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1.  Phospholipase Cζ rescues failed oocyte activation in a prototype of male factor infertility 
Fertility and Sterility  2013;99(1):76-85.
Objective
To determine the effect of infertility-linked sperm phospholipase Cζ (PLCζ) mutations on their ability to trigger oocyte Ca2+ oscillations and development, and also to evaluate the potential therapeutic utility of wild-type, recombinant PLCζ protein for rescuing failed oocyte activation and embryo development.
Design
Test of a novel therapeutic approach to male factor infertility.
Setting
University medical school research laboratory.
Patient(s)
Donated unfertilized human oocytes from follicle reduction.
Intervention(s)
Microinjection of oocytes with recombinant human PLCζ protein or PLCζ cRNA and a Ca2+-sensitive fluorescent dye.
Main Outcome Measure(s)
Measurement of the efficacy of mutant and wild-type PLCζ-mediated enzyme activity, oocyte Ca2+ oscillations, activation, and early embryo development.
Result(s)
In contrast to the wild-type protein, mutant forms of human sperm PLCζ display aberrant enzyme activity and a total failure to activate unfertilized oocytes. Subsequent microinjection of recombinant human PLCζ protein reliably triggers the characteristic pattern of cytoplasmic Ca2+ oscillations at fertilization, which are required for normal oocyte activation and successful embryo development to the blastocyst stage.
Conclusion(s)
Dysfunctional sperm PLCζ cannot trigger oocyte activation and results in male factor infertility, so a potential therapeutic approach is oocyte microinjection of active, wild-type PLCζ protein. We have demonstrated that recombinant human PLCζ can phenotypically rescue failed activation in oocytes that express dysfunctional PLCζ, and that this intervention culminates in efficient blastocyst formation.
doi:10.1016/j.fertnstert.2012.08.035
PMCID: PMC3540263  PMID: 22999959
Fertilization; male infertility; oocyte activation; phospholipase C; PLC-zeta
2.  Time Trends in Pediatric Hospitalizations for Varicella Infection Are Associated with Climatic Changes: A 22-Year Retrospective Study in a Tertiary Greek Referral Center 
PLoS ONE  2012;7(12):e52016.
Background/Aims
The transmission rate of air-borne infectious diseases may vary secondary to climate conditions. The study assessed time trends in the seasonality of hospitalized varicella cases in a temperate region in relation to climatic parameters prior to the implementation of universal varicella immunization.
Methods
A retrospective descriptive study was conducted among all pediatric and adolescent varicella patients (n = 2366) hospitalized at the “Aghia Sophia” Children's Hospital during 1982–2003 in Athens, Greece. Date of infection was computed based on hospital admission date. Seasonal and monthly trends in the epidemiology of varicella infection were assessed with time series analysis (ARIMA modeling procedure). The correlation between the frequency of varicella patients and the meteorological parameters was examined by the application of Generalized Linear Models with Gamma distribution.
Results
During 1982–2003, the occurrence of hospitalized varicella cases increased during summer (p = 0.025) and decreased during autumn (p = 0.021), and particularly in September (p = 0.003). The frequency of hospitalized varicella cases was inversely associated with air temperature (p<0.001). In contrast, the occurrence of hospitalized varicella cases was positively associated with wind speed (p = 0.009).
Conclusions
Pediatric hospitalizations for varicella infection rates have increased during summer and decreased during autumn in the examined temperate region. Time trends in hospitalized varicella cases are associated with climatic variables.
doi:10.1371/journal.pone.0052016
PMCID: PMC3532345  PMID: 23284855
3.  A dedicated surveillance network for congenital toxoplasmosis in Greece, 2006-2009: assessment of the results 
BMC Public Health  2012;12:1019.
Background
Toxoplasmosis is caused by infection with the protozoan parasite Toxoplasma gondii. Acute infections in pregnant women may be transmitted to the fetus and cause severe illness. The purpose of this study was to establish a dedicated surveillance network (DSN) for congenital toxoplasmosis (CT) in Greece, in order to assess the birth prevalence of CT.
Methods
A DSN of thirty clinicians was established for reporting CT cases from hospitals throughout Greece. The clinicians were selected on the basis that there was a high possibility the suspected cases would be referred to them from district hospitals or private clinics. Suspected cases of CT were reported on a monthly basis with a zero reporting card during a surveillance period from April 2006 to December 2009. A questionnaire was sent for any suspected case to record information including demographic parameters, clinical signs and symptoms and laboratory results. Serological and molecular confirmation of cases was performed by the Pasteur Hellenic Institute. All newborns suspected of CT received treatment and were serologically and clinically followed up for one year.
Results
The monthly response rate reached 100%, although only after reminders sent to 65% of the participant physicians. Sixty-three suspected CT cases were recorded by the DSN during the study period including fourteen confirmed and seven probable cases. Ten cases (47.6%) presented with symptoms at birth. Chorioretinitis was the most prominent manifestation, occurring in five symptomatic CT cases (50%). No other symptoms appeared by the end of the one year clinical follow up. No case was recorded by the existing surveillance system of the Hellenic Center of Disease Control and Prevention (HCDCP) during the same time period. Birth prevalence was estimated at 0.45, 0.51 and 0.51 per 10,000 births for 2007, 2008 and 2009 respectively. The incidence rate of symptomatic CT at birth was estimated at 0.10 cases per 10,000 births per year in Greece (for the period 2007–2009).
Conclusion
The DSN for CT proved to be more sensitive than the classical notification system, easy in application and very efficient in reporting rare diseases such as CT. Similar DSNs could be used to provide useful information on other rare diseases.
doi:10.1186/1471-2458-12-1019
PMCID: PMC3533839  PMID: 23173875
Congenital Toxoplasmosis; Surveillance Network; Greece
4.  Risk Factors of Antibiotic Misuse for Upper Respiratory Tract Infections in Children: Results from a Cross-Sectional Knowledge-Attitude-Practice Study in Greece 
ISRN Pediatrics  2012;2012:685302.
Background. Upper respiratory tract infections (URTIs) are common in children. The cause of URTIs is usually viral, but parents' attitudes often contribute to inappropriate prescription of antibiotics, promoting antibiotic resistance. The objective of this study was to identify possible risk factors associated with antibiotic misuse in Greece, a country with high levels of antibiotic use and antibiotic resistance. Methods. A knowledge-attitude-practice (KAP) questionnaire was developed and distributed to Greek parents caring for children who were 5-6 years old, between January and July of the same school year. Results. The sample of the study contained 5312 parents from all geographic areas of Greece. The risk factors of being a father, having low education, having immigrant status, being a single parent, having low income, having <2 or >3 children, living in the islands, and being without experience in recurrent URTIs were significantly associated to inadequate knowledge, inappropriate attitudes, and wrong practices. Conclusions. This study has identified the main groups of parents that should be targeted in future intervention programs.
doi:10.5402/2012/685302
PMCID: PMC3503327  PMID: 23209933
5.  Pertussis Epidemiology in Greece and Emerging Risk Groups during the Vaccination Era (1980–2008) 
To study the epidemiology of pertussis in Greece and epidemiologic changes throughout a period of twenty-nine years, we conducted a retrospective analysis of available data of pertussis cases for the past twenty-nine years (1980–2008) and a prospective analysis of hospitalized pertussis cases from a children's hospital in Athens for eight years (2001–2008). From 1980 through 2008, the incidence of pertussis in Greece declined from 11.2 to 0.05 cases per 100,000. Epidemic cycles occurring every 3 to 5 years were observed. Since pertussis circulation cannot be fully controlled by present immunization programs, efforts should be made to vaccinate infants at the recommended age, early diagnose, treatment as well as contact tracing of pertussis cases. Control of pertussis in social susceptible populations is necessary. A national program with adolescent and adult booster could decrease the circulation of B. pertussis. Despite an overall decrease for pertussis cases, pertussis is still a present and future challenge of public health service in Greece.
doi:10.1155/2012/303846
PMCID: PMC3457588  PMID: 23019527
6.  PLCζ causes Ca2+ oscillations in mouse eggs by targeting intracellular and not plasma membrane PI(4,5)P2 
Molecular Biology of the Cell  2012;23(2):371-380.
At fertilization the sperm causes InsP3 production and Ca2+ release by introducing phospholipase C ζ (PLCζ) into the egg. This sperm PLCζ does not cause hydrolysis of plasma membrane PIP2. Instead it hydrolyzes PIP2 found in intracellular membranes. Fertilization may involve a novel aspect of the InsP3 signaling pathway.
Sperm-specific phospholipase C ζ (PLCζ) activates embryo development by triggering intracellular Ca2+ oscillations in mammalian eggs indistinguishable from those at fertilization. Somatic PLC isozymes generate inositol 1,4,5-trisphophate–mediated Ca2+ release by hydrolyzing phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in the plasma membrane. Here we examine the subcellular source of PI(4,5)P2 targeted by sperm PLCζ in mouse eggs. By monitoring egg plasma membrane PI(4,5)P2 with a green fluorescent protein–tagged PH domain, we show that PLCζ effects minimal loss of PI(4,5)P2 from the oolemma in contrast to control PLCδ1, despite the much higher potency of PLCζ in eliciting Ca2+ oscillations. Specific depletion of this PI(4,5)P2 pool by plasma membrane targeting of an inositol polyphosphate-5-phosphatase (Inp54p) blocked PLCδ1-mediated Ca2+ oscillations but not those stimulated by PLCζ or sperm. Immunolocalization of PI(4,5)P2, PLCζ, and catalytically inactive PLCζ (ciPLCζ) revealed their colocalization to distinct vesicular structures inside the egg cortex. These vesicles displayed decreased PI(4,5)P2 after PLCζ injection. Targeted depletion of vesicular PI(4,5)P2 by expression of ciPLCζ-fused Inp54p inhibited the Ca2+ oscillations triggered by PLCζ or sperm but failed to affect those mediated by PLCδ1. In contrast to somatic PLCs, our data indicate that sperm PLCζ induces Ca2+ mobilization by hydrolyzing internal PI(4,5)P2 stores, suggesting that the mechanism of mammalian fertilization comprises a novel phosphoinositide signaling pathway.
doi:10.1091/mbc.E11-08-0687
PMCID: PMC3258180  PMID: 22114355
7.  Testosterone-induced hypertrophy, fibrosis and apoptosis of cardiac cells – an ultrastructural and immunohistochemical study 
Summary
Background
Androgen abuse is an increasing problem amongst professional and amateur athletes. Moreover, testosterone, apart from its widely accepted indications, is used for a variety of other indications such as aging and ischemia. Its actions are mainly attributed to a specific genomic mechanism through the androgen receptor, but emerging evidence reveals non-genomic effects as well. The use of androgens has been linked with several adverse effects. The purpose of this study was to examine the effects of testosterone on the morphology and the ultrastructure of the myocardium and to investigate the possible role of apoptosis.
Material/Methods
We used 12 adult male Wistar rats, separated into 2 groups. Group A consisted of 6 rats that were administered high doses of testosterone enanthate, while group B consisted of 6 male Wistar rats that received placebo (normal saline) intramuscularly. After the last day of treatment, all rats were anesthetized and sacrificed, and the hearts were removed and processed for optical and electron microscopy and immunohistochemical detection of caspase-3, an apoptosis marker.
Results
We found significant myocardial hypertrophy along with abundant ultrastructural alterations. The immunohistochemical staining of the myocardial cells for caspase-3 was positive in group A (experimental group), which is interpreted as an activation of apoptosis by testosterone treatment.
Conclusions
Testosterone abuse has serious adverse effects, including myocardial hypertrophy, myocardial fibrosis and activation of apoptosis. These findings need to be taken into account whenever androgens are prescribed to improve performance or as hormone therapy.
doi:10.12659/MSM.881930
PMCID: PMC3560513  PMID: 21873939
myocardial hypertrophy; mitochondrial edema; apoptosis; androgen abuse
8.  Novel regulation of PLCζ activity via its XY-linker 
Biochemical Journal  2011;438(Pt 3):427-432.
The XY-linker region of somatic cell PLC (phospholipase)-β, -γ, -δ and -ϵ isoforms confers potent catalytic inhibition, suggesting a common auto-regulatory role. Surprisingly, the sperm PLCζ XY-linker does not mediate auto-inhibition. Unlike for somatic PLCs, the absence of the PLCζ XY-linker significantly diminishes both in vitro PIP2 (phosphatidylinositol 4,5-bisphosphate) hydrolysis and in vivo Ca2+-oscillation-inducing activity, revealing evidence for a novel PLCζ enzymatic mechanism.
doi:10.1042/BJ20110953
PMCID: PMC3195331  PMID: 21767260
calcium oscillation; egg activation; enzyme regulation; fertilization; phospholipase Cζ (PLCζ); XY-linker; BAPTA 1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid; GST, glutathione transferase; hCG, human chorionic gonadotrophin; H-KSOM, Hepes-buffered potassium simplex optimized medium; IP3, inositol 1,4,5-trisphosphate; ORF, open reading frame; PH, pleckstrin homology; PIP2, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; SH2, Src homology 2; XYl, XY-linker
9.  Prognostic factors related to sequelae in childhood bacterial meningitis: Data from a Greek meningitis registry 
BMC Infectious Diseases  2011;11:214.
Background
Bacterial meningitis (BM) is a life-threatening disease, often related with serious complications and sequelae. Infants and children who survive bacterial meningitis often suffer neurological and other sequelae.
Methods
A total of 2,477 patients aged 1 month to 14 years old hospitalized in a Children's Hospital in Greece diagnosed with acute bacterial meningitis were collected through a Meningitis Registry, from 1974 to 2005. Clinical, laboratory and other parameters (sex, age, pathogen, duration of symptoms before and after admission) were evaluated through univariate and multivariate analysis with regard to sequelae. Analysis of acute complications were also studied but not included in the final model.
Results
The rate of acute complications (arthritis and/or subdural effusion) was estimated at 6.8% (152 out of 2,251 patients, 95%CI 5.8-7.9) while the rate of sequelae (severe hearing loss, ventriculitis, hydrocephalus or seizure disorder) among survivors was estimated at 3.3% (73 out of 2,207 patients, 95%CI 2.6-4.2). Risk factors on admission associated with sequelae included seizures, absence of hemorrhagic rash, low CSF glucose, high CSF protein and the etiology of meningitis. A combination of significant prognostic factors including presence of seizures, low CSF glucose, high CSF protein, positive blood culture and absence of petechiae on admission presented an absolute risk of sequelae of 41.7% (95%CI 15.2-72.3).
Conclusions
A combination of prognostic factors of sequelae in childhood BM may be of value in selecting patients for more intensive therapy and in identifying possible candidates for new treatment strategies.
doi:10.1186/1471-2334-11-214
PMCID: PMC3166933  PMID: 21827712
10.  Phospholipase Cζ binding to PtdIns(4,5)P2 requires the XY-linker region 
Journal of Cell Science  2011;124(15):2582-2590.
Phospholipase C-zeta (PLCζ) is a strong candidate for the mammalian sperm-derived factor that triggers the Ca2+ oscillations required for egg activation at fertilization. PLCζ lacks a PH domain, which targets PLCδ1 to the phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) substrate in the plasma membrane. Previous studies failed to detect PLCζ in the plasma membrane, hence the means of PLCζ binding to PtdIns(4,5)P2 is unclear. We find that the PLCζ XY linker, but not the C2 domain, exhibits robust binding to PtdIns(4,5)P2 or to liposomes containing near-physiological levels of PtdIns(4,5)P2. The role of positively charged residues within the XY linker was addressed by sequentially substituting alanines for three lysine residues, K374, K375 and K377. Microinjection of these mutants into mouse eggs enabled their Ca2+ oscillation-inducing activities to be compared with wild-type PLCζ. The XY-linker mutant proteins were purified and the in vitro PtdIns(4,5)P2 hydrolysis and binding properties were monitored. Successive reduction of net positive charge within the PLCζ XY linker significantly affects both in vivo Ca2+-oscillation-inducing activity and in vitro PtdIns(4,5)P2 interaction of mouse PLCζ. Our data suggest that positively charged residues within the XY linker play an important role in the PLCζ interaction with PtdIns(4,5)P2, a crucial step in generating the Ca2+ activation signal that is essential for fertilization in mammals.
doi:10.1242/jcs.083485
PMCID: PMC3138701  PMID: 21730019
Phospholipase C; Phosphoinositide signalling; Calcium oscillations; Egg activation; Fertilization
11.  Descriptive Study on Parents’ Knowledge, Attitudes and Practices on Antibiotic Use and Misuse in Children with Upper Respiratory Tract Infections in Cyprus 
Upper respiratory tract infections (URTIs) are common in children and represent a significant cause of antibiotic abuse which contributes to the development of antibiotic resistance. A survey was conducted in Cyprus in 2006 to assess parents’ and pediatricians’ Knowledge, Attitude and Practices (KAP) concerning the role of antibiotics in children with URTIs. A school-based stratified geographic clustering sampling was used and a pre-tested KAP questionnaire was distributed. A different questionnaire was distributed to paediatricians. Demographic factors associated with antibiotic misuse were identified by backward logistic regression analysis. The parental overall response rate was 69.3%. Parents (N = 1,462) follow pediatricians advice and rarely administer antibiotics acquired over the counter. Although a third expects an antibiotic prescription for URTI symptoms, most deny pressuring their doctors. Low parental education was the most important independent risk factor positively related to antibiotic misuse (OR = 2.88, 95%CI 2.02 to 4.12, p < 0.001). Pediatricians (N = 33) denied prescribing antibiotics after parental pressure but admit that parents ask for antibiotics and believe they expect antibiotic prescriptions even when not needed. In conclusion, Cypriotic parents trust their primary care providers. Although it appears that antibiotic misuse is not driven by parental pressure, the pediatricians’ view differs.
doi:10.3390/ijerph8083246
PMCID: PMC3166740  PMID: 21909304
antibiotics; bacterial resistance; KAP study; knowledge; attitudes; practices; antibiotic overuse; antibiotic misuse; questionnaire; parents
12.  Antibiotic use for upper respiratory tract infections in children: A cross-sectional survey of knowledge, attitudes, and practices (KAP) of parents in Greece 
BMC Pediatrics  2011;11:60.
Background
Upper respiratory tract infections (URTIs) are common in children. The cause of URTIs is usually viral, but parents' attitudes often contribute to inappropriate prescription of antibiotics, promoting antibiotic resistance. The objective of this study was to document and analyse parental beliefs on antibiotic use for children with URTIs in Greece, a country with high levels of antibiotic use and antibiotic resistance.
Methods
A knowledge-attitude-practice questionnaire was developed and distributed to Greek parents caring for children who were 5-6 years old, between January and July of the same school year. The sample of the study contained parents from all geographic areas of Greece.
Results
The majority of Greek parents (80%) believed that UTRIs are mostly self-limited, although 74% of them expected to receive antibiotics when such a diagnosis was given. Earache was the most common reason for which parents expected antibiotics (45%). Greek parents rarely gave antibiotics to their children without medical advice (10%) and most (88%) believed that unnecessary antibiotic use drives antibiotic resistance and they were happy to receive symptomatic therapy if instructed by their physician. Almost 70% of parents confused antibiotics with other medicines used for symptomatic therapy for a child with URTI.
Conclusion
Greek parents have a trusted relationship with their paediatrician and rarely give antibiotics without medical advice, indicating that parents contribute less than expected to antibiotic misuse. Parents also appreciate the benign course of most URTIs and the fact that unnecessary antibiotic use is harmful. More time needs to be invested in educating mostly physicians on the potential benefit from reducing antibiotic prescribing for children with URTI.
doi:10.1186/1471-2431-11-60
PMCID: PMC3141508  PMID: 21729266
13.  Male infertility-linked point mutation disrupts the Ca2+ oscillation-inducing and PIP2 hydrolysis activity of sperm PLCζ 
Biochemical Journal  2011;434(Pt 2):211-217.
A male infertility-linked human PLCζ (phospholipase Cζ) mutation introduced into mouse PLCζ completely abolishes both in vitro PIP2 (phosphatidylinositol 4,5-bisphosphate) hydrolysis activity and the ability to trigger in vivo Ca2+ oscillations in mouse eggs. Wild-type PLCζ initiated a normal pattern of Ca2+ oscillations in eggs in the presence of 10-fold higher mutant PLCζ, suggesting that infertility is not mediated by a dominant-negative mechanism.
doi:10.1042/BJ20101772
PMCID: PMC3195387  PMID: 21204786
calcium oscillation; egg activation; fertilization; infertility; phospholipase C (PLC); phospholipase Cζ (PLCζ); BAPTA, 1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid; CCD camera, charge-coupled-device camera; GST, glutathione transferase; hCG, human chorionic gonadotropin; H-KSOM, Hepes-buffered potassium simplex optimized medium; ICSI, intracytoplasmic sperm injection; IP3, inositol 1,4,5-trisphosphate; IPTG, isopropyl β-D-thiogalactopyranoside; IVF, in vitro fertilization; PIP2, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; WT, wild-type
14.  Cognitive and psychosocial development of HIV pediatric patients receiving highly active anti-retroviral therapy: a case-control study 
BMC Pediatrics  2010;10:99.
Background
The psychosocial development of pediatric HIV patients has not been extensively evaluated. The study objectives were to evaluate whether emotional and social functions are differentially associated with HIV-related complications.
Methods
A matched case-control study design was conducted. The case group (n = 20) consisted of vertically infected children with HIV (aged 3-18 years) receiving HAART in Greece. Each case was matched with two randomly selected healthy controls from a school-based population. CNS imaging and clinical findings were used to identify patients with HIV-related neuroimaging abnormalities. The Wechsler Intelligence Scale III and Griffiths Mental Abilities Scales were applied to assess cognitive abilities. The age specific Strengths and Difficulties Questionnaire was used to evaluate emotional adjustment and social skills. The Fisher's exact test, student's t-test, and Wilcoxon rank sum test were used to compare categorical, continuous, and ordinal scores, respectively, of the above scales between groups.
Results
HIV patients without neuroimaging abnormalities did not differ from patients with neuroimaging abnormalities with respect to either age at HAART initiation (p = 0.306) or months of HAART treatment (p = 0.964). While HIV patients without neuroimaging abnormalities had similar cognitive development with their healthy peers, patients with neuroimaging abnormalities had lower mean General (p = 0.027) and Practical (p = 0.042) Intelligence Quotient scores. HIV patients without neuroimaging abnormalities had an increased likelihood of both Abnormal Emotional Symptoms (p = 0.047) and Hyperactivity scores (p = 0.0009). In contrast, HIV patients with neuroimaging abnormalities had an increased likelihood of presenting with Abnormal Peer Problems (p = 0.033).
Conclusions
HIV patients without neuroimaging abnormalities are more likely to experience maladjustment with respect to their emotional and activity spheres, while HIV patients with neuroimaging abnormalities are more likely to present with compromised social skills. Due to the limited sample size and age distribution of the study population, further studies should investigate the psychosocial development of pediatric HIV patients following the disclosure of their condition.
doi:10.1186/1471-2431-10-99
PMCID: PMC3022796  PMID: 21184692
15.  Diagnostic Clinical and Laboratory Findings in Response to Predetermining Bacterial Pathogen: Data from the Meningitis Registry 
PLoS ONE  2009;4(7):e6426.
Background
Childhood Meningitis continues to be an important cause of mortality in many countries. The search for rapid diagnosis of acute bacterial meningitis has lead to the further exploration of prognostic factors. This study was scheduled in an attempt to analyze various clinical symptoms as well as rapid laboratory results and provide an algorithm for the prediction of specific bacterial aetiology of childhood bacterial meningitis.
Methodology and Principal Findings
During the 32 year period, 2477 cases of probable bacterial meningitis (BM) were collected from the Meningitis Registry (MR). Analysis was performed on a total of 1331 confirmed bacterial meningitis cases of patients aged 1 month to 14 years. Data was analysed using EPI INFO (version 3.4.3-CDC-Atlanta) and SPSS (version 15.0 - Chicago) software. Statistically significant (p<0.05) variables were included in a conditional backward logistic regression model. A total of 838 (63.0%) attributed to Neisseria meningitidis, 252 (18.9%) to Haemophilus influenzae, 186 (14.0%) to Streptococcus pneumoniae and 55 (4.1%) due to other bacteria. For the diagnosis of Meningococcal Meningitis, the most significant group of diagnostic criteria identified included haemorrhagic rash (OR 22.36), absence of seizures (OR 2.51), headache (OR 1.83) and negative gram stain result (OR 1.55) with a Positive Predictive Value (PPV) of 96.4% (95%CI 87.7–99.6). For the diagnosis of Streptococcus pneumoniae, the most significant group of diagnostic criteria identified included absence of haemorrhagic rash (OR 13.62), positive gram stain (OR 2.10), coma (OR 3.11), seizures (OR 3.81) and peripheral WBC≥15000/µL (OR 2.19) with a PPV of 77.8% (95%CI 40.0–97.2). For the diagnosis of Haemophilus influenzae, the most significant group of diagnostic criteria included, absence of haemorrhagic rash (OR 13.61), age≥1year (OR 2.04), absence of headache (OR 3.01), CSF Glu<40 mg/dL (OR 3.62) and peripheral WBC<15000/µL (OR 1.74) with a PPV of 58.5% (95%CI 42.1–73.7).
Conclusions
The use of clinical and laboratory predictors for the assessment of the causative bacterial pathogen rather than just for predicting outcome of mortality seems to be a useful tool in the clinical management and specific treatment of BM. These findings should be further explored and studied.
doi:10.1371/journal.pone.0006426
PMCID: PMC2714179  PMID: 19641629
16.  Development and assessment of a questionnaire for a descriptive cross – sectional study concerning parents' knowledge, attitudes and practises in antibiotic use in Greece 
Background
Upper Respiratory Infections (URIs) are common in children. The cause is usually viral, but parents' attitude often contributes to inappropriate antibiotic prescribing, promoting antibiotic resistance. The objective is to describe the process of developing a questionnaire to assess parents' Knowledge, Attitude and Practices (KAP) concerning the role of antibiotics when children suffer from URIs, as well as to evaluate the response rates, the completeness and the reliability (Cronbach) of the questionnaires. Finally, to note any limitations of the study.
Methods
Literature review, along with pre – testing yielded a questionnaire designed to assess the parents' KAP – level. A postal survey was set, in a national sample of 200 schools stratified by geographical region. The participants consist of a multistage geographical cluster sample of 8000 parents. The influence of demographic characteristics (i.e. sex, age, education) was analyzed. Cronbach index test and factor analysis were used to assess the reliability of the questionnaire.
Results
The response rate of the parents was 69%. Islands presented the lowest response rate while in Northern Greece the response rate was the highest. Sixty – eight point nine percent of the sample returned questionnaires fully completed, while 91.5% completed 95% of the questions. Three questions out of 70 were answered in a very low rate which was associated mostly with immigrant respondents. The section describing parents' attitude toward antibiotic use was not completed as much as the sections of knowledge or practices. The questions were factor analyzed and 10 out of the 21 extracted factors were finally evaluated, reducing the number of independent variables to 46. The reliability of the questionnaire was 0.55. However, only items that increased the Cronbach when added were eventually included in the final scales raising the internal consistency to 0.68. Limitations of the study, such as the vocabulary and form of the questionnaire and the idiocycrancy of the respondents, emerged during the analysis.
Conclusion
The response rate and the completeness of the questionnaires were higher than expected, probably attributed to the involvement of the teachers. The study findings were satisfactory regarding the development of a reliable instrument capable to measure parents' KAP characteristics.
doi:10.1186/1471-2334-9-52
PMCID: PMC2686701  PMID: 19413902
17.  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.
doi:10.1186/1471-2334-7-101
PMCID: PMC2031898  PMID: 17760993
18.  Aseptic Meningitis in Children: Analysis of 506 Cases 
PLoS ONE  2007;2(8):e674.
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/mm3 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.
doi:10.1371/journal.pone.0000674
PMCID: PMC1933255  PMID: 17668054
19.  Adherence to the screening program for HBV infection in pregnant women delivering in Greece 
Background
Hepatitis B infection (HBV) is a major Public Health Problem.
Perinatal transmission can be prevented with the identification of HBsAg(+) women and administration of immunoprophylaxis to their newborns. A national prevention programme for HBV with universal screening of pregnant women and vaccination of infants is in effect since 1998 in Greece.
Methods
To evaluate adherence to the national guidelines, all women delivering in Greece between 17–30/03/03 were included in the study. Trained health professionals completed a questionnaire on demographic data, prenatal or perinatal screening for HBsAg and the implementation of appropriate immunoprophylaxis.
Results
During the study period 3,760 women delivered. Prenatal screening for HBsAg was documented in 91.3%. Greek women were more likely to have had prenatal testing. HBsAg prevalence was 2.89% (95%CI 2.3–3.4%). Higher prevalence of HBV-infection was noted in immigrant women, especially those born in Albania (9.8%). Other risk factors associated with maternal HBsAg (+) included young maternal age and absence of prenatal testing. No prenatal or perinatal HBsAg testing was performed in 3.2% women. Delivering in public hospital and illiteracy were identifiable risk factors for never being tested. All newborns of identified HBsAg (+) mothers received appropriate immunoprophylaxis.
Conclusion
The prevalence of HBsAg in Greek pregnant women is low and comparable to other European countries. However, immigrant women composing almost 20% of our childbearing population, have significant higher prevalence rates. There are still women who never get tested. Universal vaccination against HBV at birth and reinforcement of perinatal testing of all women not prenatally tested should be discussed with Public Health Authorities.
doi:10.1186/1471-2334-6-84
PMCID: PMC1475591  PMID: 16681862

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