Characteristics of Streptococcus pneumoniae serotype 19A isolates from children in the pre and post Conjugate Vaccine Era. Single center experience 1986-2015.

BACKGROUND: The present study assessed the prevalence and characteristics of S. pneumoniae serotype 19A isolates from children with pneumococcal disease (PD), before and since introduction of pneumococcal conjugate vaccines (PCVs) in Greece. METHODS: S. pneumoniae isolates collected at one large pediatric hospital between 1986 and 2015 were serotyped by the Quellung reaction and MICs determined by Etest. Alterations of pbp genes and the presence of mefA, mefE, ermB genes were detected by polymerase chain reaction. Genotypes were assessed by multilocus sequence typing (MLST). RESULTS: Among 1875 isolates, 210 (11.2%) belonged to serotype 19A. The prevalence of PD caused by serotype 19A increased from 4.6% in the pre-PCV7 years (1986-2005) to 19.6% in the post-PCV7 years (2006-2010), peaking at 27% in 2009 (p < 0.001, 95% CI; 2.0, 18.2) with a significant upward trend (p = 0.04, 95% CI; 1.02, 12.66). Following the introduction of PCV13 in 2010, the rate decreased from 22% in 2011 to 11.4% in 2015 (p = 0.08, 95% CI; 0.92, 5.1) with a downward trend of borderline significance (p = 0.05, 95% CI; -6.8, 0.04). The multidrug resistant (MDR) serotype 19A isolates increased from 10.6% in 1986-2005 to 21.2% in 2006-2010 and to 71.8% in 2011-2015 (P < 0.001). Alterations in pbp genes were detected in all penicillin non-susceptible isolates. Of 110 erythromycin resistant isolates, 21 contained the mefE gene, 36 the ermB and 53 both the mefE and ermB genes. MLST analysis of 142 isolates revealed four dominant clonal complexes (CC); CC320, CC172, CC276 and CC199. The majority of CC320 and CC276 isolates displayed MDR phenotypes. CONCLUSION: PD caused by serotype 19A increased significantly after the introduction of PCV7 followed by a decline after PCV13 use. The vast majority of persisting 19A isolates was MDR. Surveillance studies are necessary to monitor the changes in the pneumococcal population.

Detection of bacterial pathogens in synovial and pleural fluid with the FilmArray Blood Culture Identification System.

We report the use of FilmArray Blood Culture Identification (BCID) multiplex PCR system for pathogen detection from a child with septic arthritis that Streptococcus pyogenes was identified directly from synovial fluid and a child with complicated pneumonia with pleural effusion that Streptococcus pneumoniae was identified from pleural fluid.

Molecular analysis of Streptococcus pyogenes macrolide resistance of paediatric isolates during a 7 year period (2007-13).

OBJECTIVES: The molecular characterization of paediatric group A Streptococcus (GAS) isolates regarding macrolide resistance and relevant emm types in Athens, Greece. METHODS: Pharyngeal and non-pharyngeal GAS isolates were collected during a 7 year period (2007-13) and examined for antibiotic susceptibility, macrolide resistance genes [mef(A), erm(A) and erm(B)] and relevant emm types. RESULTS: Overall, 20.4% (270/1324) of GAS isolates were resistant to macrolides. The macrolide resistance rate varied during the study period with a maximum rate observed in 2008 (29.57%) and a minimum rate observed in 2013 (10.95%) (P value for trend = 0.007). During the same period, consumption of macrolides was gradually reduced by 56.6%. No difference was observed in macrolide resistance between pharyngeal and non-pharyngeal isolates (P = 0.7). Among macrolide-resistant isolates, mef(A) was detected in 87 (32.2%), erm(A) in 136 (50.4%), erm(B) in 44 (16.3%) and both mef(A) and erm(A) in 3 (1.1%) isolates. The most prevalent emm types among macrolide-resistant isolates were emm77 (31.5%), emm4 (18.1%) and emm12 (10.7%). Ten emm types (77, 4, 12, 28, 1, 22, 11, 2, 44 and 89) accounted for 90.3% of macrolide-resistant isolates. emm types 4, 22, 44 and 77 were more prevalent in macrolide-resistant compared with macrolide-susceptible isolates, whereas emm types 1, 3, 5, 6, 75 and 89 were more prevalent in macrolide-susceptible compared with macrolide-resistant isolates. CONCLUSIONS: GAS macrolide resistance remained significant in our area during the study period. A substantial decline in the resistance rate was observed in the last year of the study, which may be related to reduced consumption of macrolides.

Pneumococcal Mastoiditis in Children Before and After the Introduction of Conjugate Pneumococcal Vaccines.

OBJECTIVES: To determine whether serotype distribution and antibiotic resistance of Streptococcus pneumoniae acute mastoiditis (AM) in children have changed in the post pneumococcal conjugate vaccines (PCVs) era. METHODS: Medical records of pneumococcal AM cases, in a tertiary pediatric hospital were reviewed from January 1999 to December 2014. S. pneumoniae isolates were serotyped using the quellung reaction and tested for antibiotic susceptibility by E-test and for macrolide resistance genes by polymerase chain reaction. RESULTS: Among 334 children with AM, S. pneumoniae was isolated from 89 (26.6%) with median age 22 months (interquartile range: 12-30 months). S. pneumoniae was recovered from ear fluid (58%), mastoid specimens (35.2%) and blood (6.8%). Resistance to penicillin, erythromycin and clindamycin was 12.4%, 49.4% and 18%, respectively. Distribution of pneumococcal serotypes before (1999-2005), after the introduction of PCV7 (2006-2010) and after PCV13 (2011-2014) was found: for the PCV7 serotypes 81%, 25% and 0% (P < 0.0001), for PCV13 additional serotypes 16.3%, 70.8% and 63.6% (P < 0.0001) and for non-PCV serotypes 2.3%, 4.1% and 36.3% (P = 0.0002), respectively. Significant increase was detected for the serotype 19A after PCV7, and this trend was not changed after PCV13 (2.3%, 50% and 50%, respectively; P < 0.0001). A significant proportion of resistant isolates to penicillin (54.5%) and erythromycin (34.8%) was identified as 19A. CONCLUSIONS: After the introduction of PCV7, a significant increase of serotype 19A and replacement of PCVs serotypes was identified. After PCV13, the overall proportion of pneumococcal mastoiditis and the incidence of serotype 19A were not significantly declined. A significant proportion of resistant isolates to penicillin and erythromycin is attributed to serotype 19A.

Rotavirus Gastroenteritis in a Neonatal Unit of a Greek Tertiary Hospital: Clinical Characteristics and Genotypes.

INTRODUCTION: Rotavirus (RV) infection in neonatal age can be mild or even asymptomatic. Several studies have reported that RV is responsible for 31%-87% of pediatric nosocomial diarrhea and causes gastroenteritis outbreaks in pediatric and neonatal units. OBJECTIVES: Study clinical characteristics, genotypes and risk factors of RV infection in neonatal age. METHODS: A prospective study was conducted from April 2009 till April 2013 in the neonatal special care unit of the largest tertiary pediatric hospital of Greece. Fecal samples and epidemiological data were collected from each neonate with gastrointestinal symptoms. RV antigen was detected with a rapid immunochromatography test. RV positive samples were further genotyped with RT PCR and sequencing using specific VP7 and VP4 primers. RESULTS: Positive for RV were 126/415 samples (30.4%). Mean age of onset was 18 days. Seventy four cases (58%) were hospital acquired. Seasonality of RV infection did not differ significantly throughout the year with the exception of 4 outbreaks. Genotypes found during the study period were G4P[8] (58.7%), G1P[8] (14.7%), G12P[8] (9.3%), G3P[8] (9.3%), G12P[6] (5.3%), G9P[8] (1.3%) and G2P[4] (1.3%). RV cases presented with: diarrhea (81%), vomiting (26.2%), fever (34.9%), dehydration (28.6%), feeding intolerance (39.7%), weight loss (54%), whilst 19% of cases were asymptomatic. Comparing community with hospital acquired cases differences in clinical manifestations were found. CONCLUSIONS: Significant incidence of nosocomially transmitted RV infection in neonatal age including asymptomatic illness exists. Genotypes causing nosocomial outbreaks are not different from community strains. Circulating vaccines can be effective in prevention of nosocomial RV infection through herd immunity.

Pasteurella multocida Infection in a neonate: evidence for a human-to-human horizontal transmission.

Pasteurella multocida is usually transmitted by animal contact; however, in a significant proportion of cases, no animal exposure can be identified. Although vertical transmission has been identified in neonates, horizontal human-to-human spread has not been documented. A case of neonatal sepsis and meningitis resulting from horizontal transmission of P. multocida is described.

Respiratory viruses involved in influenza-like illness in a Greek pediatric population during the winter period of the years 2005-2008.

Viruses are the major cause of pediatric respiratory tract infection and yet many suspected cases of illness remain uncharacterized. This study aimed to determine the distribution of several respiratory viruses in children diagnosed as having influenza-like illness, over the winter period of 2005-2008. Molecular assays including conventional and real time PCR protocols, were employed to screen respiratory specimens, collected by clinicians of the Influenza sentinel system and of outpatient pediatric clinics, for identification of several respiratory viruses. Of 1,272 specimens tested, 814 (64%) were positive for at least one virus and included 387 influenza viruses, 160 rhinoviruses, 155 respiratory syncytial viruses, 95 adenoviruses, 81 bocaviruses, 47 parainfluenza viruses, 44 metapneumoviruses, and 30 coronaviruses. Simultaneous presence of two or three viruses was observed in 173 of the above positive cases, 21% of which included influenza virus and rhinovirus. The majority of positive cases occurred during January and February. Influenza virus predominated in children older than 1 year old, with type B being the dominant type for the first season and subtypes A/H3N2 and A/H1N1 the following two winter seasons, respectively. Respiratory syncytial virus prevailed in children younger than 2 years old, with subtypes A and B alternating from year to year. This is the most comprehensive study of the epidemiology of respiratory viruses in Greece, indicating influenza, rhinovirus and respiratory syncytial virus as major contributors to influenza-like illness in children.

Inflammatory bowel disease in children: the role of a positive family history.

OBJECTIVES: The aim of this study was to evaluate any potential influence of a family history of inflammatory bowel disease (IBD) on the clinical phenotypes and the course of IBD in children. METHODS: In this retrospective study, the notes of 411 children with the diagnosis of IBD, 244 (59.4%) with ulcerative colitis, 129 (31.4%) with Crohn's disease and 38 (9.2%) with IBD unclassified, who were admitted to our department between 1 January 1981 and 31 December 2007 were reviewed. The aim was to assess the prevalence of familial IBD and its impact on the age of disease onset, clinical phenotypes according to the Montreal classification, course and outcome of disease. The control group consisted of IBD children without a family history of IBD, who were admitted to the hospital during the same time period. RESULTS: Thirty five (8.5%) children had a family history of IBD, (ulcerative colitis 6.6%, Crohn's disease 10.9% and IBD unclassified 13.2%). Sixty-eight percent of the 22 pairs of first-degree relatives were concordant for the clinical phenotype of disease. Significantly, more children with familial IBD had symptom onset and/or disease diagnosis before 5 years of age compared with sporadic IBD (P = 0.01 and P = 0.014, respectively); however, no differences were seen in sex, clinical phenotypes, need for aggressive treatment and/or surgery. CONCLUSION: Children with familial IBD had earlier onset of disease compared with those with sporadic IBD. However, this had no significant impact on the clinical phenotypes, the course and/or the outcome of disease.

Febrile seizures and primary human herpesvirus 6 infection.

Primary human herpesvirus 6 infection is acquired mainly during the first two years of life and is often associated with febrile seizures. The aim of the present study was to investigate in Greece the frequency and clinical characteristics of primary human herpesvirus 6 (HHV-6) infection in hospitalized children with febrile seizures. Children aged from 6 months to 5 years without known neurologic disease were examined for primary HHV-6 infection, by real-time polymerase chain reaction in acute-phase plasma and by indirect immunofluorescent assay for antibody titers in acute and convalescent serum. Of 65 children included in the analysis, 55 experienced the first febrile episode of seizures and 10 the second. Primary HHV-6 infection was verified in 10 of 55 children with a first febrile episode (18%), whereas none of the 10 children with a second episode of seizures had primary HHV-6 infection. Eight children were infected with HHV-6 type B and two with type A. None of the 85 control subjects had primary HHV-6 infection, but 49% had immunoglobulin G antibodies against the virus. These findings suggest that primary HHV-6 infection is frequently associated with febrile seizures in children in this geographic region and should be considered, especially for a first episode of febrile seizures.

Changing pattern in the clinical presentation of pediatric celiac disease: a 30-year study.

BACKGROUND/AIMS: The incidence of celiac disease (CD) has increased in recent years due to the recognition of atypical forms and the identification of silent cases through serological screening. Our aim was to detect temporal trends in the presentation of pediatric CD in Greece. METHODS: We reviewed the medical files of all children diagnosed with CD between 1978 and 2007 at a single academic pediatric center. Cases were classified according to the year of diagnosis. We examined demographic data, presenting symptoms, delay to diagnosis, and the prevalence of associated conditions. RESULTS: During the study period, 284 new cases of CD were diagnosed. The incidence of CD was significantly increased in recent years (p < 0.05). We observed significant trends towards older age at diagnosis (p < 0.001), longer delay to diagnosis (p < 0.05) and decreased frequency of the classical and/or gastrointestinal predominant mode of presentation (p < 0.001). In recent years, diagnosis of CD was significantly more frequent due to testing of asymptomatic children with a positive family history for CD or personal history of associated conditions (p < 0.001). CONCLUSION: We report a changing pattern in the presentation of pediatric CD in Greece. CD is diagnosed more frequently in older children, oftentimes presents with atypical symptoms, and is increasingly detected through serological screening. CD should be considered in the presence of atypical presentations.

Macrolide resistance in Streptococcus pyogenes: prevalence, resistance determinants, and emm types.

To investigate the antimicrobial resistance trends and the distribution of emm types of group A streptococci (GAS), we examined 1160 clinical isolates of GAS collected between 2003 and 2006. Susceptibilities to commonly used antimicrobial agents were determined by Etest, and macrolide resistance genes were detected by polymerase chain reaction (PCR). GAS isolates were typed by polymerase chain reaction PCR and sequencing of emm gene. The rates of resistance to erythromycin (ERY), clindamycin, azithromycin, tetracycline, and chloramphenicol were 14.9%, 1.4%, 14.9%, 18.9%, 0.6%, respectively. None of the isolates exhibited resistance to penicillin, ceftriaxone, linezolid, moxifloxacin, rifampicin, or vancomycin. Macrolide resistance increased from 12.1% in 2003 to 18.8% in 2006 (P = 0.02). Of 173 ERY-resistant GAS isolates, 93 (53.7%) harbored the mefA gene, 70 (40.4%) the ermA, and 10 (5.8%) the ermB. Eighty percent of the observed emm types are covered by the proposed 26-valent GAS vaccine. Among 173 ERY-resistant isolates, the predominant emm types were 12 (19.5%), 77 (17.9%), and 4 (16.8%), and among 770 ERY-susceptible isolates, the predominant types were 1 (18.8%), 12 (17.5%), 28 (13.8%). The observed antimicrobial resistance trends and the distribution of specific emm types have implications in guiding empiric therapy and in developing vaccine strategies to prevent GAS infections.

Wilson disease in children: analysis of 57 cases.

OBJECTIVES: Wilson disease (WD) has a wide spectrum of clinical manifestations. Affected children may be entirely asymptomatic and the diagnosis problematic. Herein we present the clinical and laboratory characteristics of 57 children with WD and point out the diagnostic difficulties in a pediatric population. PATIENTS AND METHODS: Clinical and laboratory data were collected from 57 consecutive children with WD. Evaluation included detailed physical examination, conventional laboratory testing, genetic analysis, and liver biopsy. RESULTS: The mean age at diagnosis was 9.27 +/- 3.62 years (range 4 months-18 years). Twenty patients were symptomatic, 19 were referred because of abnormal liver function test results and/or hepatomegaly, and 18 received their diagnoses after family screening. Twenty-two patients had both Kayser-Fleischer ring and decreased serum ceruloplasmin levels, 13 had urinary copper excretion after penicillamine challenge >1600 microg/24 hours, and 3 had liver copper content >250 microg/g dry weight. Of the remaining 19 patients, 17 had both low serum ceruloplasmin 75 microg/24 hours before, or >1000 microg/24 hours after penicillamine challenge. In 2 patients with equivocal cases who had serum ceruloplasmin 26 mg/dL, the diagnosis was confirmed by genetic analysis. No correlation was found between specific mutations and the disease phenotypic expression. Chelating therapy was well tolerated, and the outcome was satisfactory. CONCLUSIONS: WD in children may be obscure and requires extensive investigation to establish the diagnosis. Genetic analysis is needed in equivocal cases.

Evolution of macrolide resistance in Streptococcus pneumoniae clinical isolates in the prevaccine era.

Six hundred twelve invasive and noninvasive Streptococcus pneumoniae isolates were examined. Serogrouping was performed by the latex agglutination test and serotyping by the quellung reaction. Susceptibilities to macrolides were determined by Etest. The presence of mef(A), mef(E), and erm(B) genes were detected by polymerase chain reaction. Outpatient macrolide and lincosamide consumption was expressed in defined daily doses per 1000 inhabitants daily (DID). A significant increase in macrolide resistance rate was noted from 7.4% (14/190) in the period 1985 to 1996 to 53.7% (144/268) in 2001 to 2004 (P = 0.003). An increase in macrolide and lincosamide consumption was also observed from 4.31 +/- 0.72 in 1990 to 1996 to 6.97 +/- 1.02 DID in 2001 to 2004 (P = 0.002). Macrolide resistance was mediated by mef(E) gene in 44.5% of isolates, mef(A) in 25.6%, erm(B) in 19.8%, both erm(B) and mef(E) genes in 4.8%, and none of the examined genes in 5.3%. In the setting of increasing macrolide use, there has been a parallel increase in macrolide resistance among pneumococci in our region. The predominant resistance determinants were the mef(A) and mef(E) genes.

Meningitis registry of hospitalized cases in children: epidemiological patterns of acute bacterial meningitis throughout a 32-year period.

BACKGROUND: Bacterial meningitis remains a source of substantial morbidity and mortality in childhood. During the last decades gradual changes have been observed in the epidemiology of bacterial meningitis, related to the introduction of new polysaccharide and conjugate vaccines. The study presents an overview of the epidemiological patterns of acute bacterial meningitis in a tertiary children 's hospital during a 32-year period, using information from a disease registry. Moreover, it discusses the contribution of communicable disease registries in the study of acute infectious diseases. METHODS: In the early 1970s a Meningitis Registry (MR) was created for patients admitted with meningitis in Aghia Sofia Children's Hospital in Athens. The MR includes demographic, clinical and laboratory data as well as treatment, complications and outcome of the patients. In 2000 a database was created and the collected data were entered, analyzed and presented in three chronological periods: A (1974-1984), B (1985-1994) and C (1995-2005). RESULTS: Of the 2,477 cases of bacterial meningitis registered in total, 1,146 cases (46.3%) were classified as "probable" and 1,331 (53.7%) as "confirmed" bacterial meningitis. The estimated mean annual Incidence Rate (IR) was 16.9/100,000 for bacterial meningitis, 8.9/100,000 for Neisseria meningitidis, 1.3/100,000 for Streptococcus pneumoniae, 2.5/100,000 for Haemophilus influenzae type b (Hib) before vaccination and 0.4/100,000 for Hib after vaccination. Neisseria meningitis constituted the leading cause of childhood bacterial meningitis for all periods and in all age groups. Hib was the second most common cause of bacterial meningitis before the introduction of Hib conjugate vaccine, in periods A and B. The incidence of bacterial meningitis due to Streptococcus pneumoniae was stable. The long-term epidemiological pattern of Neisseria meningitidis appears in cycles of approximately 10 years, confirmed by a significant rise of IR in period C. The Case Fatality Rate (CFR) from all causes was 3.8%, while higher CFR were estimated for Streptococcus pneumoniae (7.5%, RR=2.1, 95% CI 1.2-3.7) and Neisseria meningitidis (4.8%, RR=1.7, 95% CI 1.1-2.5) compared to other pathogens. Moreover, overall CFR varied significantly among the three time periods (p = 0.0015), and was estimated to be higher in period C. CONCLUSION: By using the MR we were able to delineate long-term changes in the epidemiology of bacterial meningitis. Thus the MR proved to be a useful tool in the study and the prevention of communicable diseases in correlation with prevention strategies, such as vaccinations.

Aseptic meningitis in children: analysis of 506 cases.

BACKGROUND: Non-polio human enteroviruses are the leading cause of aseptic meningitis in children. The role of enterovirus PCR for diagnosis and management of aseptic meningitis has not been fully explored. METHODOLOGY/PRINCIPAL FINDINGS: A retrospective study was conducted to determine the epidemiological, clinical, and laboratory characteristics of aseptic meningitis and to evaluate the role of enterovirus PCR for the diagnosis and management of this clinical entity. The medical records of children who had as discharge diagnosis aseptic or viral meningitis were reviewed. A total of 506 children, median age 5 years, were identified. The annual incidence rate was estimated to be 17/100,000 children less than 14 years of age. Most of the cases occurred during summer (38%) and autumn (24%). The dominant clinical symptoms were fever (98%), headache (94%) and vomiting (67%). Neck stiffness was noted in 60%, and irritation in 46% of the patients. The median number of CSF cell count was 201/mm(3) with polymorphonuclear predominance (>50%) in 58.3% of the cases. Enterovirus RNA was detected in CSF in 47 of 96 (48.9%) children tested. Children with positive enterovirus PCR had shorter hospitalization stay as compared to children who had negative PCR or to children who were not tested (P = 0.01). There were no serious complications or deaths. CONCLUSIONS: Enteroviruses accounted for approximately one half of cases of aseptic meningitis. PCR may reduce the length of hospitalization and plays important role in the diagnosis and management of children with aseptic meningitis.

Evaluation of imipenem/imipenem+EDTA disk method for detection of metallo-beta-lactamase-producing Klebsiella pneumoniae isolated from blood cultures.

The objective of this study was to evaluate the imipenem (IMP) and IMP+EDTA (IMP/IMP+EDTA) disk method for the detection of metallo-beta-lactamases (MBLs) in clinical isolates of Klebsiella pneumoniae with various MIC levels to IMP. Forty-one blood isolates of K. pneumoniae with MIC to IMP ranging from < or =0.5 to > or =16 microg/ml were examined. The MICs were determined by VITEK-2 (bioMerieux Vitek two, France). Disks of 10 microg IMP with and without the addition of 0.5 M EDTA were used for the IMP/IMP+EDTA disk method. The E-test (AB Biodisk, Solna, Sweden) for MBL detection was also used. All isolates were examined for the bla (VIM-1) gene by PCR and for clonality of VIM-1-producing isolates by pulsed-field gel electrophoresis (PFGE). All isolates with MIC values of IMP < or =0.5 microg/ml exhibited no differences in inhibition zone diameters (IZD) produced by IMP and IMP+EDTA disks, whereas the isolates with MICs > or =1 microg/ml showed an increase in IZD, ranging from 8 to 26 mm. All isolates with MIC values of > or =1 microg/ml were found positive for the bla (VIM-1) gene by PCR and for MBL production by the E-test, whereas none of isolates with MICs <0.5 microg/ml was found positive by any of the tests. DNA restriction fragments generated by PFGE of VIM-1-producing isolates were classified in four main types. The IMP/IMP+EDTA disk method is simple to perform, sensitive, and specific for detection of MBL-producing K. pneumoniae clinical isolates. K. pneumoniae isolates with MICs of IMP > or =1 microg/ml and/or IZD produced by IMP disk <19 mm should be tested for MBL production.

Detection of Mycobacterium tuberculosis DNA in respiratory and nonrespiratory specimens by the Amplicor MTB PCR.

To evaluate the diagnostic performance of a commercially available Mycobacterium tuberculosis PCR assay (Amplicor MTB-ROCHE), 2296 respiratory and nonrespiratory specimens from 2296 patients with suspected tuberculosis (TB) were collected prospectively in an 8-year period. Clinical data for each patient were abstracted, and all samples were examined blindly by direct microscopy, culture, and PCR. M. tuberculosis DNA was detected in 93 of 113 culture-positive samples and in 29 of 38 samples from patients with probable TB. The lowest sensitivity was observed in pleural fluid and abscess aspirates. The sensitivity, specificity, and positive predictive value of the assay were 97.2%, 100%, and 100% for smear-positive specimens and 75.3%, 97.0%, and 47.5% for smear-negative specimens, respectively. The PCR cost per additional correct clinical decision was Euro 2826 but would have declined to Euro 308 if the test was applied only to smear-positive specimens. The overall performance of Amplicor MTB test was excellent for smear-positive, but suboptimal for smear-negative specimens.