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The purpose of this investigation was to describe the clinical and biological characteristics and evolution of invasive Fusobacterium infections in children admitted to two French paediatric tertiary care centres. Children who were admitted from 1998 to 2009 to two tertiary care centres for invasive Fusobacterium infection were included in a retrospective study. Thirty-one children with a median age of 5.7 years (interquartile range, IQR [2.3; 9.3]) were included. Nine children had an underlying condition, most commonly sickle cell disease (n = 3) or immunodeficiency (n = 3). Two children had skin effraction prior to the infection. The major sites of infection were the head and neck (n = 14) and abdomen (n = 10). Three children suffered from atypical Lemierre's syndrome. More than half of the children had a bacterial co-infection (58 %). Six children were hospitalised in an intensive care unit, and 67 % of them had a chronic underlying disease. None of the children died. Six children with negative cultures had Fusobacterium identified through 16S RNA-PCR. Fusobacterium is responsible for severe infection in children. Microbiological diagnosis might be improved by the wider use of molecular detection.

We aimed to assess the independent effect of clinical spectrum, bacterial inoculum size and physician characteristics on the sensitivity of a rapid antigen detection test (RADT) for group A streptococcus (GAS) in children. Double throat swabs were collected from 1,482 children with pharyngitis and 294 asymptomatic children in a French prospective, office-based, multicenter (n = 17) study, from October 2009 to May 2011. Patient- and physician-level factors potentially affecting RADT sensitivity were studied by univariate and multivariate multilevel analysis, with laboratory throat culture as the reference test. In children with pharyngitis and asymptomatic children, the prevalence of GAS was 38 % (95 % confidence interval 36-41 %) and 11 % (7-14 %), respectively. Overall, RADT sensitivity was 87 % (84-90 %). On stratified and multivariate multilevel analysis, RADT sensitivity was higher for children with pharyngitis than asymptomatic children (89 % vs. 41 %), children <9 than ≥ 9 years old (88 % vs. 79 %) and those with heavy than light inoculum (94 % vs. 53 %). RADT sensitivity was influenced by the physician performing the test (range 56-96 %, p = 0.01) and was higher for physicians with hospital-based clinical activity in addition to office-based practice (adjusted odds ratio 3.4 [95 % confidence interval 1.9-6.3], p < 0.001); inter-physician variations in RADT sensitivity were largely explained by this variable (proportional change in variance >99 %). The sensitivity of the RADT is independently affected by patient- and physician-level factors. Physicians who base their diagnosis of GAS pharyngitis on the results of a RADT alone should consider diagnostic accuracy monitoring and adequate training when needed.

The outcome of bacterial bloodstream infections during pregnancy has greatly improved over the last few decades. However, there are no recent data on the characteristics of bacteremia in pregnant women. The aim of this study was to describe clinical and microbiological features of bacteremia and to assess maternal and fetal outcome. This retrospective study was conducted in the obstetrics departments of five teaching hospitals in Paris, France, from 2005 to 2009. The incidence of bacteremia was 0.3%. The most common sources of bacteremia were chorioamnionitis (47%) and the most common pathogen isolated was Escherichia coli. Empirical antimicrobial therapy was inappropriate in 29% of bacteremia cases, mostly (65%) when secondary to infection with an aminopenicillin-resistant microorganism. Bacteremia during pregnancy was associated with a 10% fetal mortality. Bacteremia during pregnancy is a rare occurrence, but it is associated with an unexpectedly poor fetal outcome and a high mortality rate.

M/emm typing, based either on serotyping of the M protein or on sequencing of the emm gene, is a major tool for epidemiological studies of group A streptococci (GAS). In order to simplify M/emm typing, we designed two multiplex polymerase chain reaction (PCR) formats capable of identifying the most frequent GAS M/emm types involved in invasive infections and antimicrobial resistance. A heptaplex PCR procedure was first developed in a conventional format coupled with gel electrophoresis to identify emm types 1, 3, 4, 6, 12, 28, and 89, based on the size of the amplification products. The other method, designed to identify the same seven emm types, together with emm11, was based on a real-time PCR format coupled with high-resolution melting (HRM) analysis, allowing the rapid typing of large strain collections.

Nosocomial outbreaks of extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae are an increasing concern in neonatal intensive care units (NICUs). We describe an outbreak of ESBL-producing K. pneumoniae that lasted 5 months and affected 23 neonates in our NICU. Proton pump inhibitor and extended-spectrum cephalosporin exposure were significantly associated with the risk of ESBL-producing K. pneumoniae colonisation and/or infection. Thirty isolates recovered from clinical, screening and environmental samples in the NICU were studied by means of Raman spectroscopy, pulsed-field gel electrophoresis and repetitive extragenic palindromic polymerase chain reaction (rep-PCR). The Raman clustering was in good agreement with the results of the other two molecular methods. Fourteen isolates belonged to the Raman clone 1 and 16 to the Raman clone 3. Molecular analysis showed that all the strains expressed SHV-1 chromosomal resistance, plasmid-encoded TEM-1 and CTX-M-15 β-lactamases. Incompatibility groups of plasmid content identified by PCR-based replicon typing indicated that resistance dissemination was due to the clonal spread of K. pneumoniae and horizontal CTX-M-15 gene transfer between the two clones.

Streptococcus pneumoniae is an uncommon cause of osteoarticular infections (OAI) in children. The objective of this study was to investigate the clinical and laboratory characteristics of pneumococcal OAI before and after the introduction of the heptavalent pneumococcal conjugate vaccine (PCV7). Data were retrospectively collected from children aged <16 years who were hospitalized for pneumococcal OAI between 1997 and 2007 in four Parisian teaching hospitals. Forty-three children were included (32 with arthritis and 11 with osteomyelitis) and the median age of these children was 12.5 months (range 3 months to 14 years). Serotypes were available for 19/43 strains (44 %) from 1997 onwards and for 12/13 strains (92 %) from 2005 onwards. Seven unvaccinated children were infected with vaccine serotypes and we observed only one vaccine failure. After the introduction of PCV7, we noted an increase in short-term complications and the emergence of serotype 19A, which was penicillin-intermediate in 86 % of cases. After PCV7 introduction, serotype 19A was the most frequent serotype implicated in pediatric pneumococcal OAI. The 13-valent pneumococcal conjugate vaccine introduced in France in June 2010 should cover the emerging serotype.

Multiple locus variable number of tandem repeats (VNTR) analysis (MLVA) has been shown to provide a high level of information for epidemiological investigations and the follow-up of Pseudomonas aeruginosa chronic infection. In the present study, an automatized MLVA assay has been developed for the analysis of 16 VNTRs in two multiplex polymerase chain reactions (PCRs), followed by capillary electrophoresis. The result in the form of a code is directly usable for clustering analyses. This MLVA-16(Orsay) scheme was applied to the genotyping of 83 isolates from eight cystic fibrosis patients, demonstrating that the same genotype persisted during eight years of chronic infection in the majority of cases. Comparison with pulsed-field gel electrophoresis (PFGE) analysis showed that both methods were congruent, MLVA providing, in some cases, additional informativity. The evolution of strains during long-term infection was revealed by the presence of VNTR variants.

Invasive group A streptococcal (GAS) infections have a broad and evolving clinical spectrum, associated with various GAS genotypes and/or virulence factors that are only poorly described in children. We aimed to assess the clinical and molecular characteristics of invasive GAS infections in 28 children admitted from 2000 to 2007 at a large French pediatric tertiary care center. The GAS isolates were characterized molecularly by emm-typing and by the determination of the main virulence factors: speA, speB, speC, smeZ-1, ssa, sic, and silC. The median age of the children was 2.9 years. Osteoarticular infection (OAI) was the main clinical manifestation (n=15/28, 53%). emm-1 predominated (n=10/28), followed by emm-12, 3, and 4. No significant correlation was found between emm type and clinical manifestations, but emm-1 predominated in cases of OAI (n=7/15) and was associated with speA, speB, smeZ-1, and sic virulence factor genes. In this pediatric study, we describe a predominance of OAI associated with emm-1 GAS. Further larger international pediatric studies, including host immunity evaluation, are needed in order to better assess the pathogenesis of GAS infection in children.

The French Pediatric Infectious Diseases Group set up an active surveillance network to analyze the clinical and biological features of pneumococcal meningitis and the impact of the seven-valent pneumococcal conjugate vaccine (PCV7). From 2001 to 2005, 234 pediatric wards working with 166 microbiology laboratories enrolled all children with pneumococcal meningitis. Risk factors, signs and symptoms, vaccination status, cerebrospinal fluid analysis, treatments and case fatality rates were recorded. One hundred and sixty-nine centers (169/234) reported 616 cases, median age was 0.9 years and 67.2% of children were < or =2 years old. Underlying conditions were present in 13.1% of cases. The proportion of penicillin non-susceptible strains was 48.7%. Vancomycin plus a third-generation cephalosporin was prescribed in 92.7% of cases, and steroids were given before antibiotic treatment in 16.5% of cases. The case fatality rate was 10.8% overall and was not related to age, antibiotic susceptibility or steroid use. In children 2 to 24 months old compared to the prevaccinal period (2001-2002) a decrease of 28.4% of the number of cases was observed in 2005 (P < 0.05). Among children 2 to 24 months old, the proportion of serotypes covered by the PCV7 fell from 39/57 (68.4%) in 2001-2002 to 19/45 (42.2%) in 2005, while the proportion of non-vaccine serotypes and related serotypes increased respectively from 9/57 (15.8%) and 9/57 (15.8%) in 2001-2002 to 14/45 (31.1%) and 12/45 (26.7%) in 2005. Among 52 cases of pneumococcal meningitis that have occurred in vaccinated children (> or =1 dose) with PCV7, 7 were due by vaccine serotypes. This study provides data on underlying conditions, penicillin susceptibility, serotype evolution according to vaccination status and risk factors for mortality for pneumococcal meningitis in children from 2001-2005 in France.

In January 1996 the emergence of penicillin-susceptible, multiresistant serotype 6B Streptococcus pneumoniae isolates resistant to chloramphenicol, tetracycline, erythromycin, clindamycin and trimethoprim-sulfamethoxazole was observed in young carriers in the city of Patras, located in the southwestern region of Greece. Later, a significant spread of pneumococci with this unusual phenotype was noted in carriers living in various other areas of the country. Using restriction fragment length polymorphism of the ribosomal RNA genes, clonal relationships were found between these Greek strains and serotype 6B penicillin-susceptible, multiresistant pneumococci isolated in France between January 1992 and September 1996. The French and Greek isolates appear to have a common ancestry.

A case of staphylococcal scalded skin syndrome in a neonate caused by mother-to-child transmission through breast-feeding is reported. Staphylococcus aureus isolates from mother and child were identified using random-amplified polymorphic DNA analysis, which is a simple and rapid method for comparing strains. This study confirms the need to avoid breast-feeding in cases of breast abscess.

In the evaluation of treatment failure in group A streptococcal pharyngitis, it is essential to distinguish persistence or relapse with homologous streptococcal strains from the acquisition of new, unrelated strains. Randomly amplified polymorphic DNA analysis and restriction fragment length polymorphism analysis of total DNA were used as epidemiological tools to compare 122 pre- and post-treatment Streptococcus pyogenes isolates obtained from 61 patients. The results obtained by molecular typing showed that bacteriological failures were due to the original strains in 43 cases (70%) and to new strains in 18 cases (30%). In the present study, restriction fragment length polymorphism analysis of total DNA appeared to be more discriminative than randomly amplified polymorphic DNA analysis. Thus, molecular analysis of DNA is an effective way to distinguish recurrence from persistence or relapse and will be useful in assessing the efficacy of new antibiotic treatments.

The killing kinetics of cefuroxime against 25 Streptococcus pneumoniae isolates with penicillin MICs of < 0.1, 0.1-1.0 and 2 mg/l were studied in an in vitro model simulating the serum concentration profile in healthy adults following a single intramuscular injection of 500 mg. Cefuroxime was bactericidal against the isolates with exquisite or slightly diminished susceptibility to penicillin (-4 and -3.9 log10 cfu/ml killing, respectively) during the 6 h incubation period. In contrast, there was only a 2.8 log10 cfu/ml reduction in the initial inoculum of six of the eight isolates resistant to penicillin (MIC 2 mg/l). The two remaining penicillin-resistant isolates (cefuroxime MIC 8 mg/l) only showed a 2 log10 cfu reduction in the initial inoculum.

Time-kill kinetic studies were used to measure the bactericidal activity of amoxicillin/clavulanic acid (in a new form for pediatric use), cefaclor, cefuroxime, cefixime and erythromycin against 30 beta-lactamase producing Moraxella catarrhalis strains. Antibiotics were tested at the mean maximum serum concentration observed after administration of a standard dose and at 0.5 x Cmax, 0.33 x Cmax and 0.25 x Cmax. A 2 log10 reduction in colony counts was obtained with the Cmax of amoxicillin/clavulanic acid and cefixime after 5 h of incubation. After 24 h of incubation a decrease of 3 and 4 log was observed with cefixime and amoxicillin/clavulanic acid respectively. At 0.5 x Cmax and 0.33 x Cmax, bactericidal activity was obtained only with amoxicillin/clavulanic acid.

A combination of esterase electrophoretic typing and analysis of the restriction fragment length polymorphism of ribosomal DNA regions (ribotyping) was used to compare 27 Pseudomonas aeruginosa strains isolated before and after two-week courses of anti-pseudomonal treatment in seven cystic fibrosis patients. A total of 12 courses of therapy were studied in which ciprofloxacin, ceftazidime, azlocillin or imipenem were used alone or in combination with tobramycin. Isolates at a count of greater than or equal to 10(6) cfu/ml of sputum were collected when there was evidence of therapeutic failure on the basis of persistence of isolates whether or not they were resistant to the antibiotic used for therapy. Emergence of resistance was observed in ten cases and failure to eradicate sensitive strains in five cases. Among the 27 isolates, eight zymotypes and five ribotypes were identified. With this typing approach, resistant post-therapy isolates were found to be identical to pre-therapy isolates in all cases but one. However, in one case an additional resistant strain was isolated after therapy besides that initially present. In all five cases in which susceptibility was still observed after treatment, pre-therapy and post-therapy isolates were indistinguishable. Using this molecular typing approach, all the strains were typable. Thus combination of esterase typing and ribotyping should improve the analysis of therapeutic failure in cystic fibrosis patients.

A dynamic in vitro model was used to determine the killing kinetics of daptomycin against 15 vancomycin-resistant clinical isolates of Enterococcus faecium. Concentration profiles simulating those observed in serum following administration of both low-dose (2 mg/kg) and high-dose (6 mg/kg) daptomycin were bactericidal within 5.5 and 2.8 h, respectively. In contrast, when albumin was added to the growth medium, the corresponding bacterial killing times were slowed to greater than 24 h and 7 h; these results suggest that in the clinical setting, daptomycin dosages of approximately 6 mg/kg are required to achieve bactericidal activity against vancomycin-resistant Enterococcus faecium.

Eighty-five cerebrospinal fluid (CSF) specimens from the same number of pediatric patients with meningitis were examined to determine the bacterial count and the relationship of this count to the microscopy results, the ages of the patients and the bacterial species isolated. Bacterial counts ranged from 2 x 10 to 4 x 10(9) CFU/ml CSF. Twenty-five percent of the 85 CSF specimens positive for Haemophilus influenzae type b, Neisseria meningitidis, Streptococcus pneumoniae, Escherichia coli K1 and group B streptococci had counts of 10(7) CFU/ml or higher. Children between 1 and 6 months of age had significantly higher counts (p less than 0.05) than the other age groups. The three patients who had positive CSF cultures 24 h after the start of therapy all had initial bacterial counts of 10(7) CFU/ml or higher. The detection limit for Gram stain/microscopy was 10(5) CFU/ml. No correlation was found between bacterial count and the number of polymorphonuclear leukocytes.