Eudoxia Diza

Molecular and phylogenetic analysis of the haemagglutinin gene of pandemic influenza H1N1 2009 viruses associated with severe and fatal infections.

The objectives of this research is molecular and phylogenetic analysis of pandemic influenza A(H1N1) 2009 strains that circulated in northern Greece, focusing on severe or fatal infections, identification of sequence variations in relation with the severity of the illness and comparison of circulating viruses with the vaccine strain. A total of 1598 infections were attributed to the novel influenza A(H1N1) virus. Molecular analysis revealed a number of variations at the HA1 sequences of northern Greek circulating strains, some of which were more frequent in viruses that caused severe or fatal infections. Such mutations, the most common being D222G, demand close monitoring to continuously assess associated risks. Phylogenetic analysis confirmed the close match of the majority of circulating strains with A/California/7/09. However it also reveals a trend of 2010 strains to accumulate amino acid variations and form new plylogenetic clades. Constant molecular surveillance is important to monitor the pathogenicity of circulating strains and evaluate the vaccine efficacy.

Salivary concentration of the antimicrobial peptide LL-37 in children

OBJECTIVE Antimicrobial peptides are important components of innate immunity, especially in the unique environment of the oral cavity. Lack of the human cathelicidin LL-37 has been implicated in severe periodontitis, whilst high salivary levels of LL-37 seem to increase caries resistance. Limited data exists about the concentration of LL-37 in saliva of young children. In this study, the salivary concentration of LL-37 was examined in relation to age, gender, type of dentition (primary, mixed or permanent) and caries experience of children. DESIGN Unstimulated whole saliva was collected from 49 systemically healthy and gingivitis free children aged 2-18 years old. Their caries activity was recorded. The salivary LL-37 concentration was determined by enzyme linked immunosorbent assay (ELISA). RESULTS LL-37 was detected in all saliva samples. Its concentration varied widely, with girls exhibiting higher peptide levels than boys. A positive correlation of the LL-37 concentration was observed with age. Children with primary dentition had significantly lower peptide concentration than those with mixed or permanent dentition. Significantly lower concentrations of LL-37 were also found in children with high caries activity, compared to caries free children or to children with low to moderate caries activity. CONCLUSIONS Our results reinforce the belief that LL-37 is an important molecule of immunity in the oral environment and it seems to play a protective role against caries.

Epidemiological Study of Tick-Borne Encephalitis Virus in Northern Greece

Serum samples from 921 apparently healthy individuals living in different prefectures of Northern Greece were investigated for the presence of antibodies against tick-borne encephalitis (TBE) virus. In addition, serum and cerebrospinal fluid samples of 302 patients with central nervous system (CNS) infection were tested for the presence of specific IgG and IgM antibodies and TBE virus RNA. Two percent of the general population was found to have antibodies to the virus, with no significant differences among the age groups. Most of the seropositive individuals were male farmers, while seroprevalence varied among different prefectures (0%-5.8%). TBE was not confirmed by laboratory findings in any of the patients with CNS infection. Results of this study revealed that a flavivirus of the TBE serocomplex is circulating in Greece, yet is not a major public health problem.

Interleukin-6 and the matrix metalloproteinase response in the vitreous during proliferative vitreoretinopathy.

PURPOSE To investigate the levels of IL-6 in the vitreous of patients with RRD complicated with PVR and correlate the IL-6 levels with matrix metalloproteinase (MMP)-1,-2,-3,-8,-9 and tissue inhibitor of metalloproteinases (TIMP)-1 with respect to RRD extent, duration and PVR grade. DESIGN Cohort study. PARTICIPANTS Twenty-eight vitreous samples from 28 eyes of 28 patients with RRD complicated with PVR. METHODS Institutional study. Twenty-eight vitreous samples from 28 eyes of 28 patients with RRD complicated with PVR were collected during pars plana vitrectomy (PPV) and were compared to vitreous control samples. IL-6, MMP-1,-3,-8 and TIMP-1 levels were measured using ELISA while enzymatic activity of MMP-2, and -9 was determined employing gelatin zymography. RESULTS Protein IL-6 (p=0.030), MMP-1 (p=0.003), MMP-3 (p=0.003), TIMP-1 (p=0.001) levels as well as enzymatic activity of proMMP-9 (p=0.013), MMP-9 (p=0.017) and proMMP-2 (p=0.010), were significantly increased in PVR patients as compared to controls. IL-6 levels correlated with MMP-1 (p=0.002), proMMP-2 (p=0.006), MMP-3 (p=0.001) and TIMP-1 (p=0.006). Regression analysis revealed positive correlations between IL-6 and all MMPs and TIMP-1. CONCLUSIONS Taking into account the previously established effect of interleukins in MMP activity, the findings of this study suggest a role of IL-6 in MMP stimulation during PVR development.

Correlation of the extent and duration of rhegmatogenous retinal detachment with the expression of matrix metalloproteinases in the vitreous.

Background: Investigation of the activity of matrix metalloproteinase (MMP)-2 and -9 and protein levels of MMP-1, -3, -8, and the tissue inhibitor of MMPs (TIMP)-1 in the vitreous of patients with rhegmatogenous retinal detachment (RRD) and establishment of potential correlations of MMPs with clinical parameters. Methods: Thirty-two vitreous samples from patients with RRD and 9 vitreous samples from human organ donors (controls) were assayed for MMP-1,-3, -8, and TIMP-1 levels using enzyme-linked immunosorbent assay and MMP-2 and -9 activity employing gelatin zymography. Results: MMP-1, MMP-3, proMMP-2, proMMP-9, MMP-9, and TIMP-1 were higher in vitreous from patients with RRD as compared to organ donors. Overall, MMPs and TIMPs were differentially expressed in vitreous from RRD with respect to the duration and extent of RRD. Regression analysis for all data indicated that a model consisting of MMP-2 and TIMP-1 could estimate the extent of RRD. Conclusion: Levels of MMPs and TIMP-1 studied are elevated in vitreous during RRD. MMP-2 and TIMP-1 may have a more prominent and persistent role than other MMPs in the wound healing process of the retina during RRD. A regression model consisting of MMP-2 and TIMP-1 may prove to be of potential use in providing information for the evaluation of the extent of RRD.

Expression of matrix metalloproteinases in the subretinal fluid correlates with the extent of rhegmatogenous retinal detachment

BackgroundWe investigated the activity of matrix metalloproteinase (MMP)−2 and −9 and the protein levels of MMP−1, −3, −8 and the tissue inhibitor of MMPs (TIMP)−1 in the subretinal fluid (SRF) of patients with rhegmatogenous retinal detachment (RRD) and establishment of potential correlations with clinical parameters.MethodsThirty-seven SRF from RRD patients and nine vitreous samples from the human eye of organ donors (controls) were collected and assayed for MMP−1,−3,−8 and TIMP−1 levels using ELISA and for MMP−2 and −9 activity employing gelatin zymography.ResultsMMP−1, MMP−3, MMP−8, proMMP−2, proMMP−9, MMP−9 and TIMP−1 levels were higher in SRF compared with vitreous fluid. Overall, MMPs and TIMPs were differentially expressed in SRF with respect to duration and extent of RRD, as well as to stage of proliferative vitreoretinopathy. Regression analysis for all data indicated that a model consisting of MMP−3, MMP−8 and proMMP−9 could estimate the extent of RRD.ConclusionsMMPs and TIMP−1 levels are elevated in SRF during RRD. A regression model consisting of MMP−3, MMP−8 and proMMP−9 may be proved to be of potential use in providing information for evaluation of the extent of RRD.

Serologic Evidence of Human Granulocytic Ehrlichiosis, Greece

To the Editor: Human granulocytic ehrlichiosis (HGE), a tickborne infectious disease, was first described in 1994 (1). Several cases have been reported in the United States; reports of acute cases in Europe have been rare, although European serosurveys of the prevalence of antibodies to the HGE agent have been conducted (2–4). No similar serosurvey has been conducted in Greece, although Ixodes ricinus, thought to be the principal tick vector in Europe (5), is present in northern Greece (6). Lyme disease, which is transmitted by the same tick, has never been reported, and the seroprevalence of Lyme borreliosis in Greece is very low (7). We examined sera of 300 persons (100 men and 200 women) ages 15–78 years (mean age ± standard deviation 52.7±18.0 years), which were collected at six county hospitals in northern Greece and sent to our laboratory from April to October 2000. The participants were mostly farmers, all of whom lived in rural areas of northern Greece. All participants were healthy and had been hospitalized for routine blood tests. Each patient completed a questionnaire about medical history. The selected patients had no known history of rickettsiosis and reported no febrile or influenza-like illness during the past 6 months. Each participant provided oral consent for the serum to be used for detecting antibodies against several infectious agents related to zoonoses. The following information was recorded for each participant: age, sex, occupation, and area of residence. Serum samples were tested by indirect immunofluorescence (IFA) with commercially available antigen (Focus Technologies, Cypress, California), which uses HGE-1–infected HL60 cells. Titers >64 were considered positive. All sera were also tested for Rickettsia conorii, R. typhi, Coxiella burneti, and Ehrlichia chaffeensis by IFA and for Borrelia burgdorferi by enzyme-linked immunosorbent assay and Western blot. Sera that reacted positively to more than one of these agents were excluded. Biostatistical analysis was performed by using the statistical package SPSS for Windows 10.0.1 (Standard version, SPSS Inc., Chicago, IL). The overall prevalence of antibodies to the HGE agent was 7.3% (8.0% for men and 7.0% for women). No statistically significant differences were observed in the prevalence of antibodies in the six prefecture hospitals. Participants had no statistically significant differences in sex or age. Antibody titers to HGE were low (of 22 positive sera, 12 had titers >64 and 10 had titers >128). Several serosurveys of the prevalence of antibodies to the HGE agent have been conducted across Europe in both healthy persons and patients with suspected or confirmed Lyme borreliosis (2,3,8). Since cases of B. burgdorferi infection are rare or nonexistent in Greece and the seroprevalence of Lyme borreliosis is very low, we selected as participants 300 healthy farmers who lived in rural areas. These persons compose a group at high risk for exposure to tick bites and therefore to I. ricinus. Our prevalence is higher than those observed in Bulgaria (2.9%) and Germany (1.9%) (2,3). This finding could be attributed to the fact that the prevalence in these countries was based on blood donors, unlike our survey. However, our prevalence is substantially lower than that in Slovenia, where 15.4% of the examined population had detectable antibodies to the HGE agent and several cases of HE have been confirmed (4). Our observation that no significant differences occurred in the prevalence of antibodies to the HGE agent in the six prefectures studied could be explained by the fact that these districts are small, with little variation in environmental and climatic conditions. Even though the antibody titers to the HGE agent were low in our survey, they suggest infection at an undetermined time (9). Seven of our sera were antibody positive to both the HGE agent and at least one other rickettsial agent or B. burgdorferi. This fact, which has been observed elsewhere (9), may result from coinfection or crossreaction. These sera were excluded. Our data suggest the possibility that HGE cases exist in Greece. Since such cases have been not been reported to date, they are likely underdiagnosed. Further research is needed to clarify the presence of the HGE agent in Greece.

Correlation of matrix metalloproteinase levels with the grade of proliferative vitreoretinopathy in the subretinal fluid and vitreous during rhegmatogenous retinal detachment

Purpose:  We investigated the activity of matrix metalloproteinase (MMP)‐2 and ‐9 and their latent pro‐forms (proMMP‐2, ‐9), and protein levels of MMP‐1, ‐3, ‐8 and tissue inhibitor of MMPs (TIMP)‐1 in the subretinal fluid (SRF) and vitreous of patients with rhegmatogenous retinal detachment (RRD). Potential correlations with proliferative vitreoretinopathy (PVR) grade were determined.

Laboratory diagnosis of Mycoplasma pneumoniae respiratory tract infections in children

Mycoplasma pneumoniae is a common respiratory tractpathogen that causes up to 40% of cases of community-acquired pneumonia in children [1]. A specific diagnosis isessential because treatment of M. pneumoniae infection withβ-lactam antibiotics is ineffective. In routine laboratories,serology remains an important diagnostic tool [2, 3].However, it can only provide a retrospective diagnosis andpaired samples are required. Recently developed PCRtechniques show high levels of specificity and sensitivity forthe rapid detection of M. pneumoniae in clinical specimens[4, 5]; however, PCR alone is not always sufficient for adiagnosis [3]. In lieu of a gold-standard diagnostic method,we aimed to evaluate the laboratory methods currently usedto diagnose M. pneumoniae infection in order to find the onemost suitable for rapidly diagnosing the illness, especially inthe early phase of disease.During a 15-month period, a throat swab (viscose swab)andfirstserumspecimenweretakenonadmissionfromatotalof 75 children (42 males, 33 females; mean age 6.2 years;range 4 months–14 years) who were hospitalized fortreatment of a respiratory tract infection (RTI). According tothe guidelines of the British Thoracic Society, 53 of thesechildren had pneumonia, 16 pharyngitis and 6 tracheobron-chitis. A second serum specimen was obtained 9–24 dayslater.ThethroatspecimenswereexaminedforthepresenceofM. pneumoniae using culture in methylene blue–glucosediphasic medium (Oxoid, Basingstoke, UK), a sandwichenzyme-immunoassay for antigen detection (EIA-Ag Virion/Serion, Germany) and a PCR technique for DNA detection(extracted DNA was amplified by the primer pair P1-1 andP1-3 for the P1 adhesin gene [6]). Antibodies against M.pneumoniae were measured using the immunofluorescenceassay (IFA) for IgG and IgM (Vircell, Granada, Spain),enzyme-linked immunosorbent assay (ELISA) for IgG(Platelia BioRad, Marnes-la-Coquette, France) and IgA(Virotech, Russelsheim, Germany), and capture-ELISA forIgM (Platelia BioRad). Toconfirm thespecificityofIgMandIgAantibodydetection,theWesternblottechnique(Virotech)wasadditionallyperformed.Thismethodiscurrentlythemostspecific for detecting anti-M. pneumoniae antibodies [3]. ForIgM, a positive result requires ≥9 bands including P1protein; for IgA, >24 bands are required including P1protein. In the present study a current or recent infectionwas considered to have occurred definitely if at least two ofthe following criteria were met: (1) positive PCR and/orculture, (2) positive IgM antibodies (in the first and/or thesecond sample), (3) seroconversion or significant increase ofIgG antibodies (fourfold increase for IFA, twofold increasefor ELISA), or high IgG titers >40 AU/ml for ELISA.M. pneumoniae was detected by PCR in the throat-swabspecimens of 11 of 75 patients, by culture in three and bythe antigen detection test in only one. All culture-positivesamples were also positive by PCR. The complete resultsobtained using the various methods for the samples of eachof the 75 children studied are shown in Table 1.According to the diagnostic criteria, a definite diagnosisof current or recent M. pneumoniae infection was obtainedfor a total of 12 of the 75 pediatric patients (Table 1; patients1–12; ten with pneumonia and two with pharyngitis). Four

Accumulation of carbapenem resistance mechanisms in VIM-2-producing Pseudomonas aeruginosa under selective pressure

Pseudomonas aeruginosa has the potential to achieve resistance to carbapenems via the acquisition of carbapenemase-encoding genes, the downregulation of the OprD porin, the overexpression of efflux systems and the overproduction of cephalosporinases. One hundred and fifty carbapenem-non-susceptible isolates from 2008 to 2010 were screened for carbapenemase production, OprD porin loss, efflux pumps overexpression and inducible AmpC beta-lactamase production. For comparison reasons, the presence of the same mechanisms was also assessed in a previous collection of 30 carbapenem-non-susceptible P. aeruginosa isolated between 2003 and 2005. Results showed the accumulation of various resistance mechanisms among VIM-2 producers isolated between 2008 and 2010 with a parallel considerable increase in imipenem MIC90 and the geometric mean of the MIC values of imipenem and meropenem between the two study groups. The accumulation of carbapenem resistance mechanisms highlights the potential of this formidable pathogen for evolutionary success under antibiotic selective pressure.