emm-types, 1 and 12 (28·2% and 8·5%, respectively), the proportion of emm-type 12 remained stable during the study period, whereas emm-type 1 rates fluctuated considerably. Strains of emm-type 1 from children were associated with erythromycin susceptibility, STSS and intensive-care-unit admission, whereas emm-type 12 isolates from adults were associated with erythromycin and clindamycin resistance. Finally, specific emm-types were detected exclusively in adults or in children. In conclusion, several clinical and epidemiological differences were detected, that could prove useful in designing age-focused strategies for prevention and treatment of iGAS infections.

Blood stream infections throught the entire course of acute lymphoblastic leukemia treatment.

The incidence, type and mortality of bacteremias were evaluated in a pediatric patient cohort, during the entire course of treatment for acute lymphoblastic leukemia (ALL). Eighty-six patients with newly diagnosed ALL were studied. A bacteremic episode was defined as blood isolation of a pathogen in the presence of clinical symptomatology of septicaemia. Bacteremias were analyzed according to the treatment element being delivered and the degree of neutropenia. A central venous catheter (CVC) was inserted at diagnosis in all patients. Fifty-two episodes of bacteremias were encountered in 38/86 (44%) patients, while 48/86 patients had no positive blood culture. Three out of the 38 patients had bacteremia and CVC area infection, simultaneously. Most blood stream infections (29/52, 56%) were documented during the induction phase. Isolated Gram-positive organisms were 48%, Gram-negative 50% and 2% of the positive blood cultures represented fungaemias. The most common Gram-positive isolates were Staphylococcus species (N=22) and the commonest Gram-negative isolated pathogens were Escherichia coli and Pseudomonas aeruginosa. The majority of bacteremias (75%) occurred during neutropenia. The initial antibiotic treatment was ceftazidime or piperacillin/tazobactam and amikacin or tobramycin. CVC was not removed in the majority of bacteremias (94%). No infection related fatality was recorded. Bacteremias constituted a severe and common complication in our patient cohort. However, infection-related fatality rate was negligible, most probably due to the prompt initiation of broad coverage antimicrobial therapy.

Prevalence of emm types 1 and 12 from invasive Streptococcus pyogenes disease in Greece--results of enhanced surveillance.

Among a total of 101 isolates from the first systematic multicentre surveillance effort concerning invasive Streptococcus pyogenes disease in Greece, conducted between 2003 and 2005 and covering 38% of the population, emm types 1 and 12 were prevalent, being responsible for 27 and nine cases, respectively. The isolates from the remaining 65 cases were assigned to 26 other emm types. Erythromycin resistance (12 isolates) was primarily mef(A)-mediated, although all emm type 1 strains were susceptible. Tetracycline resistance, due mostly to tet(M), was detected in 26 isolates. Subtyping by pulsed-field gel electrophoresis yielded 50 chromosomal fingerprints, thus discriminating further among ten of the 28 observed emm types.

Spread of the Streptococcus pneumoniae Taiwan19F-14 clone among children in Greece.

Serotype 19F pneumococci were a leading cause of infections among children in Athens, Greece during 2001-2006. In total, 143 19F isolates were typed by pulsed-field gel electrophoresis (PFGE), and 38 isolates representing the main PFGE types were also characterised by multilocus sequence typing. A diversity of distinct strains belonging to sequence types 236, 1035, 274, 172 and 319 were identified, but multidrug-resistant isolates related to the Taiwan(19F)-14 clone (ST236) constituted 76.9% of the isolates. Spread of the Taiwan(19F)-14 clone explains, in part, the high incidence of antibiotic resistance observed among pneumococci reported recently from Athens.

Nationwide surveillance of Streptococcus pneumoniae in Greece: patterns of resistance and serotype epidemiology.

This nationwide study assessed the antimicrobial susceptibility and seroprevalence of Streptococcus pneumoniae in paediatric carriage isolates and in clinical isolates from adult pneumococcal disease in Greece during the years 2004-2006. Among 780 isolates recovered from the nasopharynx of children <6 years old attending day-care centres, non-susceptibility rates to penicillin, cefuroxime, ceftriaxone, erythromycin, tetracycline and trimethoprim/sulfamethoxazole were 34.7%, 25.1%, 1.0%, 33.5%, 26.4% and 44.2%, respectively. Among 89 adult clinical isolates, the respective rates were 48.3%, 46.1%, 5.6%, 48.3%, 32.6% and 40.4%. High-level resistance to penicillin, cefuroxime and ceftriaxone was recorded for 14.4%, 23.3% and 0.1% of paediatric carriage isolates, whereas for clinical adult isolates the respective rates were 25.8%, 38.2% and 2.2%. No resistance to levofloxacin and moxifloxacin was recorded, although 3.5% of paediatric carriage isolates and 23.2% of adult clinical isolates had minimum inhibitory concentrations of ciprofloxacin >2mg/L. Serotypes 19F, 14, 23F and 6B were the most prevalent among carriage and clinical isolates. The 7-valent pneumococcal conjugate vaccine was estimated to provide coverage against 71.7% of paediatric carriage isolates and 51.3% of adult clinical isolates. Resistance rates among clinical isolates from adult sources were higher than those recorded among paediatric carriage S. pneumoniae isolates and displayed an increasingly resistant profile compared with previous reports from our country, warranting continuous vigilance.

Invasive meningococcal disease in children in Greece: comparison of serogroup A disease with disease caused by other serogroups.

Although invasive meningococcal disease caused by serogroup A is not prevalent in developed countries, a considerable number of cases were recently recorded in Greece. In this study, serogroup A meningococcal disease was compared prospectively with meningococcal disease caused by other serogroups, using similar settings of testing and management during a 5-year period between 1999 and 2003. The Neisseria meningitidis serogroup was determined in 262 cases. Serogroup B predominated, accounting for 158 (60%) of the cases. Serogroup A was second most frequent (19%), followed by serogroups W135 (11%), C (8%), and Y (2%). No cases due to serogroup C were recorded during the last year of the study. Patients with serogroup A disease were older and had a milder course compared to patients infected with serogroups B or C. Toxic appearance, purpura, thrombocytopenia, abnormal coagulation tests, and the need for admission to the intensive care unit, fluid resuscitation, inotropic drugs, and mechanical ventilation were less common. Although morbidity and mortality were lower in these patients, the differences were not significant. Serogroup B is predominant in our area, and the introduction of an effective vaccine against it is a priority. Serogroup A has emerged as the second most common serogroup, but the illness associated with it is milder.

Serotypes and antimicrobial susceptibilities of 1033 pneumococci isolated from children in Greece during 2001-2004.

Pneumococci (n = 1033) isolated in the major paediatric hospitals of Athens during 2001-2004 from children with invasive infections (n = 186), non-invasive infections (n = 641) and healthy carriers (n = 206) were studied. The most prevalent serotypes were serotypes 14 (44.6%), 19F (43.5%) and 6B (22.8%) in invasive, non-invasive and carriage isolates, respectively. Among invasive isolates, the potential coverage by the seven-valent conjugate vaccine was 75.3%. Resistance rates to penicillin, amoxycillin, cefotaxime, erythromycin, co-trimoxazole, clindamycin, tetracycline and chloramphenicol were 44.6%, 2.7%, 1.2%, 43.6%, 43.5%, 12.4%, 34.7% and 5.9%, respectively. The M-phenotype accounted for 68.0% of the erythromycin-resistant isolates. All isolates were susceptible to ofloxacin.

Carriage of Neisseria meningitidis by Greek children: risk factors and strain characteristics.

Oropharyngeal swabs were cultured from 554 children aged 2-19 years attending nurseries, primary schools and secondary schools in the central Athens area. A questionnaire was completed to identify risk factors for carriage. Susceptibility to antimicrobial agents was determined by Etest. The genetic relatedness of the strains was examined by pulsed-field gel electrophoresis (PFGE), and isolate serogrouping was performed by slide agglutination. Twenty-two (4%) children were carriers of Neisseria meningitidis; seven isolates belonged to serogroup C, and five to serogroup B. One isolate was resistant to co-trimoxazole, and five showed intermediate resistance to penicillin. DNA analysis of 16 isolates revealed six distinct PFGE patterns. Clusters with indistinguishable PFGE patterns were noted in the same school. More than one serogroup was included in the same clonal group. On multivariate logistic regression analysis, only age > 12 years remained independently associated with the carrier state (odds ratio, 7.96; 95% CI, 2.24-28.33; p < 0.001). Overall, the N. meningitidis carriage rate among Greek schoolchildren increased with age, and the predominant serogroups in the Athens region were groups C and B. These findings may have important implications for future immunisation strategies with conjugate vaccines.

In vitro activity of telithromycin (HMR 3647) against Greek Streptococcus pyogenes and Streptococcus pneumoniae clinical isolates with different macrolide susceptibilities.

The susceptibilities to macrolides and telithromycin of 161 Streptococcus pyogenes and 145 Streptococcus pyogenes strains, consecutively isolated from five Greek hospitals, were determined by Etest. Moreover, mechanisms of resistance to macrolides were phenotypically and genetically determined by double disk induction test and PCR, respectively. Of the S. pneumoniae and S. pyogenes isolates, 42.8% and 30.8%, respectively, were found to be resistant to erythromycin. Of the erythromycin-resistant S. pneumoniae and S. pyogenes isolates, 57.5% and 59.5%, respectively, displayed the M phenotype and harbored the mefA/E gene. Telithromycin was found to be more active than both erythromycin and clarithromycin against both species, with considerably lower MIC50 and MIC90 values.

Predominance of two M-types among erythromycin-resistant group A Streptococci from Greek children.

In order to investigate the potential relationship between erythromycin resistance and specific M-serotypes among clinical isolates of Streptococcus pyogenes from children in Greece, we randomly selected a total of 49 erythromycin-resistant (EryR) and 21 erythromycin-susceptible (EryS) isolates from the 1158 S. pyogenes isolates from the two main children's hospitals of Athens during the period October 1997 to October 1998. The isolates were further characterized by M-serotyping, examined for their susceptibility to penicillin, vancomycin and clindamycin, and categorized into resistance phenotypes. A total of 248 (21%) S. pyogenes isolates in the two main children's hospitals of Athens during the study period were resistant to erythromycin. All 49 EryR and 21 EryS isolates were susceptible to penicillin and vancomycin. With respect to erythromycin and clindamycin resistance, phenotypes M and IR MLSB dominated, with 30 and 17 isolates, respectively, two isolates belonged to the CR MLSB phenotype. Among the erythromycin resistant isolates, two M serotypes were dominant: M22 (30%) and M84 (41%). More specifically, M22 and M84 were most prevalent in resistance phenotypes IR MLSB (65%) and M (63%), respectively. In the susceptible group, no isolate belonged to these two M-serotypes, nor was a predominant serotype found. In contrast to susceptible isolates, two distinct M-serotypes were highly represented among EryR S. pyogenes isolates and predominantly associated with two distinct phenotypes.

Fluconazole in the treatment of pediatric tinea capitis caused by Microsporum canis.

Fluconazole has been shown to be effective in the treatment of fungal scalp infections in children; however, there is limited experience of its use in Microsporum scalp infections. We studied 11 children with tinea capitis caused by Microsporum canis who received oral fluconazole at a dose of 5-7.5 mg/kg/day for 6 weeks. Mycological cure was observed in two of the 11 patients at week 4 from the start of therapy, in four patients at week 8 and in three patients at week 16. One of the remaining patients had positive mycology at week 8, but was unavailable for further evaluation. Fluconazole was effective in treating pediatric tinea capitis caused by Microsporum canis and was well tolerated. Clinical and mycological response was achieved in some patients weeks after the cessation of the administration of fluconazole.

Phenotypic and genotypic approaches to characterization of isolates of Neisseria meningitidis from patients and their close family contacts.

Characterization of isolates of Neisseria meningitidis obtained from patients with meningococcal disease or from pharyngeal swabs of asymptomatic carriers can be achieved by several approaches which provide different levels of discrimination. A total of 45 gram negative, oxidase-positive diplococcus strains isolated from 15 individuals with meningococcal disease and 30 of their family contacts were examined by three approaches: serological typing, multilocus enzyme electrophoresis (MLEE), and multilocus sequence typing (MLST). For 10 of the 15 patient and contact groups, all of the isolates were confirmed as meningococci, and the bacteria obtained from the patients and contacts, including their mother or principal caregiver in the case of children, were indistinguishable by all three methods. In the remaining five groups the isolates from the patients were distinct from those recovered from the contacts, and in three examples, in two separate groups, the contacts were shown by MLST to be carrying strains of Neisseria lactamica. The data obtained from the three techniques were consistent, although complete serological typing was possible for only a minority of isolates. Both MLEE and MLST established the genetic relationships of the isolates and identified members of known hypervirulent lineages, but MLST was faster than MLEE and had the additional advantages that it could be performed on noninfective material distributed by mail and that the results from different laboratories could be compared via the internet (http://mlst.zoo.ox.ac.uk).