Maria Exindari

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.

Porous dressings of modified chitosan with poly(2-hydroxyethyl acrylate) for topical wound delivery of levofloxacin

Absorbable and non-absorbable dressings have been fabricated into sponges via a modified thermally induced phase separation method, using a grafted derivative of chitosan with 2-hydroxyethylacrylate (CS-g-PHEA). The material was synthesized via free-radical polymerization and was characterized with FT-IR and (1)H NMR spectroscopies. The swelling ability, biocompatibility and biodegradability of the dressings were evaluated through in vitro assays while antibacterial studies were performed using three different bacterial strains, Methicillin susceptible Staphylococcus aureus (MSSA), Methicillin resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. Levofloxacin was used as model drug at different concentrations. Morphological characterization of the drug loaded dressings was performed by scanning electron microscopy, while drug-matrix interactions were evaluated by FT-IR spectroscopy. X-ray diffraction studies were carried out for the identification of the physical state for both neat and drug loaded materials. The prepared dressings showed a significant inhibition zone of the bacteria indicating the antibacterial property of the materials and loaded sponges.

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.

Influenza vaccine effectiveness against laboratory confirmed influenza in Greece during the 2013–2014 season: A test-negative study

BACKGROUND In 2013-2014 Greece experienced a resurgence of severe influenza cases, coincidental with a shift to H1N1pdm09 predominance. We sought to estimate Vaccine Effectiveness (VE) for this season using available surveillance data from hospitals (including both inpatients and outpatients). METHODS Swab samples were sent by hospital physicians to one of three laboratories, covering the entire country, to be tested for influenza using RT-PCR. The test-negative design was employed, with patients testing positive serving as cases and those testing negative serving as controls. VE was estimated using logistic regression, adjusted for age group, sex, region and calendar time, with further adjustment for unknown vaccination status using inverse response propensity weights. Additional age group stratified estimates and subgroup estimates of VE against H1N1pdm09 and H3N2 were calculated. RESULTS Out of 1310 patients with known vaccination status, 124 (9.5%) were vaccinated, and 543 patients (41.5%) tested positive for influenza. Adjusted VE was 34.5% (95% CI: 4.1-55.3%) against any influenza, and 56.7% (95% CI: 22.8-75.7%) against H1N1pdm09. VE estimates appeared to be higher for people aged 60 and older, while in those under 60 there was limited evidence of effectiveness. Isolated circulating strains were genetically close to the vaccine strain, with limited evidence of antigenic drift. CONCLUSIONS These results suggest a moderate protective effect of the 2013-2014 influenza vaccine, mainly against H1N1pdm09 and in people aged 60 and over. Vaccine coverage was very low in Greece, even among groups targeted for vaccination, and substantial efforts should be made to improve it. VE can and should be routinely monitored, and the results taken into account when deciding on influenza vaccine composition for next season.

Molecular and phylogenetic analysis and vaccine strain match of human influenza A(H3N2) viruses isolated in Northern Greece between 2004 and 2008.

Influenza A viruses are characterized by a unique genome structure, causing genetic instability, especially to the genes of haemagglutinin and neuraminidase. The objectives of this research was molecular and phylogenetic analysis of influenza A(H3N2) strains that circulated in Northern Greece since 2004, particularly the identification of sequence variations and the comparison of circulating viruses with vaccine strains. Since 2004 in Northern Greece, a total of 216 clinical samples were positive for influenza virus infections, of which 83 (38.4%) were attributed to influenza A(H3N2). Molecular analysis of the HA genes of 23 isolates showed that all circulating strains had variations at antigenic sites. Receptor binding sites were conserved in 2004-2005 and 2005-2006 strains whereas a variation was observed in all 2006-2007 strains (H195Y). Furthermore, alternative amino acids for sialic acid receptor binding sites were observed in most of the 2004-2006 isolates. Some amino acid substitutions were also observed at the neuraminidase sequences, which however had no effect on the antigenicity of the viruses. Phylogenetic analysis of each years circulating strains revealed a relatively low match with the vaccine strains A/Fujian/411/02 and A/California/7/04 for 2004-2005 and 2005-2006, respectively, whereas most 2006-2007 isolates match with the vaccine strain, A/Wisconsin/67/05. This year, unique variations were observed at antigenic and glycosylation sites of A/Serres/77/07-like stains. Constant surveillance of yearly variations is of great importance, so that vaccine strains can be evaluated.

Challenges in Antigenic Characterization of Circulating Influenza A(H3N2) Viruses during the 2011-2012 Influenza Season: an Ongoing Problem?

ABSTRACT Genetic and antigenic characterization of 37 representative influenza A(H3N2) virus strains isolated in Greece during the 2011-2012 winter season was performed to evaluate matching of the viruses with the seasonal influenza vaccine strain A/Perth/16/2009. Hemagglutinin gene sequence analysis revealed that all Greek strains clustered within the Victoria/208 genetic clade. Furthermore, substitutions in the antigenic and glycosylation sites suggested potential antigenic drift. Our hemagglutination inhibition (HI) analysis showed that the Greek viruses were Perth/16-like; however, these viruses were characterized as Victoria/208-like when tested at the United Kingdom WHO Collaborating Centre (CC) with HI assays performed in the presence of oseltamivir, a finding consistent with the genetic characterization data. Variability in the HI test performance experienced by other European laboratories indicated that antigenic analysis of the A(H3N2) virus has limitations and, until its standardization, national influenza reference laboratories should include genetic characterization results for selection of representative viruses for detailed antigenic analysis by the WHO CCs.

Genetic analysis of post-pandemic 2010–2011 influenza A(H1N1)pdm09 hemagglutinin virus variants that caused mild, severe, and fatal infections in Northern Greece

Since its appearance, influenza A(H1N1)pdm09 caused considerable morbidity and mortality in Northern Greece. Genetic analysis of post‐pandemic circulating strains scoped to investigate any correlation between genetic variations that emerged during viral evolution and severity of infection. Pharyngeal swabs/aspirates (n = 1,870) were examined with real‐time reverse transcription‐polymerase chain reaction. Hemagglutinin sequences were analyzed on 110 strains (37 fatal/73 non‐fatal cases), followed by statistical and phylogenetic analysis. Influenza A(H1N1)pdm09 was detected in 848 samples. Coexistence of clusters 3, 4, 5, 6, and 7 indicated co‐circulation of lineages in Northern Greece. Genetic analysis showed that HA sequences had 96–99% sequence similarity with the vaccine strain and that there was no association between any co‐circulating lineage and severity. Several viruses accumulated variations in HA antigenic sites. D222G was significantly associated with fatal infections, supporting its association with increased viral pathogenesis. On the other hand, four variations were associated with milder disease outcomes. Certain signature amino acid changes persisted during and/or after the pandemic, indicating their offer of selective advantages to the virus. Negative selection was observed in 70% of pandemic variations as they probably did not contribute to the virus fitness. It is of interest that persistent variations were highly identified in the vicinity of antigenic or receptor‐binding sites. Of those, K171R was associated only with fatal infections. Also of interest, only strains that were isolated from fatal infections had variations that altered both their acid–base and polarity properties. Genetic changes that may alter the antigenicity, pathogenicity and transmissibility of circulating virus variants need to be determined and closely monitored. J. Med. Virol. 87: 57–67, 2015.

Ιnfluenza A(H3N2) genetic variants in vaccinated patients in northern Greece

BACKGROUND Influenza A(H3N2) viruses predominated during the influenza 2016/2017 season and showed extensive genetic diversification. A high vaccination failure rate was noticed during the 2016/17 season in Greece, especially among the elderly. OBJECTIVES The scope of the study was to investigate the genetic characteristics of A(H3N2) circulating viruses and viruses detected in vaccinated patients. STUDY DESIGN Virus samples originated from vaccinated and unvaccinated patients, obtained at the National Influenza Centre for northern Greece. Phylogenetic analysis and comparison of the haemagglutinin gene of the viruses to the vaccine virus A/Hong Kong/4801/2014 was performed. RESULTS The majority of analysed viruses are clustering in the genetic clade 3C.2a, and in a newly emerged subclade, designated as 3C.2a1. The highest proportion of viruses detected in vaccinated patients fell into a distinct subcluster within the 3C.2a1 subclade, which is characterised by the amino acid substitutions N122D and T135K in haemagglutinin. CONCLUSIONS Viruses that belong to the 3C.2a clade are generally considered to resemble antigenically to the northern hemisphere vaccine component A/Hong Kong/4801/2014 that was recommended by WHO to be included also into the 2017/18 vaccine. However, viruses belonging to a specific 3C.2a1 subcluster was extensively circulating in northern Greece and among vaccinated individuals. Both substitutions carried by this strain were located on antigenic sites and caused losses of N-linked glycosylation sites of the virus, which could potentially affect viral antigenicity. Further studies are needed to determine the antigenicity of this variant strain and its possible implication in vaccine effectiveness.

Influenza vaccine effectiveness in preventing hospitalizations with laboratory‐confirmed influenza in Greece during the 2014–2015 season: A test‐negative study

The 2014–2015 influenza season was marked by circulation of antigenically drifted A/H3N2 strains, raising the possibility of low seasonal influenza Vaccine Effectiveness (VE). We assessed VE against hospitalization with laboratory‐confirmed influenza for the 2014–2015 season, using routine surveillance data. Non‐sentinel swab samples from Greek hospital inpatients were tested for influenza by RT‐PCR in three laboratories, covering the entire country. We estimated VE using a test‐negative design. Out of 883 patients with known vaccination status, 161 (18.2%) were vaccinated, and 392/883 patients (44.4%) tested positive for influenza, of whom 162 (41.3%) had type B and 151 (38.5%) had A/H3N2. Adjusted VE was 31.6% (95%CI: 2.9–51.8%) against any influenza, 46.8%, 95%CI: 12.5–67.6%) against type B and −1.9%, 95%CI: −69.5 to 38.7%) against A/H3N2. VE against non‐ICU hospitalization appeared to be higher, but the difference did not reach statistical significance. Circulating A/H3N2 viruses showed substantial antigenic drift, while about half of the type B strains were similar to the vaccine strain. Despite the antigenic drift of the A/H3N2 strains, the vaccine still offered substantial protection against hospitalization with laboratory‐confirmed influenza, mostly due to a surge in type B influenza late in the season. Vaccine coverage was low, even among groups targeted for vaccination, and considerable effort should be made to improve it. J. Med. Virol. 88:1896–1904, 2016.

Molecular and phylogenetic analysis of Greek measles 2010 strains

Although elimination of measles virus (MV) by 2010 was a revised target, a new epidemic has been ongoing in Greece and other European countries. The purpose of this study was the molecular and phylogenetic analysis of the Greek MV circulating strain. Twenty-four MV strains isolated from clinical samples during the 2010 outbreak were genotyped and studied in terms of nucleotide variation and phylogeny. All of the detected viruses were of the D4 genotype, which is circulating in Greece in the Roma population of Bulgarian nationality, the Greek Roma population and the Greek non-minority population, as well as in other EU countries. Phylogenetic analysis revealed that these viruses belonged to subgroup 4 of D4 MV strains. It is essential to continue epidemiological surveillance of measles in Greece to monitor the transmission pattern of the virus and the effectiveness of measles immunization, which eventually will lead to its elimination.