Decreased diversity of salivary microbiome in patients with stable decompensated cirrhosis.

BACKGROUND: In the setting of the oral-gut-liver axis, microbiome dysbiosis has been associated with decompensated cirrhosis progression. However, little is known on salivary microbiome profiles in stable decompensated patients. METHODS: We studied patients with stable decompensated cirrhosis (n =28) and matched healthy controls (n =26). There were five patients (17.8 %) with hepatocellular carcinoma (HCC). Microbiomes of the 54 salivary samples were profiled through next-generation sequencing of the 16S-rRNA region in bacteria. RESULTS: The two study groups (patients and controls) did not differ significantly concerning their baseline characteristics. The most abundant phyla were Firmicutes, Bacteroidetes, Proteobacteria, and Fusobacteria. Proposed dysbiosis ratio Firmicutes/Bacteroidetes was lower in patients than in controls (range: 0.05-2.54 vs. 0.28-2.18, p =0.4), showing no statistical significance. Phylum Deinococcus-Thermus was detected only in controls, while Phylum Planctomycetes only in patients. A-diversity analysis indicated low diversity of salivary microbiome in decompensated patients and patients with HCC, who presented specific discriminative taxa. On principal coordinate analysis (PCoA), the patients' and controls' salivary microbiomes clustered apart, suggesting differences in community composition (PERMANOVA test, p =0.008). Boruta wrapper algorithm selected the most representative genera to classify controls and patients (area under the curve =0.815). CONCLUSIONS: Patients with stable decompensated cirrhosis of various etiology and history of complications have decreased diversity of their salivary microbiome. PCoA and Boruta algorithm may represent useful tools to discriminate the salivary microbiome in patients with decompensation. Further studies are needed to establish the utility of salivary microbiome analysis, which is easier obtained than fecal, in decompensated cirrhosis. HIPPOKRATIA 2020, 24(4): 157-165.

Genetic diversity of mycobacterium tuberculosis in northern Greece.

The aim of the present study was to describe the genetic diversity of Mycobacterium tuberculosis (M.tuberculosis) strains circulating in the region of Northern Greece. A total of thirty-seven M. tuberculosis clinical isolates were analysed by the spoligotyping method. According to the results, six clusters comprising seventeen strains were detected, and the remaining twenty strains showed unique patterns. The M.tuberculosis families according to SITVITWEB were distributed as follows: Haarlem (H) (27.0%); T (24.3%); Beijing (13.5%); Latin-America and Mediterranean (LAM) (5.4%) and S (2.7%). The remaining isolates (27%) did not match any isolates within the database and they were characterized as orphans. Regarding GenoType MTBDRplus results, two strains (5.4%) were Multi-Drug-Resistant, four strains (10.8%), were isoniazid monoresistant, while the remaining thirty-one strains (83.8%) were susceptible. In conclusion, in the region of Macedonia-Thrace (Northern Greece), there was high phylogenetic diversity among M. tuberculosis isolates. Molecular tools used and data presented can have regional and national impact on tuberculosis control.

16S rRNA deep sequencing for the characterization of healthy human pharyngeal microbiome.

BACKGROUND: The recent advent of high-throughput sequencing methods enabled the study of the composition of the upper respiratory tract (URT) microbial ecosystem and its relationship with health and disease in immense detail. The aim of the present study was the characterization of the human pharyngeal microbiome of healthy individuals in Greece. MATERIALS AND METHODS: We obtained ten pharyngeal specimens from healthy volunteers, Greek resident, with Greek nationality, who were eligible to the selection criteria. The construction of DNA libraries was performed by using two primer sets that amplify selectively the corresponding hypervariable regions of the 16s region in bacteria (V2-V9). The Ion Torrent PGM platform was used for the performance of next-generation sequencing. RESULTS: In the study samples, twelve phyla were identified. The most abundant ones were Firmicutes, Proteobacteria, Bacteroidetes, followed by Actinobacteria and Fusobacteria. Seventy-nine families, 79 genera and 137 species were identified and characterized. Moreover, 17 unique differentially abundant families, 30 unique differentially abundant genera and 24 unique differentially abundant species were identified among healthy subgroups with adjusted p-values <0.05. At the genus level, Moraxella (Proteobacteria) and Gemella (Firmicutes) were detected with a statistical significance in non-smokers, while Bifidobacterium (Actinobacteria), Alloscardovia (Actinobacteria), Dialister (Firmicutes) and Filifactor (Firmicutes) were present mostly in smokers. CONCLUSIONS: The URT is colonized by a variety of protective and potentially pathogenic bacteria. This microbiome system is highly diverse and varies significantly between individuals. Geographic location and ethnicity are considered to be a strong determinants and factors affecting the diversity and abundance of the URT microbiome. Although some of the most abundant families are common irrespective of these factors, the dominance patterns are usually different between the study subjects and between the studies from other geographic locations. Unique differentially abundant families, genera and species were identified, and further studies are needed to elucidate their role. Further studies should focus on the investigation of the URT microbiome dynamics and the interaction with the host in health and disease. HIPPOKRATIA 2018, 22(1): 29-36.

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.

Severity of the two post-pandemic influenza seasons 2010-11 and 2011-12 in Northern Greece.

Since the pandemic in 2009, the two following post pandemic influenza seasons have been associated with increased morbidity and mortality rates in Northern Greece. The scope of this short report is to provide relevant epidemiological information, an evaluation of the efficacy of the seasonal vaccine and antiviral drugs. Molecular analysis revealed the close match of A(H1N1) 2009 pandemic and post-pandemic 2010-11 viruses with the vaccine virus. However, a proportion of the influenza A(H3N2) and B viruses that circulated in 2011-12 differed from the vaccine strains, possibly explaining the high hospitalization and fatality rates. There is also a trend of new virus strains to accumulate amino acid changes. Only A(H1N1) viruses resistant to oseltamivir have been detected. Constant epidemiological and molecular surveillance is essential to monitor the efficacy of the vaccine and antiviral drugs and assess the severity of each influenza season.

Laboratory surveillance of influenza virus in children under 10 years old, in northern Greece, during 2004-2010.

OBJECTIVES: Influenza viruses constitute one of the most common pathogens that cause acute respiratory disease in children. The aim of this study is to present the contribution of influenza viruses to influenza-like illness (ILI) in children (aged<10 years old) in Northern Greece during six influenza seasons (2004-2010). MATERIALS AND METHODS: 1,242 pharyngeal swabs or/and washes from children younger than 10 years old, presented as ILI infections during the last six influenza seasons (2004-2005, 2005-2006, 2006-2007, 2007-2008, 2008-2009 and 2009-2010) were examined for influenza A and B by Real-time one step RT-PCR. RESULTS: Influenza viruses were detected in 431 (34.7%) of the 1,242 specimens. In a total of 372 specimens were positive for influenza A and 58 for influenza B. The majority of the infected young patients were 6-10 years old (51.9%). CONCLUSION: Our results show that in N. Greece, influenza viruses type A and B contribute to ILI presenting infections at a rate of 34.7 % in children younger than 10 years old.

Prevalence and antimicrobial susceptibility of Ureaplasma urealyticum in asymptomatic women in Northern Greece.

AIM: This study attends to determine the prevalence and the antimicrobial susceptibility of Ureaplasma urealyticum in asymptomatic women in Northern Greece. MATERIALS-METHODS: A total of 347 cervical smears were divided into 5 groups according to age. Samples were cultured, Ureaplasma urealyticum isolation was confirmed by the detection of the urease gene and the antimicrobial susceptibility of the isolates to 9 commercially available antibiotics was determined. RESULTS: Fifty-six (16.13%) samples were found positive. Women >60 years old were rarely infected. Only 9 (16.07%) isolates were susceptible to all antimicrobials tested. Thirteen isolates were resistant and 34 intermediately resistant to ciprofloxacin, two resistant and 29 intermediately resistant to ofloxacin, three intermediately resistant to macrolides, while all were susceptible to tetracyclines. CONCLUSIONS: In Northern Greece, 16.13% of asymptomatic women seem to be carriers of Ureaplasma urealyticum mostly in reproductive ages. Almost no resistance to macrolides and tetracyclines has been noticed, while most of the isolates present resistance or moderate sensitivity to quinolones.

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.

Epidemiological and clinical characteristics of human parvovirus B19 infections during 2006-2009 in Northern Greece.

Background. Parvovirus B19 infects children and adults, often causing erythema infectiosum, polyarthritis, but also aplastic crisis in patients with chronic haemolytic anaemia, rash, fever and fetal hydrops or fetal death. This study aims at the detection of acute parvovirus B19 infections during 2006-2009 in northern Greece on epidemiological and clinical aspect.Material and methods. Specimens were obtained from 63 patients, who addressed to hospitals, suspected for acute parvovirus B19 infection (17 in 2006, 29 in 2007, 10 in 2008 and 7 in 2009). Thirty (47.6%) were children (one day - 15 years old) and 33 (52.4%) were adults (16-65 years old). The infection was shown by PCR in whole blood and/or pleural fluid and supported by detection of specific IgM antibodies in the patients' blood serum, which was performed by ELISA.Results. Twenty (31.7%) out of the 63 specimens were found to be positive: 3/17 (17.6 %) in 2006, 16/29 (55.2 %) in 2007, none in 2008 and 1/7 (14.3%) in 2009, p=0.0002. Positive children were found 10/30 (33.3%) and positive adults 10/33 (30.3%). Specific IgM antibodies were detected in all 20 positive patients. Children developed hematological disorders, mainly types of anemia (6 cases), hydrothorax/ascites (2 cases), arthritis (1 case), and liver transplant rejection (1 case). Adults were presented with pregnancy complications (2 cases), arthralgia/arthritis (4 cases), febrile syndromes (3 cases) and atypical rash (1 case).Conclusions. In conclusion, an annual variation in the circulation of parvovirus B19 was noticed, presenting an increase of acute infections in northern Greece during 2007. Regarding serious cases, although children and adults seemed equally affected, differences in clinical manifestations were observed between them, with hematological dysfunctions predominant in childhood.

Oseltamivir-resistant influenza A pandemic (H1N1) 2009 virus in Northern Greece.

Resistance to oseltamivir was observed to influenza A pandemic (H1N1) 2009 virus strains, isolated from two patients in North Greece. Investigations showed resistant viruses with the neuraminidase (NA) 275Y genotypes. Pandemic A (H1N1) 2009 virus should be closely monitored for emergence of resistant variants.

Contribution of human metapneumovirus to influenza-like infections in North Greece, 2005-2008.

Following its detection in 2001, human metapneumovirus (hMPV) has repeatedly been reported as a respiratory pathogen, especially in children. This study was aimed at determining the proportion of hMPV infections in patients with influenza-like illness (ILI) during the three influenza seasons 2005-6, 2006-7, 2007-8 in northern Greece. We collected 380 nasopharyngeal swabs or aspirates from ILI patients during the winter seasons 2005-2008 and examined them for influenza viruses and hMPV by one-step real time RT-PCR and nested RT-PCR. Influenza viruses were detected in 151 of the 380 specimens (39.7%) and hMPV in 23 of them (6.05%). Co-infections with hMPV and influenza viruses were observed in seven cases. The majority of the ILI patients (60.5%) were 0-18 years-old. However, the incidence of influenza was slightly higher in the age group of 19-60-year-olds, while the incidence of hMPV infections was higher in the age group of 0-18-year-olds. We conclude that hMPV plays an important role as a contributor in influenza-like infections, especially in children. It circulates in the community during influenza seasons and its clinical appearance can be confused with influenza symptoms. However, further research is needed to elucidate the quantitative and qualitative importance of hMPV infections.

Cluster of new influenza A(H1N1) cases in travellers returning from Scotland to Greece - community transmission within the European Union?

On 26 and 27 May, the Hellenic Centre for Disease Control and Prevention in Greece reported two confirmed cases of new influenza A(H1N1) virus infection in travellers returning from Scotland. The two cases had no apparent traceable links to an infectious source. Herein we report details of the two cases and potential public health implications.

Avian influenza A(H5N1)--current situation.

The A(H5N1) influenza virus has re-emerged in 2003 in Asia, Africa, the Pacific Region as well as Europe and since then has become endemic in some countries. The virus is usually highly pathogenic and is associated with high morbidity and overall mortality rates that reach 61%.

Influenza A(H5N1): an overview of the current situation.

Influenza viruses continue to threaten the world with a new pandemic. While currently attention is focused on the newly emerged A(H1N1) virus, the avian influenza A(H5N1) virus is still a cause of concern. Extended research is focused on the genetic evolution of the viruses, as well as their susceptibility to available antiviral drugs. One of the major priorities of the World Health Organization is to develop candidate vaccines, four of which are already licensed for use in the European Union. Since the last influenza pandemic in 1968, our knowledge of the influenza virus and its biology has greatly increased, revealing new avenues in the research for antiviral strategies and the development of effective vaccines.

Antiviral resistance of influenza A (H3N2) strains isolated in northern Greece between 2004 and 2007.

The prevention and control of influenza with vaccines and antiviral drugs is of great importance. M2 inhibitors, amantadine and rimantadine have been extensively used in some countries. The next generation of antiviral drugs, neuraminidase (NA) inhibitors oseltamivir and zanamivir, are being stockpiled for a potential influenza pandemic. The emergence of resistant strains is thus an important issue. The purpose of this study was to examine the sensitivity to M2 and NA inhibitors of Greek influenza A(H3N2) strains isolated during three influenza seasons between 2004 and 2008 and to determine the phylogenetic clades of those strains. M2 and NA sequences of 34 patient isolates were checked for known resistance mutations. In addition, haemagglutinin (HA) sequences were used to determine the phylogenetic relationship between resistant and sensitive strains. All influenza A(H3N2) strains isolated during the season 2004-5 were found susceptible to adamantanes, bearing the S31N mutation, compared to 88% of the strains isolated in 2005-6 and 75% of the strains isolated in 2006-7. Molecular analysis of the HA gene showed a correlation of the mutants with specific phylogenetic clades. No known mutations in the NA or HA gene that have been implicated in resistance to NA inhibitors were found in the A(H3N2) strains isolated in the three influenza seasons. Despite the fact that amantadine is the only drug approved for prophylaxis in Greece, it has not been extensively used. So it seems that resistant strains circulating in the area after 2005 followed the global trend of replacement of susceptible strains by resistant ones. Oseltamivir and zanamivir are currently approved only for therapeutic use in Greece and has not been extensively used either.

Rubella immunity and vaccination coverage of the population of northern Greece in 2006.

This study was prompted by two rubella outbreaks that occurred in northern Greece in the last decade (1993 and 1999) and by periodic changes to the immunisation strategy. It was designed to determine the current status of rubella immunity and vaccination coverage in this region, eight years after the last outbreak in 1999 and seven years after the last epidemiological study in the area. Among the 685 subjects studied the seroprevalence was 83.7% and the total vaccination rate was 31.3%. In people born before the introduction in 1989 of the measles/mumps/rubella (MMR) vaccine into the national immunisation programme, higher rates of rubella seropositivity (88.1%) were observed compared to those born after 1989 (77.1%). The vaccination rates for these age groups were 14.8% and 58.1%, respectively. The reason for this difference is the lack of vaccination at the time these people were children, and it underlines the need for a vaccination strategy targeting older people as well. Among women of reproductive age (16-40 years), who represented 44.8% of the study population, 13.9% were susceptible to rubella and only 18.5% were vaccinated. These results indicate that there is a great need for a comprehensive policy designed to protect mostly young adults and women of childbearing age in order to prevent congenital rubella infections. This policy should also include competent surveillance systems for rubella and congenital rubella syndrome and an evaluation of existing immunisation programmes.

Greek measles epidemic strain, 2005-2006.

The purpose of this work was the molecular study of the virus strain that caused the last measles outbreak in Greece. Twenty-four saliva specimens were obtained from selected patients serologically confirmed as measles cases between December 2005 and March 2006. Measles virus (MV) detection was performed by a nested RT-PCR. The 447-bp segment of the N gene of these MV strains was used for genotyping. The N gene sequences of the Greek MV strains were identical to each other, therefore a phylogenetic tree was constructed using one representative MV (ThesGRE/06). Our data show that the MV strain which caused the 2005-2006 outbreak in Greece belongs to genotype D6, and differs by 0.68% from the New Jersey D6 strain and by 5.5% from the MV vaccine strain Edmonston B (U03656) belonging to genotype A.

A pseudo-outbreak of human A/H5N1 infections in Greece and its public health implications.

The recent wide geographic spread of the highly pathogenic avian influenza A/H5N1 virus has important public health implications. Several wild migratory birds were confirmed to be infected with avian influenza A/H5N1 in Greece in February and March 2006. The aim of this paper is to report data from potential H5N1 human cases that presented to local hospitals during this period with a respiratory infection and expressing concern about exposure to avian influenza. A case-control investigation was conducted that included case identification with the use of a structured definition, review of epidemiological and clinical characteristics and molecular testing for avian influenza A/H5N1. The setting was the entire country of Greece during February and March 2006. The main outcomes were rates of possible cases (meeting both a clinical and an epidemiological criterion) and clinical or epidemiological characteristics differentiating them from potential cases that met either one of the criteria of a possible case, but not both. Twenty six potential patients (81% of whom met a clinical criterion, and 39% of whom met an epidemiological criterion) presented and most (85%) were admitted in local hospitals during the period of interest. The majority of cases (85%) were observed in northern Greece where most of the confirmed A/H5N1 avian cases were documented. Five of the 26 evaluated patients met the definition of a possible case. These clustered within the early period of confirmed A/H5N1 cases in wild migratory birds (P=0.05). Molecular testing was negative for all possible cases. Application of a revised case definition constructed according to newer European Union guidance resulted in the exclusion of two possible cases. Several potential A/H5N1 human cases were recently identified in Greece. Both the timing of identification and the geographical location of potential cases suggest an increased awareness on the part of the general public, as well as poor interpretation of the case definition by the clinicians.

A pseudo-outbreak of human A/H5N1 infections in Greece and its public health implications.

The recent wide geographic spread of the highly pathogenic avian influenza A/H5N1 virus has important public health implications. Several wild migratory birds were confirmed to be infected with avian influenza A/H5N1 in Greece in February and March 2006. The aim of this paper is to report data from potential H5N1 human cases that presented to local hospitals during this period with a respiratory infection and expressing concern about exposure to avian influenza. A case-control investigation was conducted that included case identification with the use of a structured definition, review of epidemiological and clinical characteristics and molecular testing for avian influenza A/H5N1. The setting was the entire country of Greece during February and March 2006. The main outcomes were rates of possible cases (meeting both a clinical and an epidemiological criterion) and clinical or epidemiological characteristics differentiating them from potential cases that met either one of the criteria of a possible case, but not both. Twenty six potential patients (81% of whom met a clinical criterion, and 39% of whom met an epidemiological criterion) presented and most (85%) were admitted in local hospitals during the period of interest. The majority of cases (85%) were observed in northern Greece where most of the confirmed A/H5N1 avian cases were documented. Five of the 26 evaluated patients met the definition of a possible case. These clustered within the early period of confirmed A/H5N1 cases in wild migratory birds (P=0.05). Molecular testing was negative for all possible cases. Application of a revised case definition constructed according to newer European Union guidance resulted in the exclusion of two possible cases. Several potential A/H5N1 human cases were recently identified in Greece. Both the timing of identification and the geographical location of potential cases suggest an increased awareness on the part of the general public, as well as poor interpretation of the case definition by the clinicians.